Molecular Biology
Depiction of the P22 bacteriophage virion as solved by electron
cryomicroscopy. This structure reveals the major components of
the infection machinery, as well as the manner in which DNA is
spooled coaxially within the icosahedral capsid. The contour level
is set so that the cross section of individual dsDNA strands can
be seen with only the outer, most ordered shell of DNA visible.
A dodecameric structure at the center of the infection machinery
functions as a pressure sensor, which during the packaging of DNA
during virus maturation, sends a termination signal to the packaging
machinery once the capsid has been fully packaged. Reconstruction
and graphics by graduate student Gabriel Lander. Work done in
the laboratory of John E. Johnson, Ph.D., in collaboration with
Bridget Carragher and Clint Potter, Department of Cell Biology,
National Research Resource for Automated Microscopy.
Kurt Wüthrich, Ph.D.
Cecil H. and Ida M. Green
Professor of Structural Biology
MOLECULAR BIOLOGY
2006
MOLECULAR BIOLOGY
H. Jane Dyson, Ph.D.
Professor
S TA F F
John H. Elder, Ph.D.***
Professor
DEPAR TMENT OF
Peter E. Wright, Ph.D.*
Professor and Chairman
Cecil H. and Ida M. Green
Investigator in Medical
Research
Ruben Abagyan, Ph.D.
Professor
Martha J. Fedor, Ph.D.*
Associate Professor
James Arthur Fee, Ph.D.
Professor of Research
Elizabeth D. Getzoff,
Ph.D.****
Professor
Carlos F. Barbas III,
Ph.D.*****
Professor
Janet and W. Keith Kellogg II
Chair
David B. Goodin, Ph.D.
Associate Professor
Rajesh Belani, Ph.D.
Adjunct Assistant Professor
Joel M. Gottesfeld, Ph.D.
Professor
Ola Blixt, Ph.D.
Assistant Professor of
Molecular Biology
Michael N. Boddy, Ph.D.
Assistant Professor
Charles L. Brooks III, Ph.D.
Professor
David A. Case, Ph.D.
Professor
Geoffrey Chang, Ph.D.*
Associate Professor
Jerold Chun, M.D., Ph.D.***
Professor
Luis De Lecea, Ph.D.**
Associate Professor
Stanford University
Palo Alto, California
Aymeric Pierre De Parseval,
Ph.D.
Assistant Professor of
Molecular Biology
Lluis Ribas De Pouplana,
Ph.D.
Adjunct Assistant Professor
Barcelona Science Park
Barcelona, Spain
Ashok Deniz, Ph.D.
Assistant Professor
David S. Goodsell Jr., Ph.D.
Associate Professor
Jennifer Harris, Ph.D.
Assistant Professor of
Biochemistry
Christian A. Hassig, Ph.D.
Adjunct Assistant Professor
Kalypsis, Inc.
San Diego, California
Peter B. Hedlund, M.D., Ph.D.
Assistant Professor of
Molecular Biology
Mirko Hennig, Ph.D.**
Assistant Professor
Medical University of South
Carolina
Charleston, South Carolina
John E. Johnson, Ph.D.
Professor
Gerald F. Joyce, M.D.,
Ph.D.*****
Professor
Dean, Faculty
Ehud Keinan, Ph.D.
Adjunct Professor
Richard A. Lerner, M.D.,
Ph.D.*****
President, Scripps Research
Lita Annenberg Hazen Professor
of Immunochemistry
Cecil H. and Ida M. Green
Chair in Chemistry
THE SCRIPPS RESEARCH INSTITUTE
153
Scott Lesley, Ph.D.
Assistant Professor of
Biochemistry
Vaughn V. Smider, Ph.D.
Assistant Professor of
Molecular Biology
Tianwei Lin, Ph.D.
Associate Professor
Robyn L. Stanfield, Ph.D.
Assistant Professor
Clare McGowan, Ph.D. †
Associate Professor
James Steven, Ph.D.
Assistant Professor
Duncan E. McRee, Ph.D.
Adjunct Associate Professor
ActiveSight
San Diego, California
Raymond C. Stevens,
Ph.D. †††
Professor
David P. Millar, Ph.D.
Associate Professor
Louis Noodleman, Ph.D.
Associate Professor
Arthur J. Olson, Ph.D.
Professor
James C. Paulson,
Professor
Ph.D. ††
Vijay Reddy, Ph.D.
Assistant Professor
Steven I. Reed,
Professor
Ph.D. †
Paul Russell, Ph.D.
Professor
Michel Sanner, Ph.D.
Associate Professor
Harold Scheraga, Ph.D.
Adjunct Professor
George W. and Grace L. Todd
Professor of Chemistry,
Emeritus
Cornell University
Ithaca, New York
Paul R. Schimmel,
Ph.D.*****
Ernest and Jean Hahn
Professor of Molecular
Biology and Chemistry
Anette Schneemann, Ph.D.
Associate Professor
Charles D. Stout, Ph.D.
Associate Professor
Peiqing Sun, Ph.D.
Associate Professor
J. Gregor Sutcliffe, Ph.D.
Professor
John A. Tainer, Ph.D.*
Professor
Fujie Tanaka, Ph.D.
Assistant Professor
Elizabeth Anne Thomas,
Ph.D.
Assistant Professor
James R. Williamson,
Ph.D.*****
Professor
Associate Dean, Kellogg
School of Science and
Technology
Ian A. Wilson, D.Phil.*
Professor
Curt Wittenberg, Ph.D. †
Professor
Kurt Wüthrich, Ph.D.
Cecil H. and Ida M. Green
Professor of Structural
Biology
Xiang-Lei Yang, Ph.D.
Assistant Professor of
Molecular Biology
Subhash C. Sinha, Ph.D.*
Associate Professor
Todd O. Yeates, Ph.D.
Adjunct Professor
University of California
Los Angeles, California
Gary Siuzdak, Ph.D.
Adjunct Associate Professor
Qinghai Zhang, Ph.D.
Assistant Professor
154 MOLECULAR BIOLOGY
2006
SERVICE FACILITIES
Ryan Burnett, Ph.D.
John Chung, Ph.D.
Manager, Nuclear Magnetic
Resonance Facilities
Zhongguo Chen, Ph.D.
Gerard Kroon
Assistant Manager, Nuclear
Magnetic Resonance
Facilities
Michael E. Pique
Director, Computer Graphics
Development
Nahid Razi, Ph.D.
Assistant Core Manager,
Consortium for Functional
Glycomics
Brian Collins, Ph.D.**
Momenta Pharmaceuticals
Boston, Massachusetts
Adrienne Elizabeth Dubin,
Ph.D.
Maria Alejandra GamezAbascal, Ph.D.
Reto Horst, Ph.D.
Julio Kovacs, Ph.D.
THE SCRIPPS RESEARCH INSTITUTE
Beatriz Gonzalez Alonso,
Ph.D.
Ronald M. Brudler, Ph.D.††††
Lintao Bu, Ph.D.
David Alvarez-Carbonell,
Ph.D.**
Memorial Sloan-Kettering
Cancer Center
New York, New York
Tommy Bui, Ph.D.
Rosa Maria Cardoso, Ph.D.
Justin E. Carlson, Ph.D.
Juliano Alves, Ph.D.
Andrew Barry Carmel, Ph.D.
Yu An, Ph.D.
Qing Chai, M.D., Ph.D.
Brigitte Anliker, Ph.D.**
Institut für Biochemie und
Molekularbiologie II
Heinrich-Heine-Universitaet
Düsseldorf, Germany
Eli Chapman, Ph.D.
Amarnath Chatterjee, Ph.D.
Andrew James Annalora,
Ph.D.
Anju Chatterji, Ph.D.**
Bayer Healthcare,
Diagnostics Division
Berkeley, California
Roger Armen, Ph.D.
Susana Chaves, Ph.D.
Richard R. Rivera, Ph.D.
Joseph W. Arndt, Ph.D.
Kenji Sugase, Ph.D.
Karunesh Arora, Ph.D.
Mabelle Ashe, Ph.D.
S TA F F S C I E N T I S T S
Koji Tamura, Ph.D.**
Tokyo University of Science
Chiba, Japan
Anton Vladislavovich
Cheltsov, Ph.D.**
Burnham Institute
La Jolla, California
Svitlana Berezhna, Ph.D.
Xiaoqin Ye, M.D., Ph.D.
Jamie Mitchell Bacher, Ph.D.
Brian M. Lee, Ph.D.**
Southern Illinois University
Carbondale, Illinois
Dirk M. Zajonc, Ph.D.**
La Jolla Institute for Allergy
& Immunology
San Diego, California
Sung-Hun Bae, Ph.D.
Peter Sobieszcsuk, Ph.D.
Core Manager, Consortium
for Functional Glycomics
S E N I O R S TA F F S C I E N T I S T
Wayne A. Fenton, Ph.D.
Ying Chuan Lin, Ph.D.
Mikhail Popkov, Ph.D.**
Amunix, Inc.
San Jose, California
Maria Martinez-Yamout, Ph.D.
Garrett M. Morris, Ph.D.
Chiaki Nishimura, Ph.D.
Jeffrey Speir, Ph.D.
Manal Swairjo, Ph.D.**
Western University of Health
Sciences
Pomona, California
Mutsuo Yamaguchi, Ph.D.
Xueyong Zhu, Ph.D.
R E S E A R C H A S S O C I AT E S
Sunny Abraham, Ph.D.**
Ambit Biosciences
San Diego, California
Melanie Ann Adams, Ph.D.
Fabio Agnelli, Ph.D.
Moballigh Ahmad, Ph.D.**
University of Illinois
Urbana-Chapaign, Illinois
Alexander Ivanov Alexandrov,
Ph.D.
SENIOR RESEARCH
A S S O C I AT E S
David Barondeau, Ph.D.**
Texas A&M Unversity
College Station, Texas
Kirk Beebe, Ph.D.
Published by TSRI Press®. © Copyright 2006,
The Scripps Research Institute. All rights reserved.
Stephen G. Aller, Ph.D.
Marcius Da Silva Almeida,
Ph.D.**
Universidade Federal do Rio
de Janeiro
Rio De Janeiro, Brazil
Wojciech Augustyniak, Ph.D.
Manidipa Banerjee, Ph.D.
Christopher Baskerville,
Ph.D.**
Veterans Administration
Medical Center
San Diego, California
Jianhan Chen, Ph.D.
Yen-Ju Chen, Ph.D.
Zhiyong Chen, Ph.D.
Srinivas Chittaboina, Ph.D.
Jungwoo Choe, Ph.D.
Chung Jen Chou, Ph.D.
Li-Chiou Chuang, Ph.D.
Lipika Basummalick, Ph.D.**
current employer unknown
relocated to Bay Area
Jean-Pierre Clamme, Ph.D.**
Université Louis Pasteur
Strasbourg
Strasbourg, France
Konstantinos Beis, Ph.D.
Linda Maria Columbus, Ph.D.
Per Bengston, Ph.D.**
Lund University
Lund, Sweden
Stephen Connelly, Ph.D.
Christine, Beuck, Ph.D.
William Henry Bisson,
Ph.D.**
Burnham Institute
La Jolla, California
Adam Corper, Ph.D.
Qizhi Cui, Ph.D. ††††
la P. Da Costa, Ph.D.
Sanjib Das, Ph.D.**
Takeda
San Diego, California
David Boehr, Ph.D.
Surya Kanta De, Ph.D.
David Bostick, Ph.D.
Robert De Bruin, Ph.D.
MOLECULAR BIOLOGY
2006
THE SCRIPPS RESEARCH INSTITUTE
155
Sohela De Rozieres, Ph.D.**
Biomatrica
San Diego, California
Bong Kwan Han, Ph.D.**
University of California
San Diego, Calfornia
Dae Hee Kim, Ph.D.
Derrick Meinhold, Ph.D.
Eda Koculi, Ph.D.
Elena Menichelli, Ph.D.
Qingdong Deng, Ph.D.**
Klypsy, Inc.
San Diego, California
Byung Woo Han, Ph.D.
Bethany Koehntop, Ph.D.
Jonathan Mikolosko, Ph.D.
Shoufa Han, Ph.D.
Milka Kostic, Ph.D.
Peter J. Mikulecky, Ph.D.
Paula Desplats, Ph.D.
Wenge Han, Ph.D.
Irina Kufareva, Ph.D.
Mauro Mileni, Ph.D.
Claire Louise Dovey, Ph.D.
Shantanu Kumar, Ph.D.
Zhanna Druzina, Ph.D.
Jason W. Harger, Ph.D.**
Illumina, Inc.
San Diego, California
Susumu Mitsumori, Ph.D.**
Shionogi Research
Laboratories
Osaka, Japan
Li-Lin Du, Ph.D.
Rodney Harris, Ph.D.
Michelle Duquette-Huber,
Ph.D.
David M. Herman, Ph.D.
Scott Eberhardy, Ph.D.**
Schering-Plough
Union, New Jersey
Stephen Edgcomb, Ph.D.
Deron Herr, Ph.D.
Jason Lanman, Ph.D.
Wen-Xu Hong, Ph.D.
Kwan Hoon Hyun, Ph.D.
Daniel Felitsky, Ph.D.
Wonpil Im, Ph.D.**
University of Kansas
Lawrence, Kansas
Jinhyuk Lee, Ph.D.**
University of Kansas
Lawrence, Kansas
Tetsuji Mutoh, Ph.D.
June Hyung Lee, Ph.D.
Hung Nguyen, Ph.D.
Kelly Lee, Ph.D.
George Nicola, Ph.D.
Edward Lemke, Ph.D.
Tadateru Nishikawa, Ph.D.
Masanori Imai, Ph.D.
Chenglong Li, Ph.D.**
Ohio State University
Columbus, Ohio
Kyoko Noguchi, Ph.D.
Liao Liang, Ph.D.
Brian V. Norledge, Ph.D.
Severn School
Severna Park, Maryland
Veli-Pekka Jaakola, Ph.D.
Kai Jenssen, Ph.D.
Vasco Liberal, Ph.D.
William M. Lindstrom, Ph.D.
Yann Gambin, Ph.D.
Hui Gao, Ph.D.**
Arena Pharmaceuticals
San Diego, California
Eric C. Johnson, Ph.D.**
Bruker BioSpin Corporation
Fremont, California
Kunheng Luo, Ph.D.
Hui-Yue Christine Lo,
Ph.D. ††††
Ann MacLaren, Ph.D.
Hope Johnson, Ph.D.
Darly Joseph Manayani, Ph.D.
Shannon E. Gardell, Ph.D.
Margaret Alice Johnson, Ph.D.
Joshua Gill, Ph.D.
Susanna Juraja, Ph.D.
Edith Caroline Glazer, Ph.D.
Christian Kannemeier, Ph.D.
Bettina Groschel, Ph.D.
Mili Kapoor, Ph.D.
Fang Guo, Ph.D.**
Department of Immonology
Scripps Research
Seongho Moon, Ph.D.**
Samsung Electronic
Seoul, South Korea
Samrat Mukhopadhyay,
Ph.D.
Glenn C. Johns, Ph.D.
Ionian Technologies
Upland, California
Elsa D. Garcin, Ph.D.
Marissa Mock, Ph.D.
Chul Won Lee, Ph.D.
Kenichi Hitomi, Ph.D.
Li Fan, Ph.D.
Pierre Henri Gaillard, Ph.D.**
Centre National de la
Recherche Scientifique
Marseille, France
Emma Langley, Ph.D.
Chang-Wook Lee, Ph.D.
Yunfeng Hu, Ph.D.
Josephine Chu Ferreon, Ph.D.
Bianca Lam, Ph.D.
Joreg Hinnerwisch, Ph.D.
Susanna V. Ekholm-Reed,
Ph.D.
Allan Chris Merrera Ferreon,
Ph.D.
Sharon Kwan, Ph.D.
Andrey Aleksandrovich
Karyakin, Ph.D.
Yang Khandogin, Ph.D.
Jeff Mandell, Ph.D.
Maria Victoria MartinSanchez, Ph.D.**
University of Geneva
Geneva, Switzerland
Santiago Cavero Martinez,
Ph.D.
Sujatha Narayan, Ph.D.
Wataru Nomura, Ph.D.
Wendy Fernandez Ochoa,
Ph.D.**
University of California
San Diego, California
Amy Odegard, Ph.D.
Lisa Renee Olano, Ph.D.
Brian L. Olson, Ph.D.
Mary O’Reilly, Ph.D.
Brian Paegel, Ph.D.
Sandeep Patel, Ph.D.**
University of Delaware
Newark, Delaware
Stephanie Pebernard, Ph.D.
Min Guo, Ph.D.
Ilja V. Khavrutskii, Ph.D.
Hanna-Stina Martinsson
Ahlzén, Ph.D.
Mahender Gurram, Ph.D.
Reza Khayat, Ph.D.
Tsutomu Matsui, Ph.D.
Bill Francesco Pedrini, Ph.D.
Robert Pejchal, Ph.D.
156 MOLECULAR BIOLOGY
Vladimir Pelmenschikov,
Ph.D.
2006
Daniela Andrea Slavin, Ph.D.
Shun-ichi Wada, Ph.D. ††††
Elisabetta Soragni, Ph.D.
Ross Walker, Ph.D.
San Diego Supercomputer
La Jolla, California
Jefferson Perry, Ph.D.
Suzanne Peterson, Ph.D.
Jessica Petrillo, Ph.D.
oran Pljevaljcic, Ph.D.
Stephanie Pond, Ph.D.
Owen Pornillos, Ph.D.**
Celgene Corporation
San Diego, California
Daniel Joseph Price, Ph.D.**
GlaxoSmithKline
Research Triangle Park,
North Carolina
John Prudden, Ph.D.
Grazia Daniela Raffa, Ph.D.**
Università di Roma La
Sapienza
Rome, Italy
THE SCRIPPS RESEARCH INSTITUTE
Holly Heaslet Soutter, Ph.D.**
Pfizer Global Research &
Development
Ann Arbor, Michigan
Greg Springsteen, Ph.D.
Furman University
Greenville, South Carolina
S.V. Ramasastry Sripada,
Ph.D.
Thomas Steinbrecher, Ph.D.
Gudrun Stengel, Ph.D.**
University of Lund
Lund, Sweden
Shih-Che Su, Ph.D.
Magnus Sundstrom, Ph.D.
Blair R. Szymczyna, Ph.D.
Jessica Williams, Ph.D.
Robert Scott Williams,
Ph.D.
Wei Zhang, Ph.D.**
Southwest Medical Center
Houston, Texas
Yong Zhao, Ph.D.
Peizhi Zhu, Ph.D.**
University of Michigan
Arbor, Michigan
Eric L. Wise, Ph.D.
S C I E N T I F I C A S S O C I AT E S
Jonathan Wojciak, Ph.D.
Enrique Abola, Ph.D.
Vance Wong, Ph.D.
Andrew S. Arvai, M.S.
Timothy I. Wood, Ph.D.**
Walter Reed Army Medical
Center
Washington, D.C
Ognian V. Bohorov, Ph.D.
Eugene Wu, Ph.D.**
Duke University Medical
Center
Durham, North Carolina
Dennis Carlton, B.S.
Vadim Cherezov, Ph.D.
Ellen Yu-Lin Tsai Chien, Ph.D.
Xiaoping Dai, Ph.D.
Marc Deller, D.Phil
Florence Muriel Tama,
Ph.D.**
University of Arizona
Tucson, Arizona
Wei Xie, Ph.D.
Atsushi Yamagata, Ph.D.
Riturparna Sinha Roy, Ph.D.
Nardos Tassew, Ph.D.**
University of Toronto
Toronto, Canada
Stanislav Rudyak, Ph.D. ††††
Hiroaki Tateno, Ph.D.
Sean Ryder, Ph.D.**
University of Massachusetts
Medical School
Worcester, Massachusetts
Rebecca E. Taurog, Ph.D.
Ewan Richardson Taylor,
Ph.D.
Yong Yao, Ph.D.**
Burnham Institute
La Jolla, California
Manami R. Saha, Ph.D.††††
Hua Tian, Ph.D.
Yongjun Ye, Ph.D.
Sanjay Adrian Saldanha,
Ph.D.
Mauricio Carrillo Tripp, Ph.D.
Kye Sook Yi, Ph.D.
Gabriela Perez-Alvarado,
Ph.D.**
Southern Illinois University
Carbondale, Illinois
Ulrich Ignaz Tschulena, Ph.D.
Yong Yin, Ph.D.
Nicholas Preece, Ph.D. ††††
Andre Schiefner, Ph.D.
Julie L. Tubbs, Ph.D.
Kenji Yoshimoto, Ph.D.
Lauren J. Schwimmer,
Ph.D.
Lin Wang, Ph.D.
Naoto Utsumi, Ph.D.
Jennifer S. Scorah, Ph.D.
Frank van Drogen, Ph.D.**
Institute für Biochemie
Zürich, Switzerland
Christopher L. Reyes, Ph.D.
Biogen Idec Research
San Diego, California
Alim Seit-Nebi, Ph.D.
Pedro Serrano-Navarro, Ph.D.
Craig McLean Shepherd,
Ph.D.
David S. Shin, Ph.D.
Develeena Shivakumar, Ph.D.
David A. Shore, Ph.D.
Published by TSRI Press®. © Copyright 2006,
The Scripps Research Institute. All rights reserved.
Ajay Vashisht, Ph.D.
Philip Arno Venter, Ph.D.
Petra Verdino, Ph.D.
William Frederick Waas,
Ph.D.
Gye Won Han, Ph.D.
Lan Xu, Ph.D.
Michael Allen Hanson, Ph.D.
Yoshiki Yamada, Ph.D.
Marcy A. Kingsbury, Ph.D.
Diane Marie Kubitz, B.A.
Qi Yan, Ph.D.**
Miramar College
San Diego, California
Padmaja Natarajan, Ph.D.
Marianne Patch, Ph.D.**
Qualcomm
San Diego, California
Naoto Yoshizuka, Ph.D.
V I S I T I N G I N V E S T I G AT O R S
Veronica Yu, Ph.D.††††
Yuan Yuan, Ph.D.
Markus Zeeb, Ph.D.
Ying Zeng, Ph.D.
Haile Zhang, Ph.D.
Qing Zhang, Ph.D.
Stephen J. Benkovic, Ph.D.
Pennsylvania State
University
University Park,
Pennsylvania
Astrid Graslund, Ph.D.
Stockholm University
Stockholm, Sweden
MOLECULAR BIOLOGY
Arne Holmgren, M.D., Ph.D.
Karolinska Institutet
Stockholm, Sweden
2006
* Joint appointment in The Skaggs
Institute for Chemical Biology
** Appointment completed; new
location shown
Barry Honig, Ph.D.
Columbia University
New York, New York
*** Joint appointment in the
Molecular and Integrative
Neurosciences Department
Arthur Horwich, M.D.
Yale University
New Haven, Connecticut
**** Joint appointments in the
Shie-Liang Hsieh, Ph.D.
National Yang-Ming
University
Taipei, Taiwan
***** Joint appointments in the
Department of Immunology and
The Skaggs Institute for
Chemical Biology
Department of Chemistry and
The Skaggs Institute for
Chemical Biology
† Joint appointment in the
Tai-Huang Huang, Ph.D.
Academica Sinica
Taipei, Taiwan
Department of Cell Biology
†† Joint appointment in the
Department of Molecular and
Experimental Medicine
Sunghoon Kim, Ph.D.
Seoul National University
Seoul, Korea
Ayori Mitsutake, Ph.D.
Keio University
Yokohama, Japan
Joseph David Ng, Ph.D.
University of Alabama,
Huntsville
Huntsville, Alabama
Victoria A. Roberts, Ph.D.
University of California
San Diego, California
Robert D. Rosenstein, Ph.D.
Lawrence Berkeley National
Laboratory
Berkeley, California
Lincoln Scott, Ph.D.
Cassia, LLC
San Diego, California
Deborah Tahmassebi, Ph.D.
University of San Diego
San Diego, California
††† Joint appointment in the
Department of Chemistry
†††† Appointment completed
THE SCRIPPS RESEARCH INSTITUTE
157
158 MOLECULAR BIOLOGY
2006
Peter E. Wright, Ph.D.
Chairman’s Overview
esearch in the Department of Molecular Biology
encompasses a broad range of disciplines, extending from structural and computational biology at
one extreme to molecular genetics at the other. During
the past year, our scientists have continued to make rapid
progress toward understanding the fundamental molecular events underlying the processes of life. Major advances
have been made in elucidating the structural biology of
signal transduction and viral assembly, in understanding mechanisms of viral infectivity, in determining the
structures of membrane proteins and multidrug transporters, in understanding the molecular basis of nucleic
acid recognition and DNA repair, and in determining the
mechanisms of protein folding and ribosome assembly.
Progress has been made in elucidating the molecular
events involved in regulation of the cell cycle, in tumor
development, in induction of sleep, in the molecular
origins of neuronal development and of CNS disorders,
in the regulation of transcription, and in the decoding
of genetic information in translation. Finally, new advances
have been made in the design of novel low molecular
weight compounds that can specifically regulate genes,
and in the area of biomolecular engineering, building
novel functions into viruses, antibodies, and zinc finger
proteins, RNA, and DNA. Progress in these and other
areas is described in detail on the following pages, and
R
THE SCRIPPS RESEARCH INSTITUTE
only a few highlights are mentioned below. The Department of Molecular Biology is also home to two major
National Institutes of Health initiatives, the Joint Center
for Structural Genomics and the Consortium for Functional Glycomics.
One of the outstanding achievements of the past year
was the determination of the structure of the intact and
infectious P22 virion by electron cryomicroscopy. Research
led by Jack Johnson has provided a remarkably detailed
view of the virion structure at an unprecedented 17-Å
resolution. The structure revealed the DNA tightly spooled
around the portal in the interior of the capsid and suggested that the virus uses a pressure-sensing mechanism
to control DNA packaging. The structure also provides
insights into the mechanisms of virion assembly and
injection of DNA into target cells.
Structural biology continues to be a major focus in
the department, and many new x-ray and nuclear magnetic resonance structures of major biomedical significance were completed during the past year. Geoffrey
Chang and colleagues reported new structural studies
of the Escherichia coli multidrug transporter EmrD,
obtaining new insights into the mechanisms by which a
diverse range of drugs are transported through the cell
membrane. Such understanding is of major importance,
given the rapidly growing problem of drug resistance in
bacteria. John Tainer and his coworkers used a combination of electron cryomicroscopy and x-ray crystallography to determine the structure of the Type IV pilus
filament of Neisseria gonorrhoeae. These studies provide new insights into assembly and disassembly mechanisms and are of importance because of the role played
by Type IV pili in allowing antibiotic resistant strains to
escape the immune system and cause persistent infections. Dr. Tainer and colleagues have also determined
new structures of DNA repair enzymes; these include
the xeroderma pigmentosum group B helicase, an
enzyme that plays an essential role in nucleotide-excision repair by removing DNA lesions caused through
exposure to ultraviolet light, and the exonuclease
domain of WRN, a protein that protects against premature aging and cancer. Defects in the gene for WRN
result in Werner’s syndrome, an inherited disease that
causes premature aging.
Research in the laboratories of Jane Dyson and Peter
Wright has provided new insights into the role of protein
conformational fluctuations in enzyme catalysis. Protein
dynamics have long been thought to play an important
role in catalysis. This new work shows how the dynamic
MOLECULAR BIOLOGY
2006
energy landscape of the enzyme dihydrofolate reductase
channels the protein through the reaction cycle. Conformational transitions between the various conformational
substates of the enzyme occur at a rate that is directly
relevant to catalysis.
Several research groups are working in areas directly
related to drug discovery and protein therapeutics. Joel
Gottesfeld and his colleagues have developed smallmolecule histone deacetylase inhibitors that reactivate
frataxin, the gene responsible for the neurodegenerative
disease Friedreich’s ataxia, a disease that is associated
with the expansion of triplet repeats in DNA. These compounds hold great promise as potential therapeutics for
Freidreich’s ataxia. Subhash Sinha, Carlos Barbas, and
Richard Lerner have developed a unique self-assembly
strategy to direct antibodies against specific cellular targets. Their novel approach has led to new compounds
targeted against metastatic breast cancer.
Many of the research groups in this department are
applying the tools of molecular and structural biology
to understand the molecular basis of human disease. In
research led by James Paulson and Ian Wilson, glycan
microarray technology is being used to identify mutations that could allow avian influenza viruses to adapt
to the human population. The glycan array is a powerful surveillance tool for mapping the pathways by which
new human pathogenic viruses can emerge. This research
has revealed a potential mutational pathway that could
switch the specificity of the highly pathogenic H5N1
avian influenza virus and allow it to adapt to humans.
Strikingly, the 3-dimensional structure of the H5N1 hemagglutinin, the protein responsible for binding the virus
to host cell receptors, bears a closer resemblance to the
hemagglutinin from the virus that caused the 1918 influenza pandemic than to that associated with more recent
influenza outbreaks.
Finally, research during the past year has greatly
advanced our understanding of the complex mechanisms
of cell-cycle regulation. Curt Wittenberg and his colleagues have identified a yeast protein that plays a central role in repressing transcription during the cell cycle.
The protein functions in a parallel manner to the important metazoan transcriptional regulator E2F. Work in
Steven Reed’s laboratory has provided new insights into
the mechanisms of multiubiquitinylation and degradation
of cyclin E, a process that is essential for the normal
regulation of the cell cycle. Misregulation of either of
these processes, transcriptional repression or cyclin
turnover, is associated with cancer.
THE SCRIPPS RESEARCH INSTITUTE
159
Molecular biology remains a field of enormous opportunity and excitement. The scientists in this department
are taking full advantage of powerful new technologies
to advance our understanding of fundamental biological
processes at the molecular level. Their discoveries will
ultimately be translated into new advances in biotechnology and in medicine.
160 MOLECULAR BIOLOGY
2006
THE SCRIPPS RESEARCH INSTITUTE
INVESTIGATORS’ R EPORTS
Structural Biology of
Immune Recognition,
Molecular Assemblies,
and Anticancer Targets
I.A. Wilson, R.L. Stanfield, J. Stevens, X. Zhu, M.A. Adams,
Y. An, K. Beis, D.A. Calarese, R.M.F. Cardoso, J.E. Carlson,
P.J. Carney, J.-W. Choe, S. Connelly, A.L. Corper, T.H. Cross,
F i g . 1 . A, Structure of the H5 A/Vietnam/1203/2004 (Viet04)
X. Dai, E.W. Debler, W.L. Densley, M.-A. Elsliger, S. Ferguson,
B.W. Han, G.W. Han, M.J. Jimenez-Dalmaroni, J.G. Luz,
J.R. Mikolosko, A. Schiefner, D.A. Shore, R.S. Stefanko,
J.A. Vanhnasy, P. Verdino, E. Wise, L. Xu, X. Xu, D.M. Zajonc
hemagglutinin trimer, represented as a ribbon diagram. The receptor binding domain, cleavage, and basic patch sites are highlighted
on one monomer. Only 2 of the 9 glycosylation sites per monomer
(positions 34 and 169 in the HA1 chain) had interpretable carbohydrates in the electron density maps. B, Glycan microarray analyses of wild-type human Viet04 hemagglutinin and mutations at
positions 226 and 228, known to be important for adaptation of
H3 viruses from avian α2-3 specificity to human α2-6 receptor
specificity. Binding to the different avian and human α2-3 and α26 sialosides on the array are highlighted.
e are working toward a better understanding
of the structure and function of a variety of
immune-related receptors and of other medically relevant proteins. We use x-ray crystallography
to determine structures for these molecules in complex
with their ligands and coreceptors. This research is instrumental for the design of future drugs and vaccines to
target these proteins.
W
INFLUENZA VIRUS
Influenza virus is a highly contagious and deadly
agent that causes acute respiratory illness. The current
H5N1 avian influenza virus has reached epizootic levels in domestic and wild birds, with worldwide debate
whether the next influenza pandemic could arise from
one of these avian strains. Hemagglutinin is the principal viral surface antigen and is responsible for binding to
host receptors through interaction with sialylated glycans.
The structure of the hemagglutinin from a highly pathogenic H5N1 influenza virus (A/Vietnam/1203/2004;
Fig. 1A) is more closely related to the human 1918 H1
hemagglutinin than to the other human, avian, and swine
hemagglutinins. We are also examining crystal structures
of (1) various influenza neuraminidases to determine the
specificity of the enzymes and their involvement in interaction/escape of the virus from current drugs and (2)
influenza viral proteins that interact with components of
the apoptosis signaling pathway.
In collaboration with O. Blixt and J. Paulson of the
Consortium for Functional Glycomics, La Jolla, California, we used their recently described glycan microarray technology to assess the propensity of the avian
receptor H5N1 A/Vietnam/1203/2004 hemagglutinin
to change from its avian receptor binding (α2-3-linked
sialic acids) to adapt to human receptors (α2-6-linked
sialic acids; Fig. 1B) and have elucidated a possible
route by which H5 viruses could gain a foothold in the
human population.
IL-2 RECEPTOR
IL-2 is a cytokine that functions as a T-cell growth
factor and a central immune system regulator. Its importance is underlined by its broad use as a therapeutic
agent against cancers of the immune system, and IL-2
antagonists are used to prevent rejection of transplanted
organs. We have determined the structure of the heterotrimeric IL-2 receptor ectodomains (IL-2Rαβγc) in
complex with IL-2 at 3.0-Å resolution (Fig. 2). Surprisingly, IL-2Rα makes no contacts with IL-2Rβ or IL-2Rγc,
and only minor changes occur in IL-2 in response to
receptor binding. Thus, our findings support the notion
that IL-2Rα delivers IL-2 to the signaling complex and
acts as a regulator of signal transduction. This research
was performed in collaboration with K.A. Smith, Cornell
University Weill Medical College, New York, New York.
T H E I N N AT E I M M U N E S Y S T E M
Toll-like receptors (TLRs) play key roles in activating immune responses during infection. The 2.1-Å
structure of the human TLR3 ectodomain revealed a
large horseshoe-shaped solenoid structure assembled
from 23 leucine-rich repeats. Seven conserved hydro-
MOLECULAR BIOLOGY
2006
THE SCRIPPS RESEARCH INSTITUTE
161
Department of Immunology, we are studying the membrane-bound part of the NADPH complex to correlate
how mutations in NADPH oxidase can cause chronic
granulomatous disease.
The nucleotide oligomerization binding domain 2
is an important intracellular receptor that recognizes
bacterial peptidoglycans. Mutations in this receptor
are associated with the inflammatory Crohn’s disease.
Structural studies are under way on the domains and
on full-length protein, in collaboration with R. Ulevitch,
Department of Immunology.
C ATA LY T I C A N T I B O D I E S
F i g . 2 . Architecture of the trimeric human IL-2 receptor (desig-
nated IL2R in the figure) signaling complex. View of the quaternary
IL-2 signaling assembly composed of α, β, and γc chains of the IL2R and IL-2, with the C terminus of the β and γ chains close to the
membrane. IL-2 binds to the elbow regions of IL-2Rβ and IL-2Rγc,
as in other cytokine receptors such as human growth hormone
receptor and erythropoietin receptor. The novel IL-2Rα chain docks
on top of this assembly but does not form any contacts with the
other 2 receptor subunits. Six N-linked carbohydrates (S1–S6) are
displayed as ball-and-stick models. S1 is wedged between D1 and
D2 of IL-2Rβ and thus contributes to the stabilization of a specific
D1/D2 interdomain angle. IL-2Rβ and IL-2Rγ form a 3-way junction with IL-2 at the heart of the quaternary high-affinity IL-2 signaling complex and provide a structural basis for the cooperativity
in assembly of the complete IL-2 signaling complex.
phobic residues in the leucine-rich repeat motif form a
tight hydrophobic core, and conserved asparagines contribute extensive hydrogen-bonding networks for solenoid
stabilization. TLR3 is largely masked by carbohydrate,
but the only glycosylation-free face may provide potential ligand-binding sites and an oligomerization interface.
We are doing biochemical analysis of the interaction
between the TLR3 ectodomain and various doublestranded RNA oligomers and structural investigations
of TLR1, TLR2, TLR6, and the TLR2 coreceptor CD36.
These projects are a collaboration with B. Beutler and
R. Ulevitch, Department of Immunology.
Neutrophils and other phagocytes play an important
role in innate immunity by serving as a first line of
defense against invading pathogens. Generation of superoxide by the phagocyte NADPH oxidase complex initiates this process by catalyzing the transfer of metabolic
electrons across the plasma membrane for reduction of
molecular oxygen. Individuals deficient in this enzymatic activity have chronic granulomatous disease,
characterized by recurrent, life-threatening bacterial
and fungal infections. In collaboration with G. Bokoch,
Abuse of cocaine is a major public health problem;
however, no treatments approved by the Food and Drug
Administration are available for cocaine abuse, addiction,
or overdose. Development of effective treatments for
cocaine abuse has been frustrated by the complex neurochemistry of cocaine addiction. Nevertheless, within
the past decade, immunotherapy for cocaine abuse has
been evaluated in preclinical and clinical trials. In collaboration with K.D. Janda, Department of Chemistry,
we determined high-resolution structures for the cocaine
catalytic antibody 7A1 for all major steps along the
catalytic reaction pathway, through cocrystallization
with substrate, products, and transition-state analogs
(Fig. 3). On the basis of this comprehensive series of
F i g . 3 . Crystal structure of the antibody 7A1 Fab′ fragment in
complex with cocaine. The secondary structure of the Fab′ and the
substrate cocaine are shown. Cocaine is trapped in the active site
and is hydrolyzed to nontoxic metabolites.
crystal structures, a catalytic mechanism has been proposed, as well as possible mutations to improve catalytic proficiency.
162 MOLECULAR BIOLOGY
2006
C O FA C T O R - C O N TA I N I N G A N T I B O D I E S
Although antibodies are generally thought to function
without use of cofactors, they are major carrier proteins
in human circulation for the biologically important
cofactor riboflavin. A riboflavin-containing bright-yellow
antibody, IgG GAR, was purified from a patient with
multiple myeloma 30 years ago and is the only available material for studies of the structure and function
of natural cofactor-containing antibodies. Our recent
3.0-Å crystal structure of GAR reveals the location in
the antibody-combining site for the riboflavin potential cofactor (Fig. 4). This research was carried out
THE SCRIPPS RESEARCH INSTITUTE
biochemical studies with our collaborators, R.A. Lerner,
K.D. Janda, P.G. Schultz, and F.E. Romesberg, Department of Chemistry.
EVOLUTION OF LIGAND RECOGNITION AND
SPECIFICITY
To enhance our understanding of how recognition
and specificity for different ligands can be accomplished
by different antibodies that have high levels of sequence
homology, we are studying the evolution of ligand-binding properties by site-directed mutagenesis. The most
active catalytic Diels-Alder antibody known to date, 1E9,
and the steroid-binding antibody DB3 are derived from
the same germ line and have 85% sequence identity.
Through sequential amino acid exchanges, the specificity of 1E9 was changed to that of DB3. Thus, only
a few binding site residues are responsible for achieving either efficient catalysis of the Diels-Alder reaction
or, when mutated, a strong steroid binder. In collaboration with D. Hilvert, ETH, Zürich, Switzerland, we are
structurally characterizing these 1E9 mutants to show
how relatively minor changes can be rationally used to
modify antibody specificity and function.
HIV TYPE 1 NEUTRALIZING ANTIBODIES
F i g . 4 . The antigen-binding site of the original yellow antibody
IgG GAR. The riboflavin cofactor is inserted into the combining site
with its isoalloxazine ring stacked between aromatic residues
TyrH33, PheH58, and TyrH100A. Together with hydrogen bonds
between the N5 atom of the ring to AsnH50 and the ribityl side
chain to ArgH52 and GluH56, these interactions reveal the structural basis for high-affinity riboflavin binding.
in collaboration with R.A. Lerner and P. Wentworth,
Jr., Department of Chemistry.
BLUE AND PURPLE FLUORESCENT ANTIBODIES
Catalytic antibodies are designed to accelerate chemical reactions by acting on the electronic ground state.
However, antibodies have been generated that can interact with and direct the photochemical behavior of the
electronically excited state of stilbene, a model compound for studies in photochemistry and photophysics.
We are exploring the structural basis of the diverse
fluorescent properties of these complexes by using x-ray
crystallography in combination with biophysical and
The search for an effective HIV type 1 vaccine has
prompted the study of the few known broadly neutralizing antibodies to HIV type 1 in complex with their
antigens, in order to structurally characterize important
viral epitopes. The potent and broadly neutralizing antibodies include 4E10 and Z13, which bind to conserved
and overlapping epitopes on the membrane-proximal
region of gp41, and 2G12, which binds to a carbohydrate cluster rich in mannose on gp120. These crystal
structures are then used as the basis for rational design
of immunogens for a candidate vaccine against HIV
type 1. This research is done in collaboration with
D. Burton, Department of Immunology; P. Dawson,
Department of Cell Biology; C.-H. Wong, Department of
Chemistry; S. Danishefsky, Sloan-Kettering Institute,
New York, New York; J.K. Scott, Simon Fraser University,
Burnaby, British Columbia; J. Moore, Cornell University,
Ithaca, New York; H. Katinger, R. Kunert, and G. Stiegler,
University für Bodenkultur, Vienna, Austria; R. Wyatt and
P. Kwong, Vaccine Research Center, National Institutes
of Health, Bethesda, Maryland; and the Neutralizing
Antibody Consortium of the International AIDS Vaccine
Initiative, New York, New York.
CLASSICAL AND NONCLASSICAL MHC AND T-CELL
RECEPTOR SIGNALING
An inflammatory joint disease with many similarities
to human rheumatoid arthritis develops spontaneously in
MOLECULAR BIOLOGY
2006
KRN T-cell receptor (TCR) transgenic mice (F1 K/B x N
mice). Class II MHC I-Ag7 presentation to KRN of selfpeptide derived from glucose-6-phosphate isomerase is a
critical step in the initiation of the disease. In collaboration with L. Teyton, Department of Immunology, we
determined the crystal structures of I-Ag7–glucose-6phosphate isomerase peptide and of the TCR KRN. We
are attempting to crystallize the KRN–I-A g7 complex
to enhance our understanding of how this autoimmune
disease is mediated at the molecular level.
The CD3 TCR coreceptor comprises several distinct
cell-surface glycoproteins that associate with TCR to
enable intracellular signal transduction upon the formation of complexes consisting of TCR and MHC-peptides.
Structural investigation into the interaction between the
TCR and CD3 subunits can aid in elucidation of the
events that lead to T-cell activation. The CD8 glycoprotein is essential for the class I MHC-restricted T-cell
response to peptide antigen, analogous to the CD4 coreceptor of class II–restricted T cells. CD8 is expressed at
the cell surface as CD8αα and CD8αβ. We have determined structures for both CD8αα and CD8αβ in complex with antibody Fab fragments. Comparison of both
forms of the CD8 coreceptor have provided insight into
how the α and β forms contribute to the functionality of
CD8. These studies are a collaboration with S. Davis,
University of Oxford, Oxford, England, and L. Teyton,
Department of Immunology.
The CD1 family is structurally related to MHC molecules, but members of the family present lipid antigens
rather than peptides to CD1-restricted TCRs. We have
determined several structures of mouse CD1d in complex with α-galacturonosyl ceramide, cis-tetracosenoyl
sulfatide, or mycobacterial phosphatidylinositol dimannoside. For each CD1d-ligand, the lipid tails are embedded in the CD1 hydrophobic binding groove, and a
restricted set of CD1d residues orient and stabilize the
various different antigenic headgroups for TCR recognition (Fig. 5). Collaborators in research on CD1 and
TCRs include D.B. Moody and M.B. Brenner, Harvard
Medical School, Boston, Massachusetts; C.-H. Wong,
Department of Chemistry; L. Teyton, Department of
Immunology; M. Kronenberg, La Jolla Institute for Allergy
and Immunology, San Diego, California; V. Kumar, Torrey
Pines Institute for Molecular Studies, San Diego, California; and W. Severn and G. Painter, Industrial Research
Ltd., Upper Hut, New Zealand.
PROTEIN TRAFFICKING
Molecular tethers play a critical role in the organization of the membrane architecture of the exocytic and
THE SCRIPPS RESEARCH INSTITUTE
163
F i g . 5 . Structure of mouse CD1d with inositol-dimannoside.
Close-up view of the binding site shows the hydrogen-bonding network between the glycolipid and CD1d. Both alkyl chains of the
ligand are deeply buried inside the binding groove (not shown),
whereas the complex inositol-dimannoside headgroup is optimally
positioned above the binding groove to directly interact with the TCR.
endocytic pathways of eukaryotic cells. In collaboration
with W. Balch, Department of Cell Biology, we have
determined the 2.0-Å structure of the Rab1 GTPaseregulated N-terminal domain of the p115 tether involved
in transport and structural organization of the Golgi complex. The structure reveals a dimeric handshakelike
assembly consisting of 2 α-solenoid chains, each with
12 novel armadillo-like, tetherin trihelical repeat elements
that form a superhelical elliptical cylinder. This structure
supports a model for binding of Rab1 on opposing membranes to promote membrane tether assembly for membrane docking and fusion and for understanding the large
family of molecular tethers.
JOINT CENTER FOR STRUCTURAL GENOMICS
The Joint Center for Structural Genomics is a large
consortium of scientists from Scripps Research; the
Stanford Synchrotron Radiation Laboratory; the University of California, San Diego; the Burnham Institute;
and the Genomics Institute of the Novartis Research
Foundation. The center is funded by the Protein Structure
Initiative of the National Institute of General Medical
Sciences. Its purpose is the high-throughput structure
determination of large protein families with no structural representatives, a biologically important group of
targets that are conserved in the central machinery of
164 MOLECULAR BIOLOGY
2006
life; the complete proteome from Thermotoga maritima;
and targets suggested by the community. To date, members of the consortium have pioneered many novel highthroughput methods, constructed a high-throughput
pipeline, and determined more than 270 nonredundant structures.
PUBLICATIONS
Almeida, M.S., Herrmann, T., Peti, W., Wilson, I.A., Wüthrich, K. NMR structure
of the conserved hypothetical protein TM0487 from Thermotoga maritima: implications for 216 homologous DUF59 proteins. Protein Sci. 14:2880, 2005.
Brunel, F.M., Zwick, M.B., Cardoso, R.M., Nelson, J.D., Wilson, I.A., Burton, D.R.,
Dawson, P.E. Structure-function analysis of the epitope for 4E10, a broadly neutralizing human immunodeficiency virus type 1 antibody. J. Virol. 80:1680, 2006.
Burton, D.R., Stanfield, R.L., Wilson, I.A. Antibody vs HIV in a clash of evolutionary titans. Proc. Natl. Acad. Sci. U. S. A. 102:14943, 2005.
Calarese, D.A., Lee, H.K., Huang, C.Y., Best, M.D., Astronomo, R.D., Stanfield,
R.L., Katinger, H., Burton, D.R., Wong, C.-H., Wilson, I.A. Dissection of the carbohydrate specificity of the broadly neutralizing anti-HIV-1 antibody 2G12. Proc.
Natl. Acad. Sci. U. S. A. 102:13372, 2005.
Cheng, H., Chong, Y., Hwang, I., Tavassoli, A., Zhang, Y., Wilson, I.A., Benkovic,
S.J., Boger, D.L. Design, synthesis, and biological evaluation of 10-methanesulfonyl-DDACTHF, 10-methanesulfonyl-5-DACTHF, and 10-methylthio-DDACTHF as
potent inhibitors of GAR Tfase and the de novo purine biosynthetic pathway.
Bioorg. Med. Chem. 13:3577, 2005.
Cheng, H., Hwang, I., Chong, Y., Tavassoli, A., Webb, M.E., Zhang, Y., Wilson,
I.A., Benkovic, S.J., Boger, D.L. Synthesis and biological evaluation of N-[4-[5(2,4-diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-2-(2,2,2-trifluoroacetyl)pentyl]benzoyl]-L-glutamic acid as a potential inhibitor of GAR Tfase and the de novo purine
biosynthetic pathway. Bioorg. Med. Chem. 13:3593, 2005.
Choe, J., Kelker, M.S., Wilson, I.A. Crystal structure of human Toll-like receptor 3
(TLR3) ectodomain. Science 309:581, 2005.
Chong, Y., Hwang, I., Tavassoli, A., Zhang, Y., Wilson, I.A., Benkovic, S.J., Boger,
D.L. Synthesis and biological evaluation of α- and γ-carboxamide derivatives of 10CF3CO-DDACTHF. Bioorg. Med. Chem. 13:3587, 2005.
DiDonato, M., Krishna, S.S., Schwarzenbacher, R., et al. Crystal structure of a
single-stranded DNA-binding protein (TM0604) from Thermotoga maritima at 2.60
Å resolution. Proteins 63:256, 2006.
Giabbai, B., Sidobre, S., Crispin, M.D., Sanchez-Ruiz, Y., Bachi, A., Kronenberg,
M., Wilson, I.A., Degano, M. Crystal structure of mouse CD1d bound to the self
ligand phosphatidylcholine: a molecular basis for NKT cell activation. J. Immunol.
175:977, 2005.
Glaser, L., Stevens, J., Zamarin, D., Wilson, I.A., Garcia-Sastre, A., Tumpey,
T.M., Basler, C.F., Taubenberger, J.K., Palese, P. A single amino acid substitution
in 1918 influenza virus hemagglutinin changes receptor binding specificity. J. Virol.
79:11533, 2005.
Han, G.W., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of an apo
mRNA decapping enzyme (DcpS) from mouse at 1.83 Å resolution. Proteins
60:797, 2005.
THE SCRIPPS RESEARCH INSTITUTE
Johnson, M.A., Peti, W., Herrmann, T., Wilson, I.A., Wüthrich, K. Solution structure of Asl1650, an acyl carrier protein from Anabaena sp PCC 7120 with a variant phosphopantetheinylation-site sequence. Protein Sci. 15:1030, 2006.
Klock, H.E., Schwarzenbacher, R., Xu, Q., et al. Crystal structure of a conserved
hypothetical protein (gi: 13879369) from mouse at 1.90 Å resolution reveals a
new fold. Proteins 61:1132, 2005.
Luz, J.G., Yu, M., Su, Y., Wu, Z., Zhou, Z., Sun, R., Wilson, I.A. Crystal structure
of viral macrophage inflammatory protein I encoded by Kaposi’s sarcoma-associated herpesvirus at 1.7 Å. J. Mol. Biol. 352:1019, 2005.
Mathews, I.I., Krishna, S.S., Schwarzenbacher, R., et al. Crystal structure of
phosphoribosylformylglycinamidine synthase II (smPurL) from Thermotoga maritima at 2.15 Å resolution. Proteins 63:1106, 2006.
Moody, D.B., Zajonc, D.M., Wilson, I.A. Anatomy of CD1-lipid antigen complexes.
Nat. Rev. Immunol. 5:387, 2005.
Peti, W., Page, R., Moy, K., O’Neil-Johnson, M., Wilson, I.A., Stevens, R.C.,
Wüthrich, K. Towards miniaturization of a structural genomics pipeline using
micro-expression and microcoil NMR. J. Struct. Funct. Genomics 6:259, 2005.
Rife, C., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of the global
regulatory protein CsrA from Pseudomonas putida at 2.05 Å resolution reveals a
new fold. Proteins 61:449, 2005.
Rife, C., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of a putative
modulator of DNA gyrase (pmbA) from Thermotoga maritima at 1.95 Å resolution
reveals a new fold. Proteins 61:444, 2005.
Shore, D.A., Teyton, L., Dwek, R.A., Rudd, P.M., Wilson, I.A. Crystal structure of
the TCR co-receptor CD8αα in complex with monoclonal antibody YTS 105.18
Fab fragment at 2.88 Å resolution. J. Mol. Biol. 358:347, 2006.
Stanfield, R.L., Gorny, M.K., Zolla-Pazner, S., Wilson, I.A. Crystal structures of
human immunodeficiency virus type 1 (HIV-1) neutralizing antibody 2219 in complex with three different V3 peptides reveal a new binding mode for HIV-1 crossreactivity. J. Virol. 80:6093, 2006.
Stanfield, R.L., Zemla, A., Wilson, I.A., Rupp, B. Antibody elbow angles are influenced by their light chain class. J. Mol. Biol. 357:1566, 2006.
Stauber, D.J., Debler, E.W., Horton, P.A., Smith, K.A., Wilson, I.A. Crystal structure of the IL-2 signaling complex: paradigm for a heterotrimeric cytokine receptor.
Proc. Natl. Acad. Sci. U. S. A. 10:2788 2006.
Stevens, J., Blixt, O., Glaser, L., Taubenberger, J.K., Palese, P., Paulson, J.C.,
Wilson, I.A. Glycan microarray analysis of the hemagglutinins from modern and
pandemic influenza viruses reveals different receptor specificities. J. Mol. Biol.
355:1143, 2006.
Stevens, J., Blixt, O., Tumpey, T.M., Taubenberger, J.K., Paulson, J.C., Wilson,
I.A. Structure and receptor specificity of the hemagglutinin from an H5N1 influenza
virus. Science 312:404, 2006.
Van Rhijn, I., Zajonc, D.M., Wilson, I.A., Moody, D.B. T-cell activation by lipopeptide antigens. Curr. Opin. Immunol. 17:222, 2005.
Wilson, I.A., Stanfield, R.L. MHC restriction: slip-sliding away. Nat. Immunol.
6:434, 2005.
Wiseman, R.L., Johnson, S.M., Kelker, M.S., Foss, T., Wilson, I.A., Kelly, J.W.
Kinetic stabilization of an oligomeric protein by a single ligand binding event. J.
Am. Chem. Soc. 127:5540, 2005.
Huang, C.C., Tang, M., Zhang, M.Y., Majeed, S., Montabana, E., Stanfield, R.L.,
Dimitrov, D.S., Korber, B., Sodroski, J., Wilson, I.A., Wyatt, R., Kwong, P.D.
Structure of a V3-containing HIV-1 gp120 core. Science 310:1025, 2005.
Wu, D., Zajonc, D.M., Fujio, M., Sullivan, B.A., Kinjo, Y., Kronenberg, M., Wilson, I.A., Wong, C.-H. Design of natural killer T cell activators: structure and function of a microbial glycosphingolipid bound to mouse CD1d. Proc. Natl. Acad. Sci.
U. S. A. 103:3972, 2006.
Jaroszewski, L., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of
Hsp33 chaperone (TM1394) from Thermotoga maritima at 2.20 Å resolution. Proteins 61:669, 2005.
Xu, Q., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of virulence
factor CJ0248 from Campylobacter jejuni at 2.25 Å resolution reveals a new fold.
Proteins 62:292, 2006.
Jin, K.K., Krishna, S.S., Schwarzenbacher, R., et al. Crystal structure of TM1367
from Thermotoga maritima at 1.90 Å resolution reveals an atypical member of the
cyclophilin (peptidylprolyl isomerase) fold. Proteins 63:1112, 2006.
Zajonc, D.M., Cantu, C. III, Mattner, J., Zhou, D., Savage, P.B., Bendelac, A.,
Wilson, I.A., Teyton, L. Structure and function of a potent agonist for the semiinvariant natural killer T cell receptor. Nat. Immunol. 6:810, 2005.
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165
Zajonc, D.M., Maricic, I., Wu, D., Halder, R., Roy, K., Wong, C.-H., Kumar, V.,
Wilson, I.A. Structural basis for CD1d presentation of a sulfatide derived from
myelin and its implications for autoimmunity. J. Exp. Med. 202:1517, 2005.
Zhang, Y., Wang, L., Schultz, P.G., Wilson, I.A. Crystal structures of apo wild-type
M jannaschii tyrosyl-tRNA synthetase (TyrRS) and an engineered TyrRS specific for
O-methyl-L-tyrosine. Protein Sci. 14:1340, 2005.
Zhu, X., Dickerson, T.J., Rogers, C.J., Kaufmann, G.F., Mee, J.M., McKenzie,
K.M., Janda, K.D., Wilson, I.A. Complete reaction cycle of a cocaine catalytic antibody at atomic resolution. Structure 14:205, 2006.
Zhu, X., Wentworth, P., Jr., Kyle, R.A., Lerner, R.A., Wilson, I.A. Cofactor-containing antibodies: crystal structure of the original yellow antibody. Proc. Natl. Acad.
Sci. U. S. A. 103:3581, 2006.
Structure and Function of
Proteins as Molecular Machines
E.D. Getzoff, M. Aoyagi, A.S. Arvai, D.P. Barondeau,
F i g . 1 . The NADPH-binding site in the crystallographic structure of
R.M. Brudler, T.H. Cross, E.D. Garcin-Hosfield, C. Hitomi,
the neuronal NOS reductase module. A triad of amino acid residues
conserved in NOS reductases and homologous flavoproteins (Tyr1322,
Ser1313, and Arg1314) stabilize the 2′ phosphate group (2′P) that
distinguishes NADPH from NADH. In contrast, Arg1400 is specific to
the calcium-regulated neuronal and endothelial NOS enzymes, in which
it performs a isozyme-specific regulatory function. In the absence
of calcium-bound calmodulin, Arg1400 helps stabilize the regulatory C-terminal tail, inhibiting nonproductive electron transfer.
K. Hitomi, C.J. Kassmann, M.E. Pique, M.E. Stroupe,
J.L. Tubbs, T.I. Wood
ur goals are to understand how proteins function as molecular machines. We use structural,
molecular, and computational biology to study
proteins of biological and biomedical interest, especially
proteins that work synergistically with coupled chromophores, metal ions, or other cofactors.
O
NITRIC OXIDE SYNTHASES
To synthesize nitric oxide, a cellular signal and
defensive cytotoxin, nitric oxide synthases (NOSs)
require calmodulin-orchestrated interactions between
their catalytic, heme-containing oxygenase module and
their electron-supplying reductase module. Crystallographic structures of wild-type and mutant NOS oxygenase dimers with substrate, intermediate, inhibitors,
cofactors, and cofactor analogs, determined in collaboration with J. Tainer, Department of Molecular Biology,
and D. Stuehr, the Cleveland Clinic, Cleveland, Ohio,
provided insights into the catalytic mechanism and
dimer stability.
Our structure-based drug design projects are aimed
at selectively inhibiting inducible NOS, to prevent inflammatory disorders, or neuronal NOS, to prevent migraines,
while maintaining blood pressure regulation by endothelial NOS. Our structure of the neuronal NOS reductase
has provided news insights into the complex regulatory
mechanisms of this enzyme family.
We have designed and assayed site-directed mutant
enzymes that support our mechanistic hypotheses for
isozyme-specific inhibition and regulation (Fig. 1). We
integrated biochemical data with our structures of NOS
oxygenase, NOS reductase, and calmodulin in complex
with peptides derived from NOS to propose a model
for the assembled holoenzyme that provides a movingdomain mechanism for electron flow from NOS reductase
to the NOS oxygenase heme. Preliminary small-angle
x-ray scattering measurements in solution provide molecular envelopes for NOS proteins that support our model.
Our model also explains how regulatory site-specific
phosphorylation and dephosphorylation activate and
inactivate nitric oxide synthesis in vivo.
PHOTOACTIVE PROTEINS AND CIRCADIAN CLOCKS
To understand in atomic detail how proteins translate sunlight into defined conformational changes for
biological functions, we are exploring the reaction
mechanisms of the blue-light receptors photoactive
yellow protein (PYP), photolyase, and cryptochrome.
PYP is the prototype for the Per-Arnt-Sim domain proteins of circadian clocks, whereas proteins of the photolyase and cryptochrome family catalyze DNA repair
or act in circadian clocks. To understand the protein
photocycle, we combined ultra-high-resolution and
time-resolved crystallographic structures of the dark
state and 2 photocycle intermediates of PYP with sitedirected mutagenesis; ultraviolet-visible spectroscopy;
166 MOLECULAR BIOLOGY
2006
time-resolved Fourier transform infrared spectroscopy;
deuterium-hydrogen exchange mass spectrometry, in
collaboration with V. Woods, University of California,
San Diego; and quantum mechanical and electrostatic
computational methods, in collaboration with L. Noodleman, Department of Molecular Biology.
Cryptochrome flavoproteins are homologs of lightdependent DNA repair photolyases that function as bluelight receptors in plants and as components of circadian
clocks in animals. We determined the first crystallographic structure of a cryptochrome, which revealed
commonalities with photolyases in DNA binding and
redox-dependent function but showed differences in
active-site and interaction-surface features. Recently, we
showed that this cryptochrome binds the same antenna
cofactor found in a photolyase homolog but uses different residues for the cofactor-binding site. New structures of photolyases from 2 other branches of the
photolyase/cryptochrome family that repair cyclobutane
pyrimidine dimers and photoproducts help us decipher
the cryptic structure, function, and evolutionary relationships of these fascinating redox-active proteins.
We are also studying clock proteins with PYP-like
and Per-Arnt-Sim domains that bind to mammalian
cryptochromes. Our goal is to determine the detailed
chemistry and atomic structure of these proteins, define
their mechanisms of action and interaction, and use
our results to understand their biological function
and regulation.
THE SCRIPPS RESEARCH INSTITUTE
targeting to specific cellular locations. By completing
the metalloprotein design cycle from prediction to highly
accurate structures, we can rigorously evaluate and
improve algorithms for the design of metal sites.
In related research, we discovered that the architecture of GFP and RFP promotes a remarkable range of
posttranslational modification chemistry. High-resolution crystallographic structures of GFP and RFP intermediates in fluorophore cyclization and oxidation lead
to a novel mechanism for the spontaneous synthesis of
this tripeptide fluorophore within the protein scaffold.
Remarkably, the same protein architectural features
that favor peptide cyclization can drive peptide hydrolysis (Fig. 2) and red shift the spectral properties of the
P R O T E I N D E S I G N A N D P O S T R A N S L AT I O N A L
M O D I F I C AT I O N C H E M I S T R Y
An ultimate goal for protein engineers is to design
and construct new protein variants with desirable catalytic or physical properties. As members of the Scripps
Research Metalloprotein Structure and Design Group,
we are testing our understanding of affinity, selectivity,
and activity of metal ions by transplanting metal sites
from structurally characterized metalloproteins into new
protein scaffolds. To aid our design efforts, we have
organized quantitative information and interactive viewing of protein metal sites at the Metalloprotein Database
and Browser (available at http://metallo.scripps.edu).
For green fluorescent protein (GFP) and the homologous red fluorescent protein (RFP), we designed, constructed, and characterized metal-ion biosensors, in
which binding of metal ions is signaled by changes in
spectroscopic properties of the naturally occurring fluorophores. Use of GFP allows optimization with random
mutagenesis, noninvasive expression in living cells, and
F i g . 2 . Spontaneous peptide hydrolysis and decarboxylation
reactions promoted by the protein architecture of GFP. A, Crystallographic structure of a designed GFP variant reveals peptide-bond
cleavage and decarboxylation chemistry at the site of GFP fluorophore synthesis. S65G and Y66S mutations converted the fluorophore tripeptide SYG sequence to GSG. The simulated annealing
omit electron density map (mesh) clearly shows the resultant break
in the polypeptide chain at this site. B, Corresponding reaction and
posttranslational products for this self-cleaving GFP variant.
MOLECULAR BIOLOGY
2006
chromophore. Decarboxylation reactions in designed variants of GFP (Fig. 2) support a role for the GFP environment in facilitating formation of radicals and 1-electron
chemistry. Together, our results provide the groundwork for the design of proteins with novel catalytic or
reporter properties.
PUBLICATIONS
Barondeau, D.P., Kassmann, C.J., Tainer, J.A., Getzoff, E.D. Understanding GFP
posttranslational chemistry: structures of designed variants that achieve backbone
fragmentation, hydrolysis, and decarboxylation. J. Am. Chem. Soc. 128:4685, 2006.
Barondeau, D.P., Tainer, J.A., Getzoff, E.D. Structural evidence for an enolate intermediate in GFP fluorophore biosynthesis. J. Am. Chem. Soc. 128:3166, 2006.
Brudler, R., Gessner, C.R., Li, S., Tyndall, S., Getzoff, E.D., Woods, V.L., Jr. PAS
domain allostery and light-induced conformational changes in photoactive yellow
protein upon I2 intermediate formation, probed with enhanced hydrogen/deuterium
exchange mass spectrometry. J. Mol. Biol. 363:148, 2006.
Panda, K., Haque, M.M., Garcin-Hosfield, E.D., Durra, D., Getzoff, E.D., Stuehr,
D.J. Surface charge interactions of the FMN module governs catalysis by nitricoxide synthase. J. Biol. Chem., in press.
Stroupe, M.E., Getzoff, E.D. The role of siroheme in sulfite and nitrite reductases.
In: Tetrapyrroles. Warren, M.J., Smith, A. (Eds.). Landes Bioscience, Georgetown,
TX, in press.
Tiso, M., Konas, D.W., Panda, K., Garcin, E.D., Sharma, M., Getzoff, E.D.,
Stuehr, D.J. C-terminal tail residue Arg1400 enables NADPH to regulate electron
transfer in neuronal nitric-oxide synthase. J. Biol. Chem. 280:39208, 2005.
Wood, T.I., Barondeau, D.P., Hitomi, C., Kassmann, C.J., Tainer, J.A., Getzoff,
E.D. Defining the role of arginine 96 in green fluorescent protein fluorophore
biosynthesis. Biochemistry 44:16211, 2005.
Structural Biology of Molecular
Interactions and Design
J.A. Tainer, A.S. Arvai, D.P. Barondeau, M. Bjoras,
B.R. Chapados, L. Craig, T.H. Cross, L. Fan, C. Hitomi,
K. Hitomi, J.L. Huffman, C.J. Kassmann, I. Li, G. Moncalian,
M.E. Pique, D.S. Shin, O. Sundheim, R.S. Williams,
T.I. Wood, A. Yamagata
ur studies reveal overall themes and common
relationships for fundamental principles and
processes of protein regulators and effectors of
DNA damage responses, reactive oxygen species, and
pathogenesis. We combine x-ray crystallography and
solution small-angle x-ray scattering methods, often at
our advanced synchrotron facility SIBLYS, to gain a
clear view of the structural chemistry that drives biology.
Further fusing these techniques with electron microscopy, we bridge the size gaps between high-resolution
macromolecular structures and lower resolution multiprotein machine complexes. We then investigate the
associated dynamic reversible interactions within cells,
O
THE SCRIPPS RESEARCH INSTITUTE
167
potential for structure-based design of inhibitors relevant
to the development of novel therapeutic agents and
chemical tools, and structural implications by biochemistry and mutagenesis. For DNA repair, we collaborate
with P. Russell and N. Boddy, Department of Molecular
Biology, to couple our structures with genetics and phenotypes. For protein design, we collaborate with E. Getzoff, Department of Molecular Biology, to understand and
control the formation of self-synthesizing chromophores
in green fluorescent protein and its homologs.
P R O T E I N M O D I F I C AT I O N S A N D F U N C T I O N
The finding that the number of protein-coding genes
in the human genome is more than 10-fold lower than
the number of proteins found in human cells by the
Human Genome Project is surprising. This huge increase
in protein diversity must primarily be due to alternative
splicing and posttranslational modification of proteins.
A particularly important and intriguing posttranslational
modification is the spontaneous peptide backbone cyclization and oxidation chemistry required to convert 3 amino
acids into a fluorophore for the family of green fluorescent proteins.
REACTIVE OXYGEN AND XENOBIOTIC CONTROL
ENZYMES
Superoxide dismutases and nitric oxide synthases are
master regulators for reactive oxygen species involved
in injury, pathogenesis, aging, and degenerative diseases.
We are characterizing the hydrogen-bonding networks
that underlie the activity of mitochondrial manganese
superoxide dismutases. For human copper, zinc superoxide dismutase, we are probing how single-site mutations cause the neurodegeneration in Lou Gehrig disease
or familial amyotrophic lateral sclerosis. For nitric oxide
synthases, we are examining the structure and chemistry that control levels of nitric oxide, which acts as
an important signal and cytotoxin with implications for
inflammatory and neurodegenerative diseases.
DNA REPAIR AND GENETIC EVOLUTION
All the information for heredity is encoded in DNA
molecules that are constantly under attack from sunlight, ionizing radiation, and other environmental carcinogens. Surprisingly, however, most DNA damage is
due to chemical reactions and free radicals that arise
from normal cellular metabolism that is necessary for
life. Thus, paradoxically, life is impossible even in the
absence of environmental toxins unless coupled to DNA
repair. Mutations that cause defects in DNA repair
systems may cause cancer and degenerative diseases
associated with aging, but fortunately the mutations
can also be exploited for cancer therapy.
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2006
THE SCRIPPS RESEARCH INSTITUTE
AGING AND THE WRN STRUCTURE
Mutation of the DNA repair protein WRN can give
rise to Werner syndrome, which is characterized by
rapid aging and cancer disorders. We have characterized
the structure of the WRN nuclease component (Fig. 1).
F i g . 2 . Conserved XPB helicase core and DNA-induced open-to-
closed conformational changes. XPB contains 4 conserved functional domains: the damage recognition domain (DRD), 2 helicase
domains (HD1 and HD2), and a thumb insert (ThM). The interaction of the helicase with DNA may induce a rotation of about 170°
of domain HD2 and ThM to form the closed conformation as
observed in the crystal structure of hepatitis C virus (HCV) NS3
helicase bound to a single-stranded DNA.
F i g . 1 . Hexameric ring model for the WRN nuclease (WRN exo)
component. A, WRN x-ray crystal structures aligned as a ring by
homology comparisons. B, DNA processing is altered in the WRN
Trp145A mutant. C, Electron density map (3σ, 5σ) of dGMP bound
to WRN exo. D, The similar internal and external dimensions of
Ku70/80 (left) and the WRN exo hexamer model (right).
This component is an editing nuclease resembling those
found in DNA polymerases. Furthermore, the editing
of DNA ends by the WRN exonuclease is stimulated
for broken DNA end joining by the Ku DNA end-binding
complex. Our findings suggest how the editing of DNA
ends during DNA damage responses can critically affect
aging and carcinogenesis.
NUCLEOTIDE EXCISION REPAIR
Nucleotide excision repair, a critical defense mechanism that removes DNA lesions caused by the mutational effects of sunlight (ultraviolet radiation) and toxic
chemicals, is also central to the success of anticancer
drugs such as cisplatin. We have focused on understanding the mechanisms of nuclear excision repair for potential improvements in cancer treatment. We determined
the crystal structure of an enzyme called xeroderma
pigmentosum group B (XPB) helicase (Fig. 2). We found
several unexpected functions of XPB helicase in nuclear
excision repair. These findings helped us address important questions about the enzyme’s role in DNA transcription and repair. XPB helicase recognizes DNA
damage that causes blockages in reading the DNA code
and aids initiation of efficient repair.
and Neisseria meningitidis. Pili play key roles in surface motility, adhesion, formation of microcolonies and
biofilms, natural transformation, and signaling. We are
characterizing structures of type IV pilin subunits: the
assembled pilus fiber, the pilus membrane protein partners, and the assembly ATPase. Pili induce a calcium
influx in host cells that plays a role in pathogenesis by
altering endocytic trafficking and lysosome homeostasis in infected cells. Because calcium is a central second messenger that regulates several signal cascades,
pilus-induced calcium bursts most likely influence
bacterial infectivity in key ways. For infections caused
by N meningitidis, these calcium bursts are expected
to activate neuronal nitric oxide synthases, resulting in
toxic levels of nitric oxide that may in part explain the
fatal effects of N meningitidis infections of the brain.
PUBLICATIONS
Ayala, I., Perry, J.P., Szczepanski, J., Tainer, J.A., Vala, M.T., Nick, H.S., Silverman, D.N. Hydrogen bonding in human manganese superoxide dismutase containing 3-fluorotyrosine. Biophys. J. 89:4171, 2005.
Ayala, P., Wilbur, J.S., Wetzler, L.M., Tainer, J.A., Snyder, A., So, M. The pilus
and porin of Neisseria gonorrhoeae cooperatively induce Ca2+ transients in
infected epithelial cells. Cell. Microbiol. 7:1736, 2005.
Barondeau, D.P., Kassmann, C.J., Tainer, J.A., Getzoff, E.D. Understanding GFP
posttranslational chemistry: structures of designed variants that achieve backbone
fragmentation, hydrolysis, and decarboxylation. J. Am. Chem. Soc. 128:4685, 2006.
Barondeau, D.P., Tainer, J.A., Getzoff, E.D. Structural evidence for an enolate
intermediate in GFP fluorophore biosynthesis. J. Am. Chem. Soc. 128:3166,
2006.
Craig, L., Volkmann, N., Arvai, A.S., Pique, M.E., Yeager, M., Egelman, E.H.,
Tainer, J.A. Type IV pilus structure by cryo-electron microscopy and crystallography: implications for pilus assembly and functions. Mol Cell. 23:651, 2006.
BACTERIAL PILI AND INFECTIOUS DISEASES
Type IV pili are essential virulence factors for many
gram-negative bacteria, such as Neisseria gonorrhoeae
Doi, Y., Katafuchi, A., Fujiwara, Y., Hitomi, K., Tainer, J.A., Ide, H., Iwai, S. Synthesis and characterization of oligonucleotides containing 2′-fluorinated thymidine glycol
as inhibitors of the endonuclease III reaction. Nucleic Acids Res. 34:1540, 2006.
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2006
Fan, L., Arvai, A., Cooper, P.K., Iwai, S., Hanaoka, F., Tainer, J.A. Conserved XPB
core structure and motifs for DNA unwinding: implications for pathway selection of
transcription or excision repair. Mol. Cell 22:27, 2006.
Fan, L., Kim, S., Farr, C.L., Schaefer, K.T., Randolph, K.M., Tainer, J.A., Kaguni,
L.S. A novel processive mechanism for DNA synthesis revealed by structure, modeling and mutagenesis of the accessory subunit of human mitochondrial DNA polymerase. J. Mol. Biol. 358:1229, 2006.
Fan, L., Perry, J.J.P., Tainer, J.A. Reactive oxygen control and DNA repair structural biology: implications for aging and neuropathology. Neuroscience, in press.
Hitomi, K., Iwaia, S., Tainer, J.A. The intricate structural chemistry of base excision repair machinery: implications for DNA damage recognition, removal, and
repair. DNA Repair (Amst.), in press.
Ivanov, I., Chapados, B.R., McCammon, J.A., Tainer, J.A. Proliferating cell nuclear
antigen loaded onto double-stranded DNA: dynamics, minor groove interactions
and functional implications. Nucleic Acids Res. in press.
Pascal, J.M., Tsodikov, O.V., Hura, G.L., Song, W., Cotner, E.A., Classen, S.,
Tomkinson, A.E., Tainer, J.A., Ellenberger, T. A flexible interface between DNA
ligase and PCNA supports conformational switching and efficient ligation of DNA.
Mol. Cell. 24:279-91, 2006.
Perry, J.J.P., Yannone, S.M., Holden, L.G., Hitomi, C., Asaithamby, A., Han, S.,
Cooper, P.K., Chen, D.J., Tainer, J.A. WRN exonuclease structure and molecular
mechanism imply an editing role in DNA end processing. Nat. Struct. Mol. Biol.
13:414, 2006.
Putnam, C.D., Hura, G.L., Tainer, J.A. Combining x-ray solution and crystal diffraction and scanning force microscopies to characterize reversible macromolecular
interactions and conformational states. Q. Rev. Biophys., in press.
Putnam, C.D., Tainer, J.A. Protein mimicry of DNA and pathway regulation. DNA
Repair (Amst.) 4:1410, 2005.
Sundheim, O., Vågbø, C.B., Bjørås, M., de Sousa, M.M.L., Talstad, V., Aas, P.A.,
Drabløs, F., Krokan, H.E., Tainer, J.A., Slupphaug, G. Human ABH3 structure and
key residues for oxidative demethylation to reverse DNA/RNA damage. EMBO J.
25:3389, 2006.
Tsutakawa, S., Tainer, J.A. Combined methods of SAXS and crystallography to characterize dynamic protein conformations at atomic resolution. J. Struct. Biol., in press.
Wood, T.I., Barondeau, D.P., Hitomi, C., Kassmann, C.J., Tainer, J.A., Getzoff,
E.D. Defining the role of arginine 96 in green fluorescent protein fluorophore
biosynthesis. Biochemistry 44:16211, 2005.
Structural Biology of Integral
Membrane Proteins
G. Chang, S. Aller, A. Chen, Y. Chen, X. He, A. Karyakin,
C.R. Reyes, P. Szewczyk, A. Ward, S. Wada, J. Yu, Y. Yin
he structural biology of integral membrane proteins
is an exciting frontier. We are interested in 5 areas:
(1) the molecular structural basis for lipid and
drug transport across the cell membrane by multidrugresistance (MDR) transporters, (2) the high-resolution
structure of yeast and mammalian MDR transporters,
(3) signal transduction by receptors, (4) the discovery
and design of potent MDR reversal agents, and (5) the
development of an in vitro cell-free system capable of
T
THE SCRIPPS RESEARCH INSTITUTE
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overproducing integral membrane proteins suitable for
biophysical study. We use several experimental methods,
including detergent/lipid protein biochemistry, 3-dimensional crystallization of integral membrane proteins,
protein x-ray crystallography, and functional analysis
of transporters.
We are addressing the molecular basis of MDR in
the treatment of infectious disease and cancer. A major
cause of MDR is drug efflux pumps imbedded in the cell
membrane. Through our structural studies on MDR
transporters, we are gaining insights into the molecular mechanics of translocating amphipathic substrates
across the cell membrane and the rational design of
powerful inhibitors.
We are combining chemistry and biology with structure for the discovery and design of potent MDR reversal
agents for cancer chemotherapy in collaboration with
M.G. Finn, Department of Chemistry; I. Urbatsch, Texas
Tech University Health Sciences Center, Lubbock, Texas;
and S. Reutz, Novartis International AG, Basel, Switzerland. In collaboration with M. Saier, University of California, San Diego, and Q. Zhang, Department of Molecular
Biology, we are probing the structures and function of
bacterial MDR transporters. In a collaboration with
R.A. Milligan, Department of Cell Biology, we are using
electron cryomicroscopy to visualize the low-resolution
structures of our transporters.
Recently, we determined the x-ray structure of an
MDR transporter called EmrD. EmrD is from the Major
Facilitator Superfamily, and it expels amphipathic compounds across the inner membrane of E coli. The structure reveals an interior that is composed mostly of
hydrophobic residues, a finding consistent with the role
of EmrD in transporting amphipathic molecules. Two
long loops extend into the inner leaflet side of the cell
membrane. This region can recognize and bind substrate
directly from the lipid bilayer. We propose that multisubstrate specificity, binding, and transport are facilitated by these loop regions and the internal cavity.
PUBLICATIONS
Yin, Y., He, X., Szewczyk, P., Nguyen, T., Chang, G. Structure of the multidrug
transporter EmrD from Escherichia coli. Science 312:741, 2006.
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Structure and Function of
Membrane-Bound Enzymes
C.D. Stout, H. Heaslet, M. Yamaguchi, V.M.M. Luna,
A. Annalora, J. Chartron, V. Sundaresan
e focus on the structure and function of membrane-bound enzymes and the development of
methods for crystallizing membrane proteins.
We study the mechanism of transhydrogenase, a mitochondrial respiratory enzyme complex that couples proton
translocation with hydride transfer. We use x-ray crystallography, biochemical and spectroscopic methods,
electron microscopy studies in collaboration with M.
Yeager, Department of Cell Biology, and nuclear magnetic resonance studies in collaboration with J. Dyson,
Department of Molecular Biology. Crystal structures of
transhydrogenase soluble domains, alone and in complex, have been determined (Fig. 1). Currently, our pri-
W
F i g . 1 . Superposition of 3 heterotrimers of transhydrogenase soluble domains observed in cocrystals. The presence of additional copies
of the smaller soluble domain (dIII, lower right) in the crystal lattice
provides a possible model for the intact enzyme in the membrane.
mary effort is to determine the structure of the intact
200-kD enzyme in its membrane-bound configuration.
We are developing applications of nanodiscs for biophysical studies of integral membrane proteins in collaboration with P. Dawson, Department of Cell Biology, and
S.G. Sligar, University of Illinois, Urbana-Champaign,
Illinois. Nanodiscs are composed of phospholipid-binding
peptides that self-assemble into discrete, water-soluble,
bilayer-containing particles. Integral membrane proteins
incorporated into these particles retain their enzymatic
activity, are amenable to biochemical assays, and may
have superior properties for crystallization in the absence
of detergents. Both transhydrogenase and cytochrome
ba 3 oxidase have been incorporated into nanodiscs.
THE SCRIPPS RESEARCH INSTITUTE
In collaboration with J.A. Fee, Department of Molecular Biology, we are studying the mechanism of action
of cytochrome ba 3 oxidase, the terminal enzyme of
respiration. The high-resolution structure of the enzyme
from Thermus thermophilus, crystallized in the presence of a detergent, has been determined. Crystallographic experiments, in concert with mutagenesis and
spectroscopy, can be used to visualize intermediates in
the reduction of oxygen to water and to define the paths
of oxygen molecules and protons into the active site.
In collaboration with E.F. Johnson, Department of
Molecular Biology; J.R. Halpert, University of Texas
Medical Branch, Galveston, Texas; and others, we are
characterizing structures of mammalian cytochrome
P450s. These membrane-associated enzymes are involved
in the biosynthesis of lipophilic hormones and specifically metabolize a remarkable diversity of exogenous
compounds and drugs. More than 60 genes for P450
occur in the human genome. High-resolution structures,
including substrate and inhibitor complexes, have been
determined for the P450s 1A2, 2C5, 2C8, 2C9, 2A6,
2A13, 3A4, and 2B4. For 2B4, 3 structures of the
enzyme in markedly different conformations provide
insight to substrate binding and membrane insertion.
A major effort to determine the basis of HIV resistance to antiviral drugs is ongoing in collaboration with
A.J. Olson and J.H. Elder, Department of Molecular
Biology; B.E. Torbett, Department of Molecular and
Experimental Medicine; and D.E. McRee, ActiveSight,
San Diego, California. One aspect of this project entails
determining the crystal structure of HIV protease-resistant mutants in complex with a wide range of inhibitors.
Additional research projects involve crystallographic collaborations on iron-sulfur enzymes, with K.S. Carroll,
University of Michigan, Ann Arbor, Michigan; electron
transfer proteins, with J.A. Fee, Department of Molecular Biology; and synthetic self-assembling peptides,
with M.R. Ghadiri, Department of Chemistry.
PUBLICATIONS
Chartron, J., Carroll, K.S., Shiau, C., Gao, H., Leary, J.A., Bertozzi, C.R., Stout,
C.D. Substrate recognition, protein dynamics, and novel iron-sulfur cluster in
Pseudomonas aeruginosa adenosine 5′-phosphosulfate reductase. J. Mol. Biol.
364:152, 2006.
Heaslet, H., Kutilek, V., Morris, G.M., Lin, Y.-C., Elder, J.H., Torbett, B.E., Stout,
C.D. Structural insights into the mechanisms of drug resistance in HIV-1 protease
NL4-3. J. Mol. Biol. 356:967, 2006.
Hillier, B.J., Sundaresan, V., Stout, C.D., Vacquier, V.D. Expression, purification,
crystallization and preliminary x-ray analysis of the olfactomedin domain from the
sea urchin cell-adhesion protein amassin. Acta Crystallogr. Sect. F Struct. Biol.
Cryst. Commun. 62(Pt. 1):16, 2006.
Johnson, E.F., Stout, C.D. Structural diversity of human xenobiotic-metabolizing cytochrome P450 monooxygenases. Biochem. Biophys. Res. Commun. 338:331, 2005.
MOLECULAR BIOLOGY
2006
Yadav, M.K., Leman, L.J., Price, D.J., Brooks, C.L. III, Stout, C.D., Ghadiri, M.R.
Coiled coils at the edge of configurational heterogeneity: structural analyses of parallel
and antiparallel homotetrameric coiled coils reveal configurational sensitivity to a single solvent-exposed amino acid substitution. Biochemistry 45:4463, 2006.
Zhao, Y., White, M.A., Muralidhara, B.K., Sun, L., Halpert, J.R., Stout, C.D.
Structure of microsomal cytochrome P450 2B4 complexed with the antifungal drug
bifonazole: insight into P450 conformational plasticity and membrane interaction.
J. Biol. Chem. 281:5973, 2006.
Cytochrome ba3 From
Thermus thermophilus: New
Windows on the Mechanisms
of Energy Transduction by
Cytochrome c Oxidases
J.A. Fee, Y. Chen
relatively small integral-membrane protein containing 2 iron and 3 copper atoms distributed in
3 redox active sites generates approximately one
third of a human’s metabolic energy. That enzyme is
cytochrome c oxidase, and its mechanism of action
remains a mystery. The enzyme was first recognized by
Charles MacMunn as “histohaematin” in the 1880s and
was studied intensely by Otto Warburg as “atmungsferment” during the 1920s and 1930s and by David
Keilin as “cytochrome” into the late 1950s. Today,
cytochrome c oxidase is still the subject of an international effort.
Cytochrome c oxidase catalyzes the following deceptively simple reaction:
A
4 cytochrome c2+ + O2 + 8 H+in → 4 cytochrome
c3+ + 2 H 2O + 4 H +out,
where the subscripts in and out refer, respectively,
to matrix and the intermembrane space of the mitochondrion or, in bacteria, to the cytoplasm and the periplasmic space. The free energy of dioxygen reduction is thus
captured as a proton gradient; the out side is positive
and the in side is negative.
During the past 5 decades, enormous progress has
been realized in understanding the chemical properties
of the 3 redox centers, and the enzyme from several
different sources has been crystalized and its structure
determined at resolutions ranging from about 3 to 1.8 Å.
Moreover, much has been learned about the pathways
of electron transfer within the enzyme and the
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171
detailed mechanisms whereby the oxygen molecule is
reduced to 2 water molecules. The outstanding questions
pertain to the flow of protons into the enzyme from its
in side, across the hydrophobic core of the membrane,
to exit on its out side. The mystery lies in how all this
scalar chemistry comes together to “pump” 4 protons
across the membrane. Although the enzyme has been
examined by using virtually every available spectroscopic
technique, no one has addressed directly the pathways
of those protons becoming either water or part of the
proton gradient.
Much of the past work was done with enzymes in a
single clade typified by the mitochondrial enzyme (derived
from an ancient bacterium) and with enzymes isolated
from common bacteria, notably Rhodobacter sphaeroides,
Paraccocus denitrificans, and Escherichia coli (the
quinol oxidase). The enzymes from these sources are
highly similar in amino acid sequence, 3-dimensional
structure, electron-transfer paths, mechanism of oxygen reduction, and, most likely, mechanisms of proton
pumping. Our research is based on a 1988 description
of a highly sequence-divergent form of the enzyme from
Thermus thermophilus, cytochrome ba3, that represents
a distinct clade of enzymes widely distributed among
archaebacteria. Respectively, these clades represent
A- and B-type oxidases.
We recently developed a homologous expression
system for cytochrome ba 3. This system allows easy
purification of the enzyme in amounts of 2–3 mg/L of
culture medium by using an N-terminal heptahistidine
tag on subunit I. The recombinant enzyme is equally
active with native, wild-type protein, and the results
of a 2.3-Å x-ray structure determination, done in collaboration with C.D. Stout, Department of Molecular
Biology, revealed the expected details at full occupancy
and at least one notable surprise.
All the A-type oxidases have a glutamic acid residue
within the hydrophobic interior of the enzyme that is
thought to be at the “end” of the D pathway of proton
transport and close to the Fe a3 -Cu B site of dioxygen
reduction (Fig. 1A). Mutation of this residue to, for example, glutamine blocks all but a small percentage of the
enzyme’s electron-transfer activity, and infrared studies
indicate that this residue “senses” changes in the chemical structure of the dioxygen reduction site. Indeed, scientists think that glutamate 286 actually donates the
pumped proton to the exit part of the molecule, becoming deprotonated with a pK a of about 9.4. However,
no direct evidence exists for this notion, and because
172 MOLECULAR BIOLOGY
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THE SCRIPPS RESEARCH INSTITUTE
Hunsicker-Wang, L.M., Pacoma, R.L., Chen, Y., Fee, J.A., Stout, C.D. A novel
cryoprotection scheme for enhancing the diffraction of crystals of recombinant cytochrome ba3 oxidase from Thermus thermophilus. Acta Crystallogr. D Biol. Crystallogr. 61(Pt. 3):340, 2005.
Developing Reagents
for Stabilization of
Membrane Proteins
Q. Zhang, M.G. Finn,* X. Ma, R.S. Roy
* Department of Chemistry, Scripps Research
ntegral membrane proteins are extremely unstable
outside the hydrophobic membrane bilayer, a situation that makes their in vitro biophysical and structural characterization difficult. An artificial environment
is therefore needed to stabilize the proteins in their
native state. We focus on developing new membranesimulating reagents for the stabilization of membrane
proteins for structural and functional studies.
Detergents, structurally similar to cell lipids, selfassemble into micellar structures and are indispensable
in dissolving integral membrane proteins into single
particles to facilitate protein crystallization. We intend
to incorporate more hydrophobicity in the interior of
detergent micelles to improve their stability and consequently their ability to stabilize integral membrane
proteins. This change is accomplished by appending
branches along the alkyl chains of detergents and, most
interestingly, by adding a short branch at the interface
between the hydrophobic tail and the hydrophilic head.
These branches may behave in 2 distinct ways like small
amphiphile additives successfully used in crystallization
of integral membrane proteins, thereby decreasing the
micellar radius and extruding water from the hydrophobic core of the micelles.
We are also working on a unique class of molecules
with facial amphiphilicity. The facial amphiphiles are
structurally distinct from the classical detergents that
have end polarity. Although not clear, the binding mode
with integral membrane proteins by the facial amphiphiles should differ from that of classical detergents.
A smaller protein–facial amphiphile complex may be
formed because of the amphiphile’s small aggregation
number, which is expected to be beneficial for obtaining well-ordered protein crystals. We have shown that
our newly designed facial amphiphiles can maintain
the full catalytic function of an ATP-binding cassette
transporter protein.
I
F i g . 1 . Cross-eyed stereo view of the Fe a3-Cu B binuclear activesite structures (leftmost Fe and Cu) of the cytochrome aa3 from R
sphaeroides (A) and the cytochrome ba 3 from T thermophilus (B),
emphasizing the overlapping position of the glutamate 286 (Glu238) in R sphaeroides and the isoleucine 235 (Ile-235) in T thermophilus. Under the influence of oxygen reduction, protons most
likely enter the protein from the upper left of the figure and exit at
the lower right.
glutamate 286 resides in a highly hydrophobic region of
the structure, its pKa most likely is much higher.
The surprise in the structure of cytochrome ba 3 is
that an isoleucine residue is isopositional with glutamate 286 as isoleucine 235, as shown in Fig. 1B. We
mutated this residue to both a glutamine and a glutamate residue in the cytochrome with no apparent loss
of electron-transfer activity. To determine if the substituted glutamate residue can be used to monitor changes
in the Fea3-Cu B pair, as it does in the A-type oxidases,
we have initiated an infrared study of these mutant proteins in collaboration with R. Gennis, University of Illinois, Urbana-Champaign, Illinois, and J. Heberle, Jülich
Research Center, Jülich, Germany. How these studies
will advance our understanding of proton-pumping
mechanisms remains unclear. What is clear is that
cytochrome ba 3 provides new openings to explore the
mechanism of the cytochrome oxidases.
PUBLICATIONS
Chen, Y., Hunsicker-Wang, L.M., Pacoma, R.L., Luna, E., Fee, J.A. A homologous
expression system for obtaining engineered cytochrome ba3 from Thermus thermophilus HB8. Protein Expr. Purif. 40:299, 2005.
Farver, O., Chem, Y., Fee, J.A., Pecht, I. Electron transfer among the CuA-, heme
b- and a3-centers of Thermus thermophilus cytochrome ba3. FEBS Lett.
580:3417, 2006.
MOLECULAR BIOLOGY
2006
The structural determination of integral membrane
proteins with our synthesized amphiphiles is being investigated in collaboration with members of the Center for
Innovative Membrane Protein Technologies of the Joint
Center for Structural Genomics at Scripps Research.
PUBLICATIONS
Bosco, D.A., Fowler, D.M., Zhang, Q., Nieva, J., Powers, E.T., Wentworth, P., Jr.,
Lerner, R.A., Kelly, J.W. Elevated levels of oxidized cholesterol metabolites in Lewy
body disease brains accelerate α-synuclein fibrillization [published correction appears
in Nat. Chem. Biol. 2:346, 2006]. Nat. Chem. Biol. 2:249, 2006.
Structural Neurobiology and
Development of Protein
Therapeutic Agents
R.C. Stevens, E.E. Abola, A.I. Alexandrov, H.M. Archer,
J.W. Arndt, G.A. Asmar-Rovira, R.R. Benoit, M.H. Bracey,
A. Brooun, Q. Chai, V.G. Cherezov, E. Chien, A. Gámez,
M.T. Griffith, C. Grittini, M.A. Hanson, V.-P. Jaakola, J. Joseph,
K. Masuda, M. Mileni, K. Moy, M. Nelson, C. Roth,
K. Saikatendu, V. Subramanian, J. Velasquez, L. Wang,
M.K. Yadav
HIGH-THROUGHPUT STRUCTURAL BIOLOGY
ut of frustration with the rate at which information on structural biology became known in the
past, we focused on developing new tools to
change the field of structural biology by accelerating the
rate of determination of protein structures. This endeavor
included pioneering microliter expression/purification
for structural studies, nanovolume crystallization, automated collection of images, and synchrotron beam line
automation. These technologies were initially tested
by staff at the Joint Center for Structural Genomics
(htpp://www.jcsg.org), where the power of the new
tools was demonstrated. Although the Joint Center for
Structural Genomics 2 has continued as a successful
second-phase structural genomics production center,
in collaboration with P. Kuhn, Department of Cell Biology, we have created 2 new technology-focused centers
funded by the National Institutes of Health.
The first center is the Joint Center for Innovative Membrane Protein Technologies (http://jcimpt.scripps.edu).
Here, in collaboration with K. Wüthrich, Q. Zhang, and
G. Chang, Department of Molecular Biology; M.G. Finn,
Department of Chemistry; and P. Kuhn and M. Yeager,
Department of Cell Biology, we do research exclusively
on eukaryotic and prokaryotic membrane proteins. The
O
THE SCRIPPS RESEARCH INSTITUTE
173
second center is the Accelerated Technologies Center
for Gene to 3D Structure (http://www.atcg3d.org). Here
we are doing collaborative work with Dr. Kuhn and with
researchers from deCODE biostructures, Bainbridge
Island, Washington; Lyncean Technologies, Palo Alto,
California; and the University of Chicago, Chicago, Illinois. In 2005, scientists at the centers showed that
high-resolution electron density maps and refined models
can be obtained from in situ diffraction of crystals grown
in microcapillaries. In 2007, the first laboratory-sized
synchrotron will be installed at Scripps Research. The
synchrotron has performance characteristics comparable to those of a synchrotron beam line in terms of
intensity and tunability and will enable us to use direct
diffraction analysis of ongoing in situ crystallization
experiments to accelerate the determination of macromolecular structures.
STRUCTURAL NEUROBIOLOGY
Although we have developed high-throughput methods to accelerate the determination of protein structures,
our primary interest is using these tools to study the
chemistry and biology of neurotransmission and of
diseases that affect neurons, particularly childhood
neurologic disorders. Our goals are to understand how
neuronal cells function on a molecular level and, on
the basis of that understanding, create new molecules
and materials that mimic neuronal signal transduction
and recognition.
BIOSYNTHESIS OF NEUROTRANSMITTERS
For neuronal signal transduction, the presynaptic cell
synthesizes neurotransmitters that then traverse the
synaptic cleft. We are using the high-throughput methods to determine the inclusive structures of complete
biochemical pathways. Specifically, we are interested
in determining the structures of all the enzymes in the
biosynthesis pathways of neurotransmitters in order to
understand the mechanistic details of each individual
enzymatic reaction at the atomic level. This approach
also allows us to determine the best path of drug discovery for the biosynthesis of neurotransmitters.
T H E R A P E U T I C A G E N T S F O R T R E AT M E N T O F
PHENYLKETONURIA
In addition to the basic hydroxylase enzymology
questions under investigation, recent clinical studies
suggest that some patients with the metabolic disease
phenylketonuria are responsive to (6R)-L-erythro-5,6,7,8tetrahydrobiopterin, the natural cofactor of phenylalanine hydroxylase. We are doing studies in collaboration
with scientists at BioMarin Pharmaceutical Inc., Novato,
174 MOLECULAR BIOLOGY
2006
THE SCRIPPS RESEARCH INSTITUTE
California, to correlate how structure can be used to
predict which patients with phenylketonuria most likely
will respond to treatment with this cofactor. Phase 3
clinical trials for the treatment of mild phenylketonuria
with the proprietary form of the cofactor, Phenoptin,
have been completed.
For classical phenylketonuria, we are developing
an enzyme replacement therapeutic agent that is being
tested in animal models. The therapy is based on administration of a modified form of phenylalanine ammonia
lyase discovered in our structural studies (Fig. 1). Last,
F i g . 1 . A, Crystal structure of phenylalanine ammonia lyase
(PAL) determined at 1.6-Å resolution. This protein structure was
engineered and chemically modified as a once-a-week injectable
therapeutic agent for treatment of phenylketonuria. B, ENU2 mice
are used as a model for phenylketonuria in preclinical studies. C
and D, A reduction in phenylalanine and immune response levels
occurs in ENU2 mice after the injection of PAL that has been
chemically modified (pegylated). These PEG-PAL formulations show
promise as therapeutic agents for treatment of phenylketonuria.
we are determining the structural basis of diseases
caused by several other enzymes involved in the biosynthesis of neurotransmitters.
NEUROTOXINS
The clostridial neurotoxins include tetanus toxin and
the 7 serotypes of botulinum toxin. We are determining
the molecular events involved in the binding, pore formation, translocation, and catalysis of botulinum neurotoxin. Although botulinum toxin is most known for
its deadly effects, it is now being used therapeutically
to treat involuntary muscle disorders such as cerebral
palsy and neuromuscular dystonias. Previously, we
determined the structure of the 150-kD holotoxin form
of the toxin, the holotoxin bound to antibodies, the
catalytic domains of several serotypes (A, B, D, F, G),
and the catalytic domain bound to substrates and inhibitors (Fig. 2). These structures are being used to under-
F i g . 2 . Serotype structures of botulinum neurotoxin (BoNT), its
light chain (LC), the closely related tetanus neurotoxin (TeNT), and
the crystal structure of 150-kD botulinum neurotoxin A bound to a
fragment of a neutralizing monoclonal antibody.
stand and redesign the toxin’s mechanism of action
and to determine additional therapeutic applications of
the toxin.
CANNABINOID SIGNALING
In collaboration with B.F. Cravatt, Department of
Cell Biology, we solved the structure of fatty acid amide
hydrolase, a degradative integral membrane enzyme
responsible for setting intracellular levels of endocannabinoids, to 2.8 Å. Fatty acid amide hydrolase is intimately
associated with CNS signaling processes such as retrograde synaptic transmission, a process that is also
modulated by the illicit substance δ-9-tetrahydrocannabinol. With our knowledge of the 3-dimensional structure, we are trying to understand how the enzyme works
at a basic level and how it might be the basis for potential drug discovery.
PUBLICATIONS
Arndt, J.W., Chai, Q., Christian, T., Stevens, R.C. Structure of botulinum neurotoxin type D light chain at 1.65 Å resolution: repercussions for VAMP-2 substrate
specificity. Biochemistry 45:3255, 2006.
MOLECULAR BIOLOGY
2006
Arndt, J.W., Jacobson, M.J., Abola, E.E., Tepp, W.H., Johnson, E.A., Stevens, R.C.
A structural perspective of the sequence variability within botulinum neurotoxin
subtypes A1-A4. J. Mol. Biol. 362:733, 2006.
Blau, N., Koch, R., Matalon, R., Stevens, R.C. Five years of synergistic scientific
effort on phenylketonuria therapeutic development and molecular understanding.
Mol. Genet. Metab. 86(Suppl. 1):S1, 2005.
Collins, B., Stevens, R.C., Page, R. Crystallization optimum solubility screening:
using crystallization results to identify the optimal buffer for protein crystal formation.
Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 61(Pt. 12):1035, 2005.
DiDonato, M., Krishna, S.S., Schwarzenbacher, R., et al. Crystal structure of a
single-stranded DNA-binding protein (TM0604) from Thermotoga maritima at 2.60
Å resolution. Proteins 63:256, 2006.
Han, G.W., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of an apo
mRNA decapping enzyme (DcpS) from mouse at 1.83 Å resolution. Proteins
60:797, 2005.
THE SCRIPPS RESEARCH INSTITUTE
175
Scriver, C.R., Hurtubise, M., Prevost, L., Phommarinh, M., Konecki, D., Erlandsen,
H., Stevens, R.C., Waters, P.J., Ryan, S., McDonald, D., Sarkissan, C. A PAH
gene knowledge base: content, informatics, utilization. In: PKU and BH4: Advances
in Phenylketonuria and Tetrahydrobiopterin Research. Blau, N. (Ed.). SPS Publications, Heilbrun, Germany, 2006, p. 434.
Swaminathan, S., Stevens, R.C. Three-dimensional protein structures of botulinum
neurotoxin light chains serotypes A, B, and E. In: Treatments from Toxins: The
Therapeutic Potential of Clostridial Neurotoxins. Foster, K.A., Hambleton, P., Shone,
C.C. (Eds.). CRC Press: Boca Raton, FL, in press.
Xu, Q., Schwarzenbacher, R., Krishna, S.S., et al. Crystal structure of acireductone
dioxygenase (ARD) from Mus musculus at 2.06 Å resolution. Proteins 64:808, 2006.
Xu, Q., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of virulence
factor CJ0248 from Campylobacter jejuni at 2.25 Å resolution reveals a new fold.
Proteins 62:292, 2006.
Yadav, M.K., Gerdts, C.J., Sanishvili, R., Smith, W.W., Roach, L.S., Ismagilov, R.F.,
Kuhn, P., Stevens, R.C. In situ data collection and structure refinement from microcapillary protein crystallization. J. Appl. Crystallogr. 38:900, 2005.
Han, G.W., Krishna, S.S., Schwarzenbacher, R., et al. Crystal structure of the
ApbE protein (TM1553) from Thermotoga maritima at 1.58 Å resolution. Proteins
64:1083, 2006.
Jaroszewski, L., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of
Hsp33 chaperone (TM1394) from Thermotoga maritima at 2.20 Å resolution.
Proteins 61:669, 2005.
Jin, K.K., Krishna, S.S., Schwarzenbacher, R., et al. Crystal structure of TM1367
from Thermotoga maritima at 1.90 Å resolution reveals an atypical member of the
cyclophilin (peptidylprolyl isomerase) fold. Proteins 63:1112, 2006.
Joseph, J.S., Saikatendu, K.S., Subramanian, V., Neuman, B.W., Brooun, A.,
Griffith, M., Moy, K., Yadav, M.K., Velazquez, J., Buchmeier, M.J., Stevens, R.C.,
Kuhn, P. Crystal structure of non-structural protein-10 (nsp10) from the SARS
coronavirus reveals a novel fold with two zinc-binding motifs. J. Virol. 80:7894,
2006.
Klock, H.E., Schwarzenbacher, R., Xu, Q., et al. Crystal structure of a conserved
hypothetical protein (gi: 13879369) from mouse at 1.90 Å resolution reveals a
new fold. Proteins 61:1132, 2005.
Matalon, R., Michals-Matalon, K., Koch, R., Grady, J., Tyring, S., Stevens, R.C.
Response of patients with phenylketonuria in the US to tetrahydrobiopterin. Mol.
Genet. Metab. 86(Suppl. 1):S17, 2005.
Mathews, I.I., Krishna, S.S., Schwarzenbacher, R., et al. Crystal structure of
phosphoribosylformylglycinamidine synthase II (smPurL) from Thermotoga maritima at 2.15 Å resolution. Proteins 63:1106, 2006.
Mathews, I.I., Krishna, S.S., Schwarzenbacher, R., et al. Crystal structure of
phosphoribosylformyl-glycinamidine synthase II, PurS subunit (TM1244) from
Thermotoga maritima at 1.90 Å resolution. Proteins 65:249, 2006.
Pérez, B., Desviat, L.R., Gómez-Puertas, P., Martínez, A., Stevens, R.C., Ugarte, M.
Kinetic and stability analysis of PKU mutations identified in BH4-responsive patients.
Mol. Genet. Metab. 86(Suppl. 1):S11, 2005.
Peti, W., Page, R., Moy, K., O’Neil-Johnson, M., Wilson, I.A., Stevens, R.C.,
Wüthrich, K. Towards miniaturization of a structural genomics pipeline using
micro-expression and microcoil NMR. J. Struct. Funct. Genomics 6:259, 2005.
Ratia, K., Saikatendu, K.S., Santarsiero, B.D., Barretto, N., Baker, S.C., Stevens,
R.C., Mesecar, A.D. Severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme. Proc. Natl. Acad. Sci. U. S. A.
103:5717, 2006.
Saikatendu, K.S., Joseph, J.S., Subramanian, V., Clayton, T., Griffith, M., Moy, K.,
Velasquez, J., Neuman, B.W., Buchmeier, M.J., Stevens, R.C., Kuhn, P. Structural
basis of severe acute respiratory syndrome coronavirus ADP-ribose-1′′-phosphate
dephosphorylation by a conserved domain of nsP3. Structure 13:1665, 2005.
Schwarzenbacher, R., McMullan, D., Krishna, S.S., et al. Crystal structure of a
glycerate kinase (TM1585) from Thermotoga maritima at 2.70 Å resolution reveals
a new fold. Proteins 65:243, 2006.
High-Throughput Approaches to
Protein Structure and Function
S.A. Lesley, M. Deller, D. Carlton, H. Johnson, Y. Elias,
T. Clayton
enomic information from the large number of
sequenced species has provided as many questions as it has answered. Evaluating protein
structure and function is of primary importance for understanding the basic biology of the cell and is a challenge
in the context of the genome. To address this challenge,
we have established high-throughput approaches for
evaluating structural and functional diversity of proteins.
We have developed the capacity to clone, express, purify,
and crystallize large numbers of proteins in parallel as
part of our structural genomics effort with the Joint Center for Structural Genomics, and we hope to apply these
same tools to characterize the molecular basis of the
specificity of enzyme substrates.
The goals of the Joint Center for Structural Genomics
are to develop a high-throughput and cost-effective structure pipeline and to use the pipeline to determine novel
protein folds and explore protein structure-function relationships. We have used this approach in the extensive
study of the thermophilic bacterium Thermotoga maritima and for targets from mouse and other bacterial
genomes. Our technologies have enabled us to perform
comprehensive structural studies of these proteomes.
To date, these efforts have resulted in more than 300
novel protein structures from the center.
Functional studies of selected targets have been
performed. For example, in collaboration with A. Kohen,
University of Iowa, Iowa City, we explored the mecha-
G
176 MOLECULAR BIOLOGY
2006
nism of thymidylate synthase from T maritima. This
protein has a novel fold and a unique flavin-dependent
biochemical mechanism. The gene for thymidylate synthase is an essential one, and the protein is an important
potential antibacterial target because of its structural
dissimilarity with the human protein.
We have also developed the method of deuterium
exchange by mass spectrometry in collaboration with
V. Woods, University of California, San Diego, to characterize protein regions with highly flexible regions
that interfere with crystallization. Subsequent elimination of these regions dramatically improves crystallization and has resulted in structures for several
problematic structures.
Expression of membrane proteins continues to be
one of the most difficult challenges in studying this
important protein class. Our structural genomics efforts
in collaboration with S. Eshaghi, Karolinska Institutet,
Stockholm, Sweden, have led to the structure of the
integral membrane protein CorA, a magnesium transporter from T maritima. In collaboration with P. Schultz,
Department of Chemistry, we are exploring the use of
unnatural amino acids to enhance the purification and
crystallization of integral membrane proteins.
THE SCRIPPS RESEARCH INSTITUTE
Jin, K.K., Krishna, S.S., Schwarzenbacher, R., et al. Crystal structure of TM1367
from Thermotoga maritima at 1.90 Å resolution reveals an atypical member of the
cyclophilin (peptidylprolyl isomerase) fold. Proteins 63:1112, 2006
Klock, H.E., Schwarzenbacher, R., Xu, Q., et al. Crystal structure of a conserved
hypothetical protein (gi: 13879369) from mouse at 1.90 Å resolution reveals a
new fold. Proteins 61:1132, 2005.
Klock, H.E., White, A., Koesema, E., Lesley, S.A. Methods and results for semiautomated cloning using integrated robotics. J. Struct. Funct. Genomics 6:89, 2005.
Kreusch, A., Han, S., Brinker, A., Zhou, V., Choi, H., He, Y., Lesley, S.A., Caldwell, J., Gu, X. Crystal structures of a new class of HSP90 inhibitors, dihydroxyphenylpyrazoles. Bioorg. Med. Chem. Lett. 15:1475, 2005.
Kreusch, A., Han, S., Brinker, A., Zhou, V., Choi, H.S., He, Y., Lesley, S.A., Caldwell,
J., Gu, X.J. Crystal structures of human HSP90α complexed with dihydroxyphenylpyrazoles. Bioorg. Med. Chem. Lett. 15:1475, 2005.
Lesley, S.A., Wilson, I.A. Protein production and crystallization at the Joint Center
for Structural Genomics. J. Struct. Funct. Genomics 6:71, 2005.
Levin, I., Miller, M.D., Schwarzenbacher, R., et al. Crystal structure of an indigoidine synthase A (IndA)-like protein (TM1464) from Thermotoga maritima at 1.90
Å resolution reveals a new fold. Proteins 59:864, 2005.
Mason, A., Agrawal, N., Washington, M.T., Lesley, S.A., Kohen, A. A lag-phase in
the reduction of flavin dependent thymidylate synthase (FDTS) revealed a mechanistic missing link. Chem. Commun. (Camb.) 1781, 2006, Issue 16.
Mathews, I., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA) from Thermotoga maritima
at 2.0 Å resolution reveals a new fold. Proteins 59:869,2005.
Mathews, I.I., Krishna, S.S., Schwarzenbacher, R., et al. Crystal structure of phosphoribosylformylglycinamidine synthase II (smPurL) from Thermotoga maritima at
2.15 Å resolution. Proteins 63:1106, 2006.
PUBLICATIONS
Arndt, J.W., Schwarzenbacher, R., Page, R., et al. Crystal structure of an α/β serine hydrolase (YDR428C) from Saccharomyces cerevisiae at 1.85 Å resolution.
Proteins 58:755, 2005.
McMullan, D., Canaves, J.M., Quijano, K., Abdubek, P., Nigoghossian, E., Haugen, J., Klock, H.E., Vincent, J., Hale, J., Paulsen, J., Lesley, S.A. High-throughput protein production for x-ray crystallography and use of size-exclusion
chromatography to validate computational biological unit predictions. J. Struct.
Funct. Genomics 6:135, 2005.
Chamberlain, P.P., Sandberg, M.L., Sauer, K., Cooke, M.P., Lesley, S.A., Spraggon,
G. Structural insights into enzyme regulation for inositol 1,4,5-trisphosphate 3kinase B. Biochemistry 44:14486, 2005.
Page, R., Deacon, A.M., Lesley, S.A., Stevens, R.C. Shotgun crystallization strategy for structural genomics, II: crystallization conditions that produce high resolution structure for T maritima proteins. J. Struct. Funct. Genomics 6:209, 2005.
Columbus, L., Lipfert, J., Klock, H., Millett, I., Doniach, S., Lesley, S.A. Expression, purification, and characterization of Thermotoga maritima membrane proteins
for structure determination. Protein Sci. 15:961, 2006.
Rife, C., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of the global
regulatory protein CsrA from Pseudomonas putida at 2.05 Å resolution reveals a
new fold. Proteins 61:449, 2005.
DiDonato, M., Krishna, S.S., Schwarzenbacher, R., et al. Crystal structure of a
single-stranded DNA-binding protein (TM0604) from Thermotoga maritima at 2.60
Å resolution. Proteins 63:256, 2006.
Rife, C., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of a putative
modulator of DNA gyrase (pmbA) from Thermotoga maritima at 1.95 Å resolution
reveals a new fold. Proteins 61:444, 2005.
Eshaghi, S., Niegowski, D., Kohl, A., Martinez Molina, D., Lesley, S.A., Nordlund, P.
Crystal structure of a divalent metal ion transporter CorA at 2.9 Å resolution [published correction appears in Science 313:1389, 2006]. Science 313:354, 2006.
Wang, Y., Klock, H., Yin, H., Wolff, K., Bieza, K., Niswonger, K., Matzen, J.,
Gunderson, D., Hale, J., Lesley, S., Kuhen, K., Caldwell, J., Brinker, A. Homogeneous high-throughput screening assays for HIV-1 integrase 3β-processing and
strand transfer activities. J. Biomol. Screen. 10:456, 2005.
Han, G.W., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of an apo
mRNA decapping enzyme (DcpS) from mouse at 1.83 Å resolution. Proteins
60:797, 2005.
Han, G.W., Schwarzenbacher, R., Page, R., et al. Crystal structure of an alanineglyoxylate aminotransferase from Anabaena sp. at 1.70 Å resolution reveals a noncovalently linked PLP cofactor. Proteins 58:971, 2005.
Han, S., Zhou, V., Pan, S., Liu, Y., Hornsby, M., McMullan, D., Klock, H., Lesley,
S.A., Gray, N., Caldwell, J., Gu, X.J. Identification of coumarin derivatives as a novel
class of allosteric MEK1 inhibitors. Bioorg. Med. Chem. Lett. 15:5467, 2005.
Jaroszewski, L., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of
Hsp33 chaperone (TM1394) from Thermotoga maritima at 2.20 Å resolution.
Proteins 61:669, 2005.
Xu, Q., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of a formiminotetrahydrofolate cyclodeaminase (TM1560) from Thermotoga maritima at
2.80 Å resolution reveals a new fold. Proteins 58:976, 2005.
Xu, Q., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of virulence
factor CJ0248 from Campylobacter jejuni at 2.25 Å resolution reveals a new fold.
Proteins 62:292, 2006.
MOLECULAR BIOLOGY
2006
Nuclear Magnetic Resonance
Spectroscopy in Protein
Structural Biology and Structural
Genomics
M. Almeida, W. Augustyniak, L. Columbus, M. Geralt,
R. Horst, M. Johnson, B. Pedrini, W.J. Placzek, P. Serrano,
K. Wüthrich
ur research program focuses on 2 areas. First,
in a collaboration with A. Horwich, Yale University, New Haven, Connecticut, who is a visiting
scientist at Scripps Research, we are investigating structural and mechanistic aspects of the function of GroEtype chaperonin systems in Escherichia coli. This
research concerns the process of protein folding in
healthy and diseased organisms and thus is directly
related to the currently extensively discussed protein
misfolding diseases. Because of the large size of the
GroE-type supramolecular structures, this project
depends on continuous improvement of existing solution nuclear magnetic resonance (NMR) techniques
and development of new techniques.
Second, we develop and apply NMR methods for
use in structural genomics. We participate in the Joint
Center for Structural Genomics (JCSG), the JCSG Center for Innovative Membrane Protein Technologies, and
the Consortium for Functional and Structural Proteomics
of SARS-CoV–Related Proteins (FSPS). On the one hand,
we explore the use of automated microscale NMR equipment for the screening of recombinant protein preparations for folded globular domains. On the other hand,
we use NMR spectroscopy to determine the structures of
selected proteins from the proteomes under study. The
following sections highlight our research on proteins
from the severe acute respiratory syndrome–associated
coronavirus (SARS-CoV) proteome, which is pursued
under the auspices of the FSPS (http://sars.scripps.edu)
and the JCSG (http://www.jcsg.org).
O
THE SARS-COV PROBLEM
In 2003, a major global outbreak of SARS was
caused by a newly emerged coronavirus. The coronavirus genome is composed of a single plus-strand
RNA of about 30 kb and is the largest genome among
known RNA viruses. About two thirds of the genome
is devoted to encoding the replicase polyprotein, which
is cleaved by viral proteases to release the mature
THE SCRIPPS RESEARCH INSTITUTE
177
nonstructural proteins that perform enzymatic functions
of the virus within the host cell. These functions include
RNA-processing steps and other functions that are currently unknown. The genome also encodes viral structural proteins, which form part of the mature viral
particle along with genomic RNA.
Several of the replicase proteins of coronaviruses
have little or no apparent relationship to other known
proteins, and little is known about how they function
during viral infection. In addition, the reasons for the
severe signs and symptoms caused by SARS-CoV in
comparison with other human coronaviruses, which
usually cause much less severe infections, are currently unknown. We are using NMR spectroscopy for
structural and functional investigations of SARS-CoV
proteins to gain information about the viral life cycle
and to identify possible new antiviral strategies.
NONSTRUCTURAL PROTEIN 1
Nonstructural protein 1 (nsp1) is the leader protein
of the SARS-CoV genome and the first to be translated
and cleaved by the viral protease to its mature form.
It has little apparent relationship to proteins of other
coronaviruses and may perform a function unique to
SARS-CoV. We used NMR spectroscopy to investigate
the solution structure and dynamics of nsp1. The protein adopts a new 3-dimensional fold, with a distorted,
6-stranded β-barrel covered by an α-helix. This stable,
folded globular domain carries a long, flexibly disordered polypeptide “tail” at the C terminus. We used
bioinformatics techniques to search for local structural
features that might provide insight into the functional
properties of this protein. We detected a possible protease active site on one end of the β-barrel, indicating
that nsp1 may be a previously unrecognized viral protease. Follow-up studies indicated that formation of a
functional active site may require the presence of the
long C-terminal tail, or of other protein cofactors.
NONSTRUCTURAL PROTEIN 3
The viral element nsp3 is a large protein of about
2000 amino acid residues that most likely includes
multiple functional domains. We designed smaller constructs of this protein encompassing predicted individual
domains, and used 1-dimensional 1H NMR screening
to identify those domains that were independently folded.
We then determined the solution structure of the N-terminal domain, nsp3a. Unexpectedly, we found that its
structure is similar to that of the α/β roll fold of ubiquitin. This structural motif is most commonly found in
proteins of eukaryotes that are involved in cellular sig-
178 MOLECULAR BIOLOGY
2006
naling pathways. Therefore, the nsp3a domain may be
used by the virus to interact with signaling proteins of
the host cell and to interfere with cellular pathways in
order to increase virulence.
We are also investigating a possible second function of this protein in RNA processing. Using NMR spectroscopy and mass spectrometry, we identified RNA
molecules that bind to nsp3a. We are studying these
interactions to determine possible enzymatic or scaffolding functions.
THE SCRIPPS RESEARCH INSTITUTE
PUBLICATIONS
Almeida, M.S., Herrmann, T., Peti, W., Wilson, I.A., Wüthrich, K. NMR structure
of the conserved hypothetical protein TM0487 from Thermotoga maritima: implications for 216 homologous DUF59 proteins. Protein Sci. 14:2880, 2005.
Columbus, L., Peti, W., Etezady-Esfarjani, T., Herrmann, T., Wüthrich, K. NMR
structure determination of the conserved hypothetical protein TM1816 from Thermotoga maritima. Proteins 60:552, 2005.
Horst, R., Bertelsen, E.B., Fiaux, J., Wider, G., Horwich, A.L., Wüthrich, K.
Direct NMR observation of a substrate protein bound to the chaperonin GroEL.
Proc. Natl. Acad. Sci. U. S. A. 102:12748, 2005.
Peti, W., Herrmann, T., Zagnitko, O., Grzechnik, S.K., Wüthrich, K. NMR structure of the conserved hypothetical protein TM0979 from Thermotoga maritima.
Proteins 59:387, 2005.
NONSTRUCTURAL PROTEIN 7
The viral component nsp7 is highly conserved
between the different coronaviruses and probably performs an essential core function in this virus family.
Interestingly, the solution structure of nsp7 also shows
a new fold that was not previously observed in any
known protein structure. The structure consists of 4
helices, and although many 4-helix bundle proteins are
known, nsp7 does not form a bundle. Rather 3 helices
are assembled into a flat sheet, with the helices antiparallel, and the fourth helix is stacked across one side of
this sheet (Fig. 1). The other surface of the flat sheet
Peti, W., Johnson, M.A., Herrmann, T., Neuman, B.W., Buchmeier, M.J., Nelson,
M., Joseph, J., Page, R., Stevens, R.C., Kuhn, P., Wüthrich, K. Structural genomics of the severe acute respiratory syndrome coronavirus: nuclear magnetic resonance structure of the protein nsp7. J. Virol. 79:12905, 2005.
Peti, W., Page, R., Moy, K., O’Neil-Johnson, M., Wilson, I.A., Stevens, R.C.,
Wüthrich, K. Towards miniaturization of a structural genomics pipeline using
micro-expression and microcoil NMR. J. Struct. Funct. Genomics 6:259, 2005.
Nuclear Magnetic Resonance
of 3-Dimensional Structure
and Dynamics of Proteins
in Solution
P.E. Wright, H.J. Dyson, R. Burge, J. Ferreon, N. Greenman,
T.-H. Huang, B.B. Koehntop, M. Kostic, B. Lee, C.W. Lee,
M. Landes, M. Martinez-Yamout, T. Nishikawa, K. Sugase, J.
Wojciak, M. Zeeb, E. Manlapaz, L.L. Tennant, J. Chung,
D.A. Case, J. Gottesfeld, R. Evans,* M. Montminy*
* Salk Institute, La Jolla, California
F i g . 1 . Ensemble of 20 conformers representing the polypeptide
backbone in the solution structure of SARS-CoV nsp7. The 3
helices α2, α3, and α4 assemble into a flat sheet, with the α1
helix stacked diagonally across one surface of this sheet. Reprinted
with permission from Peti, W., et al. J. Virol. 79:12905, 2005.
e use multidimensional nuclear magnetic
resonance (NMR) spectroscopy to investigate the structures, dynamics, and interactions of proteins in solution. Such studies are essential
for understanding the mechanisms of action of these
proteins and for elucidating structure-function relationships. The focus of our current research is protein-protein and protein–nucleic acid interactions involved in the
regulation of gene expression.
Copyright 2005 American Society for Microbiology.
T R A N S C R I P T I O N FA C T O R – N U C L E I C A C I D C O M P L E X E S
contains hydrophobic and negatively charged patches,
which most likely are sites for protein-protein interactions. Currently, we are using NMR spectroscopy to
identify interactions with other SARS-CoV proteins. Such
interactions could be targeted for the design of inhibitory
molecules with antiviral activity.
NMR methods are being used to determine the
3-dimensional structures and intramolecular dynamics
of zinc finger motifs from several eukaryotic transcriptional regulatory proteins, both free and complexed with
target nucleic acid. Zinc fingers are among the most
abundant domains in eukaryotic genomes. They play a
central role in the regulation of gene expression at both
W
MOLECULAR BIOLOGY
2006
the transcriptional and the posttranscriptional level,
mediated through their interactions with DNA, RNA,
or protein components of the transcriptional machinery. The C2H2 zinc finger, first identified in transcription
factor IIIA (TFIIIA), is used by numerous transcription
factors to achieve sequence-specific recognition of DNA.
There is growing evidence, however, that some C2H2 zinc
finger proteins control gene expression both through
their interactions with DNA regulatory elements and, at
the posttranscriptional level, by binding to RNA.
The best-characterized example of a C2H 2 zinc finger protein that binds specifically to both DNA and to
RNA is TFIIIA, which contains 9 zinc fingers. We showed
previously that different subsets of zinc fingers are
responsible for high-affinity binding of TFIIIA to DNA
(fingers 1–3) and to 5S RNA (fingers 4–6). To obtain
insights into the mechanism by which the TFIIIA zinc
fingers recognize both DNA and RNA, we have used
NMR methods to determine the structures of the complex formed by zf1-3 (a protein containing fingers 1–3)
with DNA and by zf4-6 (a protein consisting of fingers
4–6) with a fragment of 5S RNA.
Three-dimensional structures were determined previously for the complex of zf1-3 with the cognate 15-bp
oligonucleotide duplex. The structures contain several
novel features and reveal that prevailing models of DNA
recognition, which assume that zinc fingers are independent modules that contact bases through a limited
set of amino acids, are outmoded.
In addition to its role in binding to and regulating
the 5S RNA gene, TFIIIA also forms a complex with
the 5S RNA transcript. NMR structures of the complex
formed by zinc fingers 4–6 with a truncated form of 5S
RNA have been completed and give important insights
into the structural basis for 5S RNA recognition. Finger 4 of the protein recognizes both the structure of
the RNA backbone and the specific bases in the loop E
motif of the RNA, in a classic lock-and-key interaction.
Fingers 5 and 6, with a single residue between them,
undergo mutual induced-fit folding with the loop A
region of the RNA, which is highly flexible in the absence
of the protein.
NMR studies of 2 alternate splice variants of the
Wilms tumor zinc finger protein are in progress. These
proteins differ only through insertion of 3 additional
amino acids (the tripeptide lysine-threonine-serine) in
the linker between fingers 3 and 4, yet have marked
differences in their DNA-binding properties and subcellular localization. 15N relaxation measurements indi-
THE SCRIPPS RESEARCH INSTITUTE
179
cate that the insertion increases the flexibility of the
linker between fingers 3 and 4 and abrogates binding of
the fourth zinc finger to its cognate site in the DNA
major groove, thereby modulating DNA-binding activity.
The x-ray structure of the DNA complex has been determined, providing insights into the mechanism by which
disease-causing mutations interfere with DNA binding.
NMR studies of the RNA complex are in progress. We
have also determined the structure of a novel zinc
finger protein that binds to double-stranded RNA and
have begun experiments to define the mechanism of
RNA recognition.
Several novel zinc binding motifs have recently been
identified that mediate gene expression at the posttranscriptional level by regulating mRNA processing and
metabolism. Regulatory proteins of the TIS11 family
bind specifically, through a pair of novel CCCH zinc
fingers, to the adenosine-uridine–rich element in the
3′ untranslated region of short-lived cytokine, growth factor, and proto-oncogene mRNAs and control expression by promoting rapid degradation of the message. We
recently determined the NMR structure of the complex
formed between the tandem zinc finger domain of TIS11d
and its binding site on the adenosine-uridine–rich element. This structure showed sequence-specific recognition of single-stranded RNA through formation of a
network of hydrogen bonds between the polypeptide
backbone and the Watson-Crick edges of the bases.
PROTEIN-PROTEIN INTERACTIONS IN
T R A N S C R I P T I O N A L R E G U L AT I O N
Transcriptional regulation in eukaryotes relies on
protein-protein interactions between DNA-bound factors
and coactivators that, in turn, interact with the basal
transcription machinery. The transcriptional coactivator CREB-binding protein (CBP) and its homolog p300
play an essential role in cell growth, differentiation, and
development. Understanding the molecular mechanisms
by which CBP and p300 recognize their various target
proteins is of fundamental biomedical importance. CBP
and p300 have been implicated in diseases such as
leukemia, cancer, and mental retardation and are novel
targets for therapeutic intervention.
We previously determined the structure of the kinaseinducible activation domain of the transcription factor
CREB bound to its target domain (the KIX domain) in
CBP. Ongoing work is directed toward mapping the interactions between KIX and the transcriptional activation
domains of the proto-oncogene c-Myb and of the mixedlineage leukemia protein. The solution structure of the
180 MOLECULAR BIOLOGY
2006
ternary complex between KIX, c-Myb and the mixedlineage leukemia protein has been completed and provides insights into the structural basis for the ability of
the KIX domain to interact simultaneously and allosterically with 2 different effectors. Our work has also provided new understanding of the thermodynamics of the
coupled folding and binding processes involved in interaction of KIX with transcriptional activation domains.
We are using R 2 relaxation dispersion experiments to
elucidate the mechanism by which folding of the kinaseinducible activation domain of CREB is coupled to binding to its KIX target domain. These experiments reveal
formation of a transient and largely unfolded encounter
complex, which then folds on the surface of the KIX
domain to form the helical structure observed in the
fully bound state.
Recently, we determined the structure of the complex between the hypoxia-inducible factor Hif-1α and
the CH1 domain of CBP. The interaction between Hif-1α
and CBP/p300 is of major therapeutic interest because
of the central role Hif-1α plays in tumor progression
and metastasis; disruption of this interaction leads to
attenuation of tumor growth. A protein named CITED2
functions as a negative feedback regulator of the hypoxic
response by competing with Hif-1α for binding to the
CH1 domain of CBP. We determined the structure of the
complex formed between CITED2 and the CH1 domain
and were able to show that the CH1 domain is folded
into a stable 3-dimensional structure even in the absence
of binding partners. The intrinsically unstructured Hif-1α
and CITED2 domains use partly overlapping surfaces
of the CH1 motif to achieve high-affinity binding and
compete effectively with each other for CBP/p300. We
are continuing to map the multiplicity of interactions
between CBP/p300 domains and their numerous biological targets to understand the complex interplay of
interactions that mediate key biological processes in
health and disease.
PUBLICATIONS
De Guzman, R.N., Goto, N.K., Dyson, H.J., Wright, P.E. Structural basis for cooperative transcription factor binding to the CBP coactivator. J. Mol. Biol. 355:1005, 2006.
Kostic, M., Matt, T., Martinez-Yamout, M.A., Dyson, H.J., Wright, P.E. Solution
structure of the Hdm2 C2H2C4 RING, a domain critical for ubiquitination of p53.
J. Mol. Biol. 363:433, 2006.
Lee, B.M., Xu, J., Clarkson, B.K., Martinez-Yamout, M.A., Dyson, H.J., Case, D.A.,
Gottesfeld, J.M., Wright, P.E. Induced fit and “lock and key” recognition of 5S
RNA by zinc fingers of transcription factor IIIA. J. Mol. Biol. 357:275, 2006.
THE SCRIPPS RESEARCH INSTITUTE
Folding of Proteins and
Protein Fragments
P.E. Wright, H.J. Dyson, C. Nishimura, D. Felitsky, Y. Yao,
J. Chung, L.L. Tennant, V. Bychkova,* T. Uzawa,**
S. Takahashi**
* Institute of Protein Research, Puschino, Russia
** Kyoto University, Kyoto, Japan
he molecular mechanism by which proteins fold
into their 3-dimensional structures remains one
of the most important unsolved problems in structural biology. Nuclear magnetic resonance (NMR) spectroscopy is uniquely suited to provide information on
the structure of transient intermediates formed during
protein folding. Previously, we used NMR methods to
show that many peptide fragments of proteins have a
tendency to adopt folded conformations in water solution. The presence of transiently populated folded structures, including reverse turns, helices, nascent helices,
and hydrophobic clusters, in water solutions of short
peptides has important implications for initiation of protein folding. Formation of elements of secondary structure
probably plays an important role in the initiation of protein folding by reducing the number of conformations
that must be explored by the polypeptide chain and by
directing subsequent folding pathways.
T
A P O M Y O G L O B I N F O L D I N G PAT H WAY
A major program in our laboratory is directed
toward a structural and mechanistic description of
the apomyoglobin folding pathway. Previously, we
used quenched-flow pulse-labeling methods in conjunction with 2-dimensional NMR spectroscopy to map
the kinetic folding pathway of the wild-type protein.
With these methods, we showed that an intermediate
in which the A, G, and H helices and part of the B helix
adopt hydrogen-bonded secondary structure is formed
within 6 milliseconds of the initiation of refolding. Folding then proceeds by stabilization of additional structure
in the B helix and in the C and E helices. We are using
carefully selected myoglobin mutants and both optical
stopped-flow spectroscopy and NMR methods to further
probe the kinetic folding pathway. For some of the
mutants studied, the changes in amino acid sequence
resulted in changes in the folding pathway of the protein. These experiments are providing novel insights
into both the local and the long-range interactions that
stabilize the kinetic folding intermediate. Of particular
importance, long-range interactions have been observed
MOLECULAR BIOLOGY
2006
that indicate nativelike packing of some of the helices
in the kinetic molten globule intermediate.
Apomyoglobin provides a unique opportunity for
detailed characterization of the structure and dynamics
of a protein-folding intermediate. Conditions were previously identified under which the apomyoglobin molten
globule intermediate is sufficiently stable for acquisition
of multidimensional heteronuclear NMR spectra. Analysis
of 13C and other chemical shifts and measurements of
polypeptide dynamics provided unprecedented insights
into the structure of this state.
The A, G, and H helices and part of the B helix are
folded and form the core of the molten globule. This
core is stabilized by relatively nonspecific hydrophobic
interactions that restrict the motions of the polypeptide
chain. Fluctuating helical structure is formed in regions
outside the core, although the population of helix is low
and the chain retains considerable flexibility. The F helix
acts as a gate for heme binding and only adopts stable structure in the fully folded holoprotein.
The acid-denatured (unfolded) state of apomyoglobin
is an excellent model for the fluctuating local interactions
that lead to the transient formation of unstable elements
of secondary structure and local hydrophobic clusters
during the earliest stages of folding. NMR data indicated
substantial formation of helical secondary structure in
the acid-denatured state in regions that form the A and
H helices in the folded protein and also revealed nonnative structure in the D and E helix regions.
Because the A and H regions adopt stabilized helical
structure in the earliest detectable folding intermediate,
these results lend strong support to folding models in
which spontaneous formation of local elements of secondary structure plays a role in initiating formation of
the A-[B]-G-H molten globule folding intermediate. In
addition to formation of transient helical structure, formation of local hydrophobic clusters has been detected by
using 15N relaxation measurements. Significantly, these
clusters are formed in regions where the average surface area buried upon folding is large. In contrast to
acid-denatured unfolded apomyoglobin, the urea-denatured state is largely devoid of structure, although residual hydrophobic interactions have been detected by using
relaxation measurements.
We measured residual dipolar couplings for unfolded
states of apomyoglobin by using partial alignment in
strained polyacrylamide gels. These data provide novel
insights into the structure and dynamics of the unfolded
polypeptide chain. We have shown that the residual
THE SCRIPPS RESEARCH INSTITUTE
181
dipolar couplings arise from the well-known statistical
properties of flexible polypeptide chains. Residual dipolar couplings provide valuable insights into the dynamic
and conformational propensities of unfolded and partly
folded states of proteins and hold great promise for charting the upper reaches of protein-folding landscapes.
To probe long-range interactions in unfolded and
partially folded states of apomyoglobin, we introduced
spin-label probes at several sites throughout the polypeptide chain. These experiments led to the surprising
discovery that structures with nativelike topology exist
within the ensemble of conformations formed by the
acid-denatured state of apomyoglobin. They also indicated that the packing of helices in the molten globule
state is similar to that in the native folded protein.
The view of protein folding that results from our
work on apomyoglobin is one in which collapse of the
polypeptide chain to form increasingly compact states
leads to progressive accumulation of secondary structure and increasing restriction of fluctuations in the
polypeptide backbone. Chain flexibility is greatest at
the earliest stages of folding, in which transient elements
of secondary structure and local hydrophobic clusters
are formed. As the folding protein becomes increasingly
compact, backbone motions become more restricted,
the hydrophobic core is formed and extended, and
nascent elements of secondary structure are progressively stabilized. The ordered tertiary structure characteristic of the native protein, with well-packed side
chains and relatively low-amplitude local dynamics,
appears to form rather late in folding.
We recently introduced a variation on the classic
quench-flow technique, which makes use of the capabilities of modern NMR spectrometers and heteronuclear NMR experiments, to study the proteins labeled
along the folding pathway in an unfolded state in an
aprotic organic solvent. This method allows detection
of many more amide proton probes than in the classic
method, which required formation of the fully folded
protein and the measurement of its NMR spectrum in
water solutions. This method is particularly useful in
documenting changes in the folding pathway that result
in the destabilization of parts of the protein in the molten
globule intermediate. We recently showed that selfcompensating mutations designed to change the amino
acid sequence such that the average area buried upon
folding is significantly changed while the 3-dimensional
structure of the final folded state remains the same.
These studies showed that the average area buried
182 MOLECULAR BIOLOGY
2006
THE SCRIPPS RESEARCH INSTITUTE
upon folding is an accurate predictor of those parts of
the apomyoglobin molecule that will fold first and participate in the molten globule intermediate. Quench-flow
hydrogen exchange experiments performed on a series
of hydrophobic core mutants indicated that the overall
helix-packing topology of the kinetic folding intermediate is like that of the native protein, despite local nonnative interactions in packing of the G and H helices.
Finally, using a rapid mixing device, we have reduced the
dead time of the kinetic refolding experiments and have
shown that a compact helical intermediate is formed
within 400 microseconds after initiation of apomyoglobin refolding.
FOLDING-UNFOLDING TRANSITIONS IN CELLULAR
M E TA B O L I S M
Many species of bacteria sense and respond to their
own population density by an intricate autoregulatory
mechanism known as quorum sensing; the bacteria
release extracellular signal molecules, called autoinducers, for cell-cell communication within and between
bacterial species. A number of bacteria appear to use
quorum sensing for regulation of gene expression in
response to fluctuations in cell population density.
Processes regulated in this way include symbiosis,
virulence, competence, conjugation, production of antibiotics, motility, sporulation, and formation of biofilms.
We determined the 3-dimensional solution structure
of a complex composed of the N-terminal 171 residues
of the quorum-sensing protein SdiA of Escherichia coli
and an autoinducer molecule, N-octanoyl-1-homoserine lactone (HSL) (Fig. 1). The SdiA-HSL system shows
the “folding switch” behavior associated with quorumsensing factors produced by other bacterial species. In
the presence of HSL, the SdiA protein is stable and folded
and can be produced in good yields from an E coli expression system. In the absence of the autoinducer, the protein is expressed into inclusion bodies. Samples of the
SdiA-HSL complex can be denatured but cannot be
refolded in aqueous buffers. The solution structure of
the complex provides a likely explanation for this behavior. The autoinducer molecule is tightly bound in a deep
pocket in the hydrophobic core and is bounded by specific hydrogen bonds to the side chains of conserved
residues. The autoinducer thus forms an integral part
of the hydrophobic core of the folded SdiA.
F i g . 1 . Folding of protein and protein fragments. Ribbon dia-
gram showing the lowest energy structure of the complex between
HSL and E coli SdiA.
basis of their interactions, but unfolded proteins are
impossible to characterize structurally by x-ray crystallography, and spectroscopic methods of all kinds are
limited. It is necessary to explore unfolded proteins
under conditions that approximate their physiologic
milieu: in solution, at physiologic pHs and salt concentrations, and in the presence of specific cofactors.
Structural insights will be obtained not only from the
delineation of 3-dimensional structures but also from
the description of conformational ensembles and of the
motions of polypeptide chains under various conditions.
To gain new insights into the structural basis for the
ability of unfolded and partly folded proteins to function
in living systems, we are studying the interactions of
“client” proteins and cochaperones with a well-known
eukaryotic chaperone, Hsp90. Some of the protein components are much larger than have traditionally been
studied by using solution NMR. However, we have
designed a set of experiments that will allow us to draw
valid conclusions about the extent and role of disorder in
Hsp90 interactions. In particular, we will apply techniques recently developed in our laboratory for the
analysis of hydrogen-deuterium exchange from unstable
partially folded proteins by trapping the 2H-labeled
species in the aprotic solvent dimethyl sulfoxide. This
powerful new technique will be used to probe the structure, stability, and interactions of client proteins and
cochaperones with Hsp90.
CHAPERONE–COCHAPERONE–CLIENT PROTEIN
INTERACTIONS
Understanding the role of unfolded states in cellular
processes will require an understanding of the structural
PUBLICATIONS
Dyson, H.J., Wright, P.E. According to current textbooks, a well-defined threedimensional structure is a prerequisite for the function of the protein: is this correct? IUBMB Life 58:107, 2006.
MOLECULAR BIOLOGY
2006
Dyson, H.J., Wright, P.E., Scheraga, H.A. The role of hydrophobic interactions in
initiation and propagation of protein folding. Proc. Natl. Acad. Sci. U. S. A.
103:13057, 2006.
Kamikubo, Y., Kroon, G., Curriden, S.A., Dyson, H.J., Loskutoff, D.J. The
reduced, denatured somatomedin B domain of vitronectin refolds into a stable, biologically active form. Biochemistry 45:3297, 2006.
Martinez-Yamout, M.A., Venkitakrishnan, R.P., Preece, N.E., Kroon, G., Wright,
P.E., Dyson, H.J. Localization of sites of interaction between p23 and Hsp90 in
solution. J. Biol. Chem. 281:14457, 2006.
Nishimura, C., Dyson, H.J., Wright, P.E. Identification of native and non-native structure in kinetic folding intermediates of apomyoglobin. J. Mol. Biol. 355:139, 2006.
Papadopoulos, E., Oglecka, K., Mäler, L., Jarvet, J., Wright, P.E., Dyson, H.J.,
Gräslund, A. NMR solution structure of the peptide fragment 1-30, derived from
mouse Doppel protein, in DHPC micelles. Biochemistry 45:159, 2006.
Yao, Y., Martinez-Yamout, M.A., Dickerson, T.J., Brogan, A.P., Wright, P.E., Dyson,
H.J. Structure of the Escherichia coli quorum sensing protein SdiA: activation of the
folding switch by acyl homoserine lactones. J. Mol. Biol. 355:262, 2006.
Yao, Y., Martinez-Yamout, M.A., Dyson, H.J. Backbone and side chain 1H, 13C
and 15N assignments for Escherichia coli SdiA1-171, the autoinducer-binding
domain of a quorum sensing protein [letter]. J. Biomol. NMR 31:373, 2005.
Nuclear Magnetic Resonance
Studies of the Structure and
Dynamics of Enzymes
H.J. Dyson, P.E. Wright, S.H. Bae, D. Boehr, G. Kroon,
M. Martinez-Yamout, N.E. Preece, S.C. Sue, L.M. Tuttle,
Y. Yao, L.L. Tennant, J. Chung, C.L. Brooks, S.J. Benkovic,*
A. Holmgren,** E.A. Komives***
* Pennsylvania State University, University Park, Pennsylvania
** Karolinska Institutet, Stockholm, Sweden
*** University of California, San Diego, California
e use site-specific information on structure
and dynamics obtained via nuclear magnetic
resonance (NMR) to further the understanding of protein function. We focus on the mechanism of
enzymes and the relationship between dynamics and
function in a number of medically important systems.
W
DYNAMICS IN ENZYME ACTION
Dynamic processes are implicit in the catalytic
function of all enzymes. We use state-of-the-art NMR
methods to elucidate the dynamic properties of several
enzymes. New methods have been developed for analysis
of NMR relaxation data for proteins that tumble anisotropically and for analysis of slow timescale motions.
Dihydrofolate reductase plays a central role in folate
metabolism and is the target enzyme for a number of
antibacterial and anticancer agents. 15N relaxation experiments on dihydrofolate reductase from Escherichia
coli revealed a rich diversity of backbone dynamical
THE SCRIPPS RESEARCH INSTITUTE
183
features for a broad range of timescales (picoseconds
to milliseconds).
A major focus is on the characterization of all intermediates in the dihydrofolate reductase reaction cycle.
We have identified functionally important motions in
loops that control access to the active site of dihydrofolate reductase on timescales similar to those of the
hydride transfer chemistry and the rate-determining
step of product release. These motions differ in amplitude and timescale depending on the presence of substrate and/or cofactor in the active site, priming the
nicotinamide ring of the cofactor and the pterin ring of
the substrate for hydride transfer. In addition, measurements of the population distribution of aliphatic sidechain rotamers provided evidence for coupled motion
of active-site side chains that could enhance the catalytic process.
Most recently, we used relaxation dispersion measurements to obtain direct information on microsecondmillisecond timescale motions in dihydrofolate reductase,
allowing us to characterize the structures of excited
states involved in some of these catalysis-relevant processes. Fluctuations between these states, which involve
motions of the nicotinamide ring of the cofactor into and
out of the active site, occur on a timescale that is directly
relevant to the structural transitions involved in progression through the catalytic cycle (Fig. 1).
Dihydrofolate reductase is also the test system for
a series of experiments to address the question, If all
of the chemistry goes on at the active site, what is the
purpose of the rest of the enzyme? We are using chimeric
mutants, synthesized by our collaborator S.J. Benkovic,
Pennsylvania State University, by using a library approach.
The purpose of these experiments is to test the hypothesis
that local variations in amino acid sequence, 3-dimensional structure, and polypeptide chain dynamics strongly
influence the local interactions that mediate enzyme
catalysis and may constitute the essential circumstance
that allows enzymes to achieve high turnover rates as
well as exquisite specificity in their reactions. A combination of NMR structure and dynamics measurements,
single-molecule fluorescence measurements, and analysis of the catalytic steps in these mutant proteins will
provide new insights into the role of the protein in
enzyme catalysis.
S T R U C T U R E A N D D Y N A M I C S O F P R I O N VA R I A N T S
Onset of prion diseases is caused by conversion of
the cellular prion protein PrPC into an abnormally folded
isoform, PrPSc, that has the same primary structure as
184 MOLECULAR BIOLOGY
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F i g 1 . Schematic diagram showing the energy landscape of dihydrofolate reductase catalysis. Ground state (larger) and higher energy
(smaller) structures of each intermediate in the cycle, modeled on
published x-ray structures are shown. For each intermediate in the
catalytic cycle, the higher energy conformations detected in the
relaxation dispersion experiments resemble the ‘ground-state’ conformations of adjacent intermediates. Rate constants for the interconversion between the complexes, measured by pre–steady state enzyme
kinetics at 298 K, pH6 are indicated with gray arrows, while the
rates measured in relaxation dispersion experiments are shown with
black arrows. From Boehr et al., Science 313:1638, 2006. Reprinted
with permission from AAAS.
PrPC but a totally different 3-dimensional conformation.
The abnormally folded (“scrapie”) form of the protein is
associated with several diseases, including scrapie in
sheep, bovine spongiform encephalopathy (mad cow
disease), and human Creutzfeldt-Jakob disease and
other inherited prion diseases. We are gathering information on the mechanism of PrPSc formation that can be
obtained from structural and dynamic studies of mutant
prion proteins corresponding to inherited prion diseases.
Individuals carrying familial mutations such as
P102L (P101L in our study) are more susceptible than
those without such mutations to prion disease. On the
other hand, sheep or humans carrying Q167R and/or
Q218K mutations are resistant to scrapie and CreutzfeldtJakob disease, respectively. We are using the proteaseresistant cores of wild-type and mutant mouse prion
proteins to study the structural and dynamic basis of
PrP C -to-PrP Sc conversion in inherited prion diseases.
The core is sufficient to transmit infectivity.
DYNAMICS AND THE FUNCTION OF IΚB α
It is becoming increasingly clear that the function
of many systems in living cells depends not only on the
structures of the components but also on their flexibility. Numerous examples exist in which components of
THE SCRIPPS RESEARCH INSTITUTE
an important biological interaction are unstructured or
partly structured. In addition, even those interacting
molecules that can be classified as “folded” have areas
of mobility. Often, these areas are located precisely in
the active site of an enzyme or in the binding site of
an interacting molecule.
A central molecular interaction in cellular control is
the interaction between the nuclear transcription factor
NF-κB and its inhibitor IκBα. IκBα consists of a series
of ankyrin repeats, which appear to have differential
mobility. Using hydrogen-deuterium exchange and mass
spectrometry, our collaborator E.A. Komives, University
of California, San Diego, found that the second, third,
and fourth ankyrin repeats of IκBα are well folded,
whereas the fifth and sixth repeats, apparently with
exactly the same structure, are highly dynamic. These
observations prompt a number of questions: Are the
motions inferred from the hydrogen-deuterium mass
spectrometry experiments also reflected in the backbone and side-chain dynamics of the protein, as measured by NMR relaxation? Are the motions still present
in the IκBα–NF-κB complex? Are they necessary for
complex formation, so that if they are damped out, for
example, by site-directed mutagenesis at appropriate
positions, is the formation of the complex disfavored? To
answer these questions, we are doing a series of NMR
experiments on IκBα and its complexes with NF-κB.
PUBLICATIONS
Boehr, D.D., Dyson, H.J., Wright, P.E. An NMR perspective on enzyme dynamics.
Chem. Rev. 106:3055, 2006.
Boehr, D.D., McElheny, D., Dyson, H.J., Wright, P.E. The dynamic energy landscape of dihydrofolate reductase catalysis. Science 313:1638, 2006.
Ring Assemblies Mediating
ATP-Dependent Protein Folding
and Unfolding
A.L. Horwich, W.A. Fenton, E. Chapman, E. Koculi,
J. Hinnerwisch
arge ring assemblies function in many cellular
contexts as compartments within a compartment,
where actions can be carried out on a substrate
bound in the central space inside an oligomeric ring
by a high local concentration of surrounding active sites.
Both protein folding and unfolding are carried out in
an ATP-dependent fashion by such assemblies (Fig. 1).
L
MOLECULAR BIOLOGY
2006
F i g . 1 . Protein folding and unfolding by chaperone ring assemblies. In protein folding mediated by the chaperonin GroEL (left),
the energy of binding ATP and the cochaperonin GroES is used to
produce rigid body movements of a GroEL ring that eject a bound
nonnative substrate polypeptide into a GroES-encapsulated central
cavity, switched from hydrophobic (shaded) to hydrophilic wall
character, where productive folding proceeds. The free energy provided by a set of hydrogen bonds formed between the γ-phosphate
of ATP and the nucleotide pocket is critical to producing a power
stroke of apical domain movement that can eject the substrate
polypeptide into the folding chamber. In contrast, in ClpA-mediated
unfolding (right), this chaperone seems to use ATP hydrolysis by its
D2 ATPase domain to drive a forceful distalward movement of a
loop facing its central channel, exerting mechanical force on a
bound protein that is proposed to exert an unfolding action.
We are studying the essential double-ring components
known as chaperonins that assist protein folding to the
native state. We are focusing on the bacterial chaperonin GroEL. We have also been examining an opposite
number, an “unfoldase,” the bacterial heat-shock protein
100 ring assembly known as ClpA. During the past year,
we continued to investigate the structural correlates of
the ATPase cycle of GroEL and the polypeptide-binding
mechanisms of ClpA.
GroEL STRUCTURE AND ALLOSTERY
GroEL is an allosteric and highly cooperative machine
with positive cooperativity of ATP binding among the
subunits of a ring and negative cooperativity of binding
between the 2 rings. A long-standing question has been
how the information on nucleotide binding and ATP-vsADP state is transmitted from one ring to the other to
produce these changes in affinity that lie at the heart
of the chaperonin protein-folding cycle.
In collaboration with N.A. Ranson, University of
Leeds, Leeds, England, and H.R. Saibil, University of
London, London, England, we completed a electron
cryomicroscopy reconstruction of the transient complex
composed of GroEL, its cochaperonin GroES, and ATP by
taking advantage of a mutant form of GroEL (D398A).
The mutant form binds ATP normally but hydrolyzes it at
about 2% of the normal rate, permitting the complex to
be captured by rapid freezing on electron cryomicroscopy
grids. By comparing this structure (7.7-Å resolution)
THE SCRIPPS RESEARCH INSTITUTE
185
with a similarly obtained GroEL-GroES-ADP structure
(8.7-Å resolution), we discovered that the differences
occur mainly in the trans (unliganded) ring.
Information on the nucleotide state of the cis ring
appears to be transmitted through the positioning of
helix D, which extends from the nucleotide-binding site
to the inter-ring interface, where it interacts with the
corresponding helix in the trans ring. ATP binding
changes the relationship of these helices from that of
an unliganded ring, resulting in a series of changes in
the interface and the position of the trans helix that
lead to a closing of the nucleotide pocket and a reduction in exposure of the substrate-binding hydrophobic
residues in the trans apical domain. Hydrolysis of ATP
to ADP reverses these changes, opening both the trans
apical domains and the trans nucleotide pocket to permit ligand binding and the formation of a new folding
chamber on this ring.
T H E T R A J E C T O R Y O F P R O T E I N F O L D I N G AT G r o E L
Another question of interest regarding protein folding
by GroEL is whether, and how, GroEL affects the folding pathway taken by a substrate protein. Most likely
some action beyond simply preventing aggregation is
occurring, because some substrate proteins cannot fold
spontaneously without GroEL and GroES function. In
collaboration with R. Horst and K. Wüthrich, Department of Molecular Biology, we are studying the folding
pathways of human dihydrofolate reductase during both
spontaneous and GroEL-assisted folding. Using hydrogen-deuterium exchange during refolding of 15N-labeled
human dihydrofolate reductase and nuclear magnetic resonance analysis of the final native form, we are examining pathways taken inside and outside the chaperonin.
ROLE OF THE N-DOMAINS OF ClpA
We and others have shown that the N-domains of
ClpA are not required for action on substrates bearing
the ssrA degradation tag, but removal of the N-domains
slows the unfolding and degradation of such substrates.
We explored this observation further and found that
whereas binding and unfolding of ssrA-tagged substrate
are not affected by removal of the N-domains, removal
of the domains results in diminished stability of a complex composed of ClpA and ClpP, a double-ring protease
that cooperates with ClpA to degrade certain proteins.
Consequently, proteins unfolded and translocated by
ClpA interact with the protease component and hence
escape degradation. In contrast, substrate proteins bearing a RepA degradation tag appear to be completely
dependent on the N-domains for initial binding to ClpA.
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LOOPS IN ClpA
In collaboration with A. Deniz, Department of
Molecular Biology, we are examining the role of the
recently identified loops in ClpA that mediate ssrA binding and substrate protein translocation and associated
unfolding. We have designed a series of cysteine substitution variants of ClpA and ClpP that can be labeled with
fluorescent reporters and used in single-molecule experiments designed to observe the putative motion of these
loops during the substrate translocation/unfolding cycle
of ClpA. Such experiments may enable us to correlate
movement with ATP hydrolysis and to determine whether
such movement is coordinated among the 6 subunits
of a ClpA ring or is random.
PUBLICATIONS
Hinnerwisch, J., Reid, B.G., Fenton, W.A., Horwich, A.L. Roles of the N-domains
of the ClpA unfoldase in binding substrate proteins and in stable complex formation
with the ClpP protease. J. Biol. Chem. 280:40838, 2005.
Horst, R., Wider, G., Fiaux, J., Bertelsen, E.B., Horwich, A.L., Wüthrich, K. Proton-proton Overhauser NMR spectroscopy with polypeptide chains in large structures. Proc. Natl. Acad. Sci. U. S. A. 103:15445, 2006.
Ranson, N.A., Clare, D.K., Farr, G.W., Houldershaw, D., Horwich, A.L., Saibil,
H.R. Allosteric signaling of ATP hydrolysis in GroEL-GroES complexes. Nat. Struct.
Mol. Biol. 13:147, 2006.
Chemical Regulation of
Gene Expression
D. Alvarez, R. Burnett, C.J. Chou, D. Herman, K. Jenssen,
S. Ku, E. Soragni, J. Puckett,* S. Tsai,* M. Farkas,*
P.B. Dervan,* J.M. Gottesfeld
* California Institute of Technology, Pasadena, California
he ability to control gene expression at will has
been a longstanding goal in molecular biology and
human medicine. We focus on pyrrole-imidazole
polyamides, a class of small molecules that can be programmed by chemical synthesis to recognize a wide
range of DNA sequences. The following is a summary
of our recent efforts to develop polyamides as therapeutic agents for human disease and to identify another
class of small molecules that offer promise in the treatment of neurodegenerative diseases.
T
B L O C K I N G C A N C E R C E L L P R O L I F E R AT I O N W I T H
A P O LYA M I D E - C H L O R A M B U C I L C O N J U G AT E
The nitrogen mustard chlorambucil is a common
DNA alkylator used to treat a variety of lymphatic cancers. Because chlorambucil alkylates DNA at all potentially available guanine residues in the genome, coupling
of chlorambucil to a polyamide will increase the DNA-
THE SCRIPPS RESEARCH INSTITUTE
sequence specificity and perhaps decrease unwanted
side effects while retaining the ability of the compound
to kill cancer cells. We recently found that a specific
polyamide-chlorambucil conjugate called 1R-Chl alters
the morphology and growth characteristics of colon
carcinoma cells in culture and causes the cells to arrest
in the G2/M stage of the cell cycle, without any apparent cytotoxic effects.
Cells treated with 1R-Chl do not grow in soft agar
and do not form tumors in nude mice, indicating that
polyamide-treated cells are no longer tumorigenic. The
compound blocks proliferation of metastatic colon carcinoma cells in immunocompromised mice, and no
apparent toxic effects occur at doses required for a therapeutic effect. Importantly, this gene-targeted small
molecule requires no delivery vehicle because the molecule is cell permeable and localizes in the nucleus of
various cancer cell lines. Using microarray analysis, we
found that the gene target of 1R-Chl is the gene for
histone H4c, a member of the gene family that encodes
a critical component of cellular chromatin and a gene
that is highly expressed in a wide range of cancer cells.
Reduction in histone H4 protein by polyamide treatment
was confirmed in cells treated with 1R-Chl, which caused
chromatin decondensation.
To confirm that downregulation of histone H4c
transcription is the primary event leading to cell-cycle
arrest by 1R-Chl, we turned to short interfering RNAs
directed toward H4c mRNA. Unlike 1R-Chl, which
arrests cells at the G2/M phase of the cell cycle, the
H4c short interfering RNA arrests cells at the G 1 /S
phase. However, G 2/M arrest by 1R-Chl and downregulation of the H4c gene can be confirmed in other
tumorigenic cell lines. We found that 1R-Chl causes
extensive DNA damage in colon cancer cells, leading
to phosphorylation of histone H2A.X at serine 139 and
recruitment of the DNA repair protein Nbs1 to discrete
sites in the genome. These events are hallmarks of the
cellular DNA damage response pathway. Control polyamide-Chl conjugates that lack binding sites in the H4c
gene and have no antiproliferative effects by themselves
can cause G2/M cell-cycle arrest when used in combination with short interfering RNAs to histone mRNAs.
On the basis of these findings, we propose that
1R-Chl exerts its antiproliferative effect through a novel
2-hit mechanism. The highly transcribed H4c gene in
several cancer cell lines is a primary target for DNA
alkylation by 1R-Chl, resulting in downregulation of
H4c transcription and histone H4 protein. Loss of his-
MOLECULAR BIOLOGY
2006
tone protein leads to a transition from condensed to open
chromatin, exposing otherwise hidden binding sites for
1R-Chl. These sites are then alkylated by 1R-Chl, causing widespread DNA damage and a cascade of events
leading to G2/M arrest and loss of tumorigenicity.
Our findings indicate how a single molecule can target cancer cells because of a specific gene expression
profile and block cancer cell proliferation. Ongoing studies are aimed at the development of 1R-Chl as a potential human cancer therapeutic agent.
P O LYA M I D E S A S A C T I VAT O R S O F G E N E E X P R E S S I O N
The neurodegenerative disease Friedreich’s ataxia
is caused by gene silencing through expansion of GAATTC triplet repeats in the first intron of a nuclear gene
that encodes the essential mitochondrial protein frataxin.
Normal frataxin alleles have 6–34 repeats whereas alleles from patients with Friedreich’s ataxia have 66–1700
repeats. Longer repeats cause a more profound frataxin
deficiency and are associated with earlier onset and
increased severity of the disease. Two models have
been proposed to account for gene silencing by expanded
GAA-TTC repeats: unusual DNA structures and repressive heterochromatin.
Molecules that reverse formation of unusual DNA
structures and/or heterochromatin in the gene for frataxin
most likely increase transcription through expanded
GAA-TTC repeats, thereby relieving the deficiency in
frataxin mRNA and protein in cells from patients with
Friedreich’s ataxia. We found that polyamides targeting GAA-TTC repeats partially alleviated transcription
repression of frataxin in a cell line derived from white
blood cells from a patient with Friedreich’s ataxia. These
molecules also increased frataxin protein levels in these
cells, and microarray studies showed that a limited
number of genes in the human genome were affected
by polyamides targeting GAA-TTC repeat DNA.
We hypothesize that polyamides might act as a thermodynamic “sink” and lock GAA-TTC repeats into doublestranded B DNA. Such an event would disfavor duplex
unpairing, which is necessary for formation of the
unusual DNA structures associated with expanded triplet
repeats. Alternatively, polyamides may relieve heterochromatin-mediated repression by opening the chromatin
domain containing frataxin. To explore this last hypothesis, we turned to another class of small molecules.
H I S T O N E D E A C E T Y L A S E I N H I B I T O R S T H AT
R E V E R S E F R ATA X I N S I L E N C I N G
We used antibodies to the various modification states
of the core histones and chromatin immunoprecipita-
THE SCRIPPS RESEARCH INSTITUTE
187
tion methods to examine the chromatin structure of the
gene for frataxin in normal cells and in cell lines derived
from patients with Friedreich’s ataxia. We found that gene
silencing at expanded frataxin alleles was accompanied by hypoacetylation of histones H3 and H4 and
methylation of histone H3 at lysine 9, consistent with a
heterochromatin-mediated repression mechanism.
These findings suggest that histone deacetylase inhibitors, compounds that reverse heterochromatin, might
activate frataxin. We identified a commercial histone
deacetylase inhibitor, BML-210, that partially reverses
silencing in the Friedreich’s ataxia cell line. On the basis
of the structure of this compound, we synthesized and
assayed a series of derivatives of BML-210 and identified histone deacetylase inhibitors that reverse frataxin
silencing in primary lymphocytes from patients with
Friedreich’s ataxia. These molecules act directly on the
histones associated with frataxin, increasing acetylation
at particular lysine residues on histones H3 and H4.
Unlike many triplet-repeat diseases (e.g., the polyglutamine expansion diseases such as Huntington’s disease
and the spinocerebellar ataxias), expanded GAA-TTC
triplets do not alter the coding potential of frataxin.
Thus, gene activation would be of therapeutic benefit.
Studies in animals are under way to explore the bioavailability and efficacy of these histone deacetylase inhibitors.
PUBLICATIONS
Alvarez, D., Chou, C.J., Latella, L., Zeitlin, S.G., Ku, S., Puri, P.L., Dervan, P.B.,
Gottesfeld, J.M. A two-hit mechanism for pre-mitotic arrest of cancer cell proliferation by a polyamide-alkylator conjugate. Cell Cycle 5:1537, 2006.
Burnett, R., Melander, C., Puckett, J.W., Son, L.S., Wells, R.D., Dervan, P.B.,
Gottesfeld, J.M. DNA sequence-specific polyamides alleviate transcription inhibition associated with long GAA-TTC repeats in Friedreich’s ataxia. Proc. Natl. Acad.
Sci. U. S. A. 103:11497, 2006.
Herman, D., Jenssen, K., Burnett, R., Soragni, E., Perlman, S.L., Gottesfeld, J.M.
Histone deacetylase inhibitors reverse gene silencing in Friedreich’s ataxia. Nat.
Chem. Biol. 2:551, 2006.
Lee, B.M., Xu, J., Clarkson, B.K., Martinez-Yamout, M.A., Dyson, H.J., Case,
D.A., Gottesfeld, J.M., Wright, P.E. Induced fit and “lock and key” recognition of
5S RNA by zinc fingers of transcription factor IIIA. J. Mol. Biol. 357:275, 2006.
Trzupek, J.D., Gottesfeld J.M., Boger D.L. Alkylation of duplex DNA in nucleosome
core particles by duocarmycin SA and yatakemycin. Nat. Chem. Biol. 2:79, 2006.
Nucleic Acid Dynamics
D.P. Millar, J. Gill, G. Pljevaljc̆ić, S. Pond, G. Stengel,
N. Tassew, E.J.C. Van der Schans
T
he focus of our research is the assembly and
conformational dynamics of nucleic acid–based
macromolecular machines and assemblies. We
188 MOLECULAR BIOLOGY
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use single-molecule fluorescence methods to investigate a range of systems, including ribozymes, ribonucleoprotein complexes, and DNA polymerases. Our
studies reveal the dynamic structural rearrangements
that occur during the assembly and function of these
macromolecular machines.
RIBOZYMES
RNA conformation plays a central role in the mechanism of ribozyme catalysis. The hairpin ribozyme is a
small nucleolytic ribozyme that serves as a model system for studies of RNA folding and catalysis. The hairpin ribozyme consists of 2 internal loops, 1 of which
contains the scissile phosphodiester bond, displayed
on 2 arms of a 4-way multihelix junction.
To attain catalytic activity, the ribozyme must fold
into a compact conformation in which the 2 loops
become connected by a network of tertiary hydrogen
bonds. We monitor the formation of this docked structure by using fluorescence resonance energy transfer
(FRET) and ribozyme constructs labeled with donor and
acceptor dyes within the loop-bearing arms. By measuring FRET at the level of single ribozyme molecules,
we reveal subpopulations of compact and extended conformers that are not detected in ensemble experiments.
Using this approach, we found that the ribozyme populates an intermediate state in which the 2 loops are
in proximity but tertiary interactions have yet to form.
This quasi-docked state forms rapidly (submillisecond
timescale), but the subsequent formation of the tertiary
contacts between the 2 loops occurs much more slowly.
The hairpin ribozyme is an ideal system for exploring
this fundamental mechanism of the formation of RNA
tertiary structure.
R I B O N U C L E O P R O T E I N A S S E M B LY
The Rev protein from HIV type 1 is a key regulatory
protein that controls the transition from early to late
patterns of viral gene expression. Rev binds to a highly
structured region within the viral mRNA, known as the
Rev response element (RRE), where it forms an oligomeric ribonucleoprotein complex. The formation of this
complex inhibits splicing and facilitates export of the
viral RNA from the nucleus to the cytoplasm. Because
of its critical role in the viral life cycle, the Rev-RRE
complex provides a novel target for the development
of therapeutic drugs.
To dissect the mechanism of assembly of ribonucleoprotein complexes, we use single-molecule fluorescence
imaging methods to monitor the progressive formation
of oligomeric complexes of Rev on individual RRE mole-
THE SCRIPPS RESEARCH INSTITUTE
cules immobilized on a solid surface. We also use single-pair FRET to probe changes in the conformation of
the RRE during the assembly process. We are using the
results of these mechanistic studies to develop novel
fluorescence-based methods for high-throughput screening of libraries of chemical compounds. The new screening tools are being used to identify small molecules that
block binding of Rev to the RRE or prevent the subsequent Rev-Rev oligomerization.
D N A P O LY M E R A S E S
DNA polymerases are remarkable for their ability to
synthesize DNA at rates approaching several hundred
base pairs per second while maintaining an extremely
low frequency of errors. To elucidate the origin of polymerase fidelity, we are using single-molecule fluorescence methods to examine the dynamic interactions
that occur between a DNA polymerase and its DNA
and nucleotide substrates. The FRET method is being
used to observe conformational transitions of the
enzyme-DNA complex that occur during selection and
incorporation of an incoming nucleotide substrate.
Our results reveal that binding of a correct nucleotide
substrate induces a slow conformational change within
the polymerase, causing the “fingers” subdomain to close
over the DNA primer terminus and incoming nucleotide.
Our studies are providing new insights into the dynamic
structural changes responsible for nucleotide recognition
and selection by DNA polymerases. Single-pair FRET
methods are also being used to monitor the movement
of the DNA primer/template between the separate polymerizing and editing sites of the enzyme. This active-site
switching of DNA plays a key role in the proofreading
process used to remove misincorporated nucleotides
from the newly synthesized DNA. The advantage of
single-molecule observations is that they eliminate the
need to synchronize a population of molecules, allowing these dynamic processes to be directly observed.
PUBLICATIONS
Bailey, M.F., Van der Schans, E.J.C., Millar, D.P. Dimerization of the Klenow fragment of Escherichia coli DNA polymerase I is linked to its mode of DNA binding.
Biochemistry, in press.
Tian, F., Debler, E.W., Millar, D. P., Deniz, A.A., Wilson, I.A., Schultz, P.G. Multicolor fluorescent antibodies. Angew. Chemie, in press.
MOLECULAR BIOLOGY
2006
Single-Molecule Biophysics
A.A. Deniz, S.Y. Berezhna, J.P. Clamme, A.C.M. Ferreon,
Y. Gambin, E. Lemke, S. Mukhopadhyay, P. Zhu
e develop and use state-of-the-art singlemolecule fluorescence methods to address
key biological questions. Single-molecule and
small-ensemble methods offer key advantages over traditional measurements, allowing us to directly observe
the behavior of individual subpopulations in mixtures
of molecules and to measure kinetics of structural transitions of stochastic processes under equilibrium conditions. We use these methods to study multiple structural
states or reaction pathways during the folding and
assembly of biomolecules.
A major goal is to apply single-molecule methods to
studies of protein folding and aggregation. Using relatively simple model systems, we are addressing several
fundamental questions about folding mechanisms. Partially folded or misfolded protein structures are also
thought to play important cellular roles, and these states
also can be studied by using single-molecule methods.
In this context, we are examining the interplay between
folding and aggregation of Sup35, a yeast prion protein,
in collaboration with S.L. Lindquist, Whitehead Institute
for Biomedical Research, Cambridge, Massachusetts,
and of α-synuclein, a protein implicated in the pathogenesis of Parkinson’s disease and other neurodegenerative diseases.
In addition, we have developed a single-molecule
fluorescence quenching method that will be useful for
measuring distances shorter than 30 Å in proteins and
RNA, a scale at which the resolution of single-pair fluorescence resonance energy transfer (FRET) is low. This
method is being used to monitor structural properties of
Sup35 as a function of the aggregation process.
To better study the folding, assembly, and activity
of larger and multicomponent biological complexes, we
are developing new multicolor single-molecule FRET
methods. As part of this continuing goal, we have been
improving our recently developed diffusion 3-color single-molecule FRET method for simultaneously measuring more than a single intramolecular or intermolecular
distance. In collaboration with J.R. Williamson, Department of Molecular Biology, we are using these novel
methods to study the detailed mechanisms of assembly
of fragments of the bacterial ribosome. Most recently,
we began adding microfluidics capabilities to our exper-
W
THE SCRIPPS RESEARCH INSTITUTE
189
imental repertoire, to further facilitate studies of molecular structure, folding, and function.
Finally, using high-sensitivity fluorescence imaging,
we are beginning to study and compare the pathways
of nuclear and cytoplasmic RNA interference. In studies
done in collaboration with P.G. Schultz, Department of
Chemistry, our observations of the localization of small
interfering RNA in live cells provide evidence for a yetto-be-determined mechanism that directs the RNA to
cellular compartments containing the target RNA.
PUBLICATIONS
Berezhna, S.Y., Supekova, L., Supek, F., Schultz, P.G., Deniz, A.A. siRNA in
human cells selectively localizes to target RNA sites. Proc. Natl. Acad. Sci. U. S. A.
103:7682, 2006.
Zhu, P., Clamme, J.-P., Deniz, A.A. Fluorescence quenching by TEMPO: a sub-30
Å single-molecule ruler. Biophys. J. 89:L37, 2005.
Computer Modeling of Proteins
and Nucleic Acids
D.A. Case, M. Crowley, Q. Cui, F. Dupradeau,* S. Moon,
D. Nguyen, V. Pelmentschikov, D. Shivakumar, R.C. Walker,
W. Zhang, J. Ziegler**
* Université Jules Verne, Amiens, France
** Universität Bayreuth, Bayreuth, Germany
omputer simulations offer an exciting approach to
the study of many aspects of biochemical interactions. We focus primarily on molecular dynamics simulations (in which Newton’s equations of motions
are solved numerically) to model the solution behavior
of biomacromolecules. Recent applications include
detailed analyses of electrostatic interactions in short
peptides (folded and unfolded), proteins, and oligonucleotides in solution.
In addition, molecular dynamics methods are useful in refining solution structures of proteins by using
constraints derived from nuclear magnetic resonance
(NMR) spectroscopy, and we continue to explore new
methods in this area. Our developments are incorporated
into the Amber molecular modeling package, designed
for large-scale biomolecular simulations, and into other
software, including Nucleic Acid Builder, for developing
3-dimensional models of unusual nucleic acid structures;
SHIFTS, for analyzing chemical shifts in proteins and
nucleic acids; RNAmotif, for finding structural motifs
in genomic sequence databases; and DOCK, for placing inhibitors into enzyme active sites.
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NMR AND THE STRUCTURE AND DYNAMICS OF
PROTEINS AND NUCLEIC ACIDS
Our overall goal is to extract the maximum amount
of information about biomolecular structure and dynamics from NMR experiments. To this end, we are studying
the use of direct refinement methods for determining
biomolecular structures in solution, going beyond distance constraints to generate closer connections between
calculated and observed spectra. We are also using
quantum chemistry to study chemical shifts and spinspin coupling constants. Other types of data, such as
chemical shift anisotropies, direct dipolar couplings in
partially oriented samples, and analysis of cross-correlated relaxation, are also being used to guide structure
refinement. In recent structural studies, we focused on
the binding of zinc finger proteins with RNA and on
structural influences on amide proton chemical shifts.
NUCLEIC ACID MODELING
Another project centers on the development of novel
computer methods to construct models of “unusual”
nucleic acids that go beyond traditional helical motifs.
We are using these methods to study circular DNA, small
RNA fragments, and 3- and 4-stranded DNA complexes,
including models for recombination sites. We continue
to develop efficient computer implementations of continuum solvent methods to allow simplified simulations
that do not require a detailed description of the solvent (water) molecules; this approach also provides a
useful way to study salt effects.
Recent efforts have made second derivatives of these
energies available, so that normal mode analyses of
nucleic acids with dozens to hundreds of nucleotides
can be analyzed and the predictions compared with
those of simpler, elastic continuum models. These
efforts provide a new avenue for developing and testing low-resolution models that can be used for large
molecular assemblies.
D Y N A M I C S A N D E N E R G E T I C S O F N AT I V E A N D
N O N N AT I V E S TAT E S O F P R O T E I N S
Analysis methods similar to those described for
nucleic acids are also being used to estimate thermodynamic properties of “molten globules” and unfolded
states of proteins. These studies are an extension of our
earlier work on the folding of peptide fragments of proteins. A key feature is the development of computational
methods that can be used to model pH and salt dependence of complex conformational transitions such as
unfolding events. A second aspect of this research is a
detailed interpretation of NMR results for protein non-
THE SCRIPPS RESEARCH INSTITUTE
native states through molecular dynamics simulations
and the construction of models for molecular motion
and disorder.
All of these modeling activities are based on molecular mechanics force fields, which provide estimates of
energies as a function of conformation. We continue to
work on improvements in force fields; recently, we
focused on adding aspects of electronic polarizability,
going beyond the usual fixed-charge models, and on
methods for handling arbitrary organic molecules that
might be considered potential inhibitors in drug discovery efforts. Overall, the new models should provide a
better picture of the noncovalent interactions between
peptide groups and the groups’ surroundings, leading
ultimately to more faithful simulations.
V I B R AT I O N A L A N A LY S I S O F I R O N - S U L F U R C L U S T E R S
IN PROTEINS
A wide variety of proteins contain iron-sulfur clusters
at their active sites; these proteins participate in electrontransport chains and in important enzymatic reactions
such as the reduction of atmospheric nitrogen to ammonia by nitrogenase. Advances in synchrotron radiation
sources now make it possible to probe the vibrational
behavior of these clusters by using nuclear resonance
vibrational spectroscopy (NRVS). This technique senses
the coupling of a nuclear (Mossbauer) excitation to
molecular vibrations. The result is a set of vibrational frequencies and intensities that indicate what sorts of deformations can take place. When the molecular structure is
known, this information can contribute to the understanding of oxidation-reduction behavior and electron
transfer kinetics. In situations in which the cluster structure is not known, NRVS data might useful as a “fingerprint” to help identify the structure.
We have been using quantum chemistry calculations to help understand NRVS spectra. Figure 1 shows
a early example, comparing calculated and experimental spectra for a simple iron-sulfur “cubane” structure,
a cluster type found in hundreds of known proteins. The
calculations (shown as a dashed line) are in excellent
agreement with experimental data (solid line), both in
terms of frequencies and in terms of intensities. We are
extending these calculations to models for the active
site of nitrogenase, where the structure of the complex
is still uncertain. If calculations like these can be used
to closely track the experimental results, NRVS will be
an important new tool for characterizing the active sites
of metalloenzymes.
MOLECULAR BIOLOGY
2006
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191
Rizzo, R.C., Aynechi, T., Case, D.A., Kuntz, I.D. Estimation of absolute free energies of hydration using continuum methods: accuracy of partial charge models and
optimization of nonpolar contributions. J. Chem. Theory Comput. 2:128, 2006.
Steinbrecher, T., Case, D.A., Labahn, A. A multistep approach to structure-based
drug design: studying ligand binding at the human neutrophil elastase. J. Med.
Chem. 49:1837, 2006.
Wang, J., Wang, W., Kollman, P.A., Case, D.A. Automatic atom type and bond
type perception in molecular mechanical calculations. J. Mol. Graphics Model.
25:247, 2006.
Xiao, Y., Fisher, K., Smith, M.C., Newton, W.E., Case, D.A., George, S.J., Wang, H.,
Sturhahn, W., Alp, E.E., Zhao, J., Yoda, Y., Cramer, S.P. How nitrogenase shakes: initial information about P-cluster and FeMo-cofactor normal modes from nuclear resonance vibrational spectroscopy (NRVS). J. Am. Chem. Soc. 128:7608, 2006.
Xiao, Y., Koutmos, M., Case, D.A., Coucouvanis, D., Wang H., Cramer, S.P.
Dynamics of an [Fe4S4(SPh)4]2– cluster via IR, Raman, and nuclear resonance
vibrational spectroscopy (NRVS): analysis using 36S substitution, DFT calculations,
and empirical force fields. Dalton Trans. 2192, 2006, Issue 18.
Quantum Chemistry of RedoxActive Metalloenzymes
L. Noodleman, D.A. Case, W.-G. Han, V. Pelmenschikov,
J.A. Fee, L. Hunsicker-Wang,* T. Lovell,** T. Liu***
* Trinity University, San Antonio, Texas
** AstraZeneca R&D, Mölndal, Sweden
*** University of Maryland, College Park, Maryland
F i g . 1 . Calculated and experimental NRVS spectra for an ironsulfur cluster.
PUBLICATIONS
Baker, N.A., Bashford, D., Case, D.A. Implicit solvent electrostatics in biomolecular simulation. In: New Algorithms for Macromolecular Simulation. Leimkuhler, B.,
et al. (Eds.). Springer, New York, 2006, p. 263. Lecture Notes in Computational
Science and Engineering, Vol. 49.
Brown, R.A., Case, D.A. Second derivatives in generalized Born theory. J. Comput.
Chem. 27:1662, 2006.
Case, D.A., Cheatham, T.E. III, Darden, T., Gohlke, H., Luo, R., Merz, K.M., Jr.,
Onufriev, A., Simmerling, C., Wang, B., Woods, R. The Amber biomolecular simulation programs. J. Comput. Chem. 26:1668, 2005.
Dixit, S.B., Beveridge, D.L., Case, D.A., Cheatham, T.E. III, Giudice, E., Lankas,
R., Lavery, R., Maddocks, J.H., Osman, R., Sklenar, H., Thayer, K.M., Varnai, P.
Molecular dynamics simulations of the 136 unique tetranucleotide sequences of
DNA oligonucleotides, II: sequence context effects on the dynamical structures of
the 10 unique dinucleotide steps. Biophys. J. 89:3721, 2005.
Dupradeau, F.-Y., Case, D.A., Yu, C., Jimenez, R., Romesberg, F.E. Differential
solvation and tautomer stability of a model base pair within the minor and major
grooves of DNA. J. Am. Chem. Soc. 127:15612, 2005.
Lee, B.M., Xu, J., Clarkson, B.K., Martinez-Yamout, M.A., Dyson, H.J., Case,
D.A., Gottesfeld, J.M., Wright, P.E. Induced fit and “lock and key” recognition of
5S RNA by zinc fingers of transcription factor IIIA. J. Mol. Biol. 357:275, 2006.
Mathews, D.H., Case, D.A. Nudged elastic band calculation of minimal energy
pathways for the conformational change of a GG noncanonical pair. J. Mol. Biol.
357:1683, 2006.
Moon, S., Case, D.A. A comparison of quantum chemical models for calculating
NMR shielding parameters in peptides: mixed basis set and ONIOM methods combined with a complete basis set extrapolation. J. Comput. Chem. 27:825, 2006.
e use a combination of modern quantum
chemistry (density functional theory, DFT)
and classical electrostatics to describe the
energetics, reaction pathways, and spectroscopic properties of metalloenzymes.
Critical biosynthetic and regulatory processes may
involve catalytic transformations of fairly small molecules or groups by transition-metal centers. The ironmolybdenum cofactor center of nitrogenase catalyzes
the multielectron reduction of molecular nitrogen to 2
molecules of ammonia plus molecular hydrogen. We
are continuing our work on the catalytic cycle of this
enzyme, following up on our earlier work on the structure and oxidation state of the cofactor complex in the
“resting enzyme” before multielectron reduction and
nitrogen binding.
On the basis of DFT calculated vs experimental
physical properties, including redox potentials, cluster
geometries, and Mössbauer isomer shifts, the core
cluster has a MoFe 7S 9X prismane active site, where
the central X most likely is nitride and the “resting
cluster oxidation state” is Mo(IV)Fe(II) 4Fe(III) 3. If the
central ligand is nitride, as we have proposed, this
ligand is not a substrate or a reaction product of the
catalytic cycle. Instead, nitride is inserted into a cen-
W
192 MOLECULAR BIOLOGY
2006
tral vacancy site of a more open iron-molybdenum cofactor precursor, probably in a noncatalytic deamination
process that occurs before insertion of the cluster into
the iron-molybdenum protein.
Class I ribonucleotide reductases are aerobic enzymes
that catalyze the reduction of ribonucleotides to deoxyribonucleotides, providing the required building blocks
for DNA replication and repair. These ribonucleotideto-deoxyribonucleotide reactions occur via a long-range
radical (or proton-coupled electron transfer) propagation mechanism initiated by a fairly stable tyrosine radical, “the pilot light.” When this pilot light goes out, the
tyrosine radical is regenerated by a high-oxidation-state
Fe(III)-Fe(IV)-oxo enzyme called intermediate X. We are
using DFT and electrostatics calculations in combination
with analysis of Mössbauer, electron nuclear double resonance, and magnetic circular dichroism spectroscopies
to search for a proper structural and electronic model
for intermediate X.
We have also examined the mechanism of formation
of intermediate X, starting from an earlier Fe(III)2-µperoxo intermediate (Fig. 1). Spectroscopic and quantum
chemical DFT evidence indicates that the formation of
intermediate X is proton catalyzed. On the basis of calculations of spectroscopic parameters and energies, we
propose that intermediate X contains a dioxo bridging
the Fe(III)-Fe(IV) in an asymmetric diamond structure.
The Fe(IV) site is farther from and the Fe(III) site is closer
to the redox-active tyrosine 122. Figure 2 shows the
molecular orbitals corresponding to the lowest energy
Fe(IV) d→d optical excitation. The 3 Fe(IV) d→d bands
that we predict on the basis of DFT vertical self-consistent reaction field methods are in excellent agreement
with the bands observed by using magnetic circular
dichroism spectroscopy. Further exploration of the
tyrosine radical activation and subsequent catalytic
cycle are planned.
THE SCRIPPS RESEARCH INSTITUTE
Noodleman, L., Han, W.-G. Structure, redox, pKa, spin: a golden tetrad for understanding metalloenzyme energetics and reaction pathways. J. Biol. Inorg. Chem.
11:674, 2006.
PUBLICATIONS
Han, W.-G., Liu, T., Lovell, T., Noodleman, L. Active site structure of class I
ribonucleotide reductase intermediate X: a density functional theory analysis of
structure, energetics, and spectroscopy. J. Am. Chem. Soc. 127:15778, 2005.
Han, W.-G., Liu, T., Lovell, T., Noodleman, L. Density functional theory study of
Fe(IV) d-d optical transitions in active-site models of class I ribonucleotide reductase intermediate X with vertical self-consistent reaction field methods. Inorg.
Chem. 45:8533, 2006.
Han, W.-G., Liu, T., Lovell, T., Noodleman, L. DFT calculations of 57Fe Mössbauer
isomer shifts and quadrupole splittings for iron complexes in polar dielectric media:
applications to methane monooxygenase and ribonucleotide reductase. J. Comput.
Chem. 27:1292, 2006.
Han, W.-G., Liu, T., Lovell, T., Noodleman, L. Seven clues to the origin and structure of class-I ribonucleotide reductase intermediate X. J. Inorg. Biochem.
100:771, 2006.
F i g . 1 . A feasible path showing how ribonucleotide reductase
intermediate X is formed by the reaction of oxygen with the
reduced ribonucleotide reductase–R2 di-iron center. Reproduced
with permission from J. Am. Chem. Soc. 127:15778, 2005.
Copyright 2005 American Chemical Society.
MOLECULAR BIOLOGY
2006
F i g . 2 . Our proposed model for the active site of class I ribonucleotide reductase intermediate X. Molecular orbital plots show the
lowest energy Fe(IV) d→d optical excitation.
Theoretical and Computational
Molecular Biophysics
C.L. Brooks III, C. An, R. Armen, I. Borelli, D. Bostick,
D. Braun, L. Bu, J. Chen, M.F. Crowley, O. Guvench, R. Hills,
W. Im,* J. Khandogin, I. Khavrutskii, J. Lee, J. Magee,**
R. Manige, M. Michino, A. Mitsutake,*** H.D. Nguyen,
S. Patel,**** D.J. Price, V. Reddy, H.A. Scheraga,*****
C. Shepard, F. Tama,† I.F. Thorpe, M.C. Tripp, R. Wheeler,††
C. Wildman, K. Yoshimoto
* Kansas University, Lawrence, Kansas
** University of Manchester, Manchester, England
*** Kelo University, Tokyo, Japan
**** University of Delaware, Newark, Delaware
***** Cornell University, Ithaca, New York
†
University of Arizona, Tucson, Arizona
††
University of Oklahoma, Norman, Oklahoma
nderstanding the forces that determine the
structure of proteins, peptides, nucleic acids,
and complexes containing these molecules and
the processes by which these structures are adopted
is essential to complete our knowledge of the molecular
nature of structure and function. To address such questions, we use statistical mechanics, molecular simulation, statistical modeling, and quantum chemistry.
Creating atomic-level models to simulate biophysical processes (e.g., protein folding or binding of a ligand
to a biological receptor) requires (1) the development
of new potential energy functions that accurately represent the atomic interactions and (2) the use of quan-
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193
tum chemistry to aid in determining the parameters
for the models. Calculation of thermodynamic properties
requires the development and implementation of new
theoretical and computational approaches that connect
averages over atomistic descriptions to experimentally
measurable thermodynamic and kinetic properties.
Interpreting experimental results at more microscopic
levels is fueled by the development and investigation of
theoretical models for the processes of interest. Massive
computational resources are needed to realize these
objectives, and this motivates our efforts aimed at the
efficient use of new computer architectures, including
large supercomputers, Linux Beowulf clusters, computational grids, and Internet-based volunteer supercomputers. Each of the objectives and techniques mentioned
represents an ongoing development area within our
research program in computational biophysics. The following are highlights of a few specific projects.
FOLDING, STRUCTURE, AND FUNCTION OF
MEMBRANE-BOUND PROTEINS
Folding, insertion, assembly, and stability of membrane proteins are directly governed by the unique
hydrophilic and hydrophobic environment provided by
biological membranes. Modeling this heterogeneous
environment is both an obstacle and an essential requisite to experimental and computational studies of the
structure and function of membrane proteins. Because
of the biological importance and marked presence of
membrane proteins in known genomes (i.e., about 30%
of all proteins), one aim of modern molecular biophysics
should be the development of methods that can be used
in experimental studies to understand the structure and
function of these systems. We recently developed theoretical methods that enable the exploration of protein insertion and folding in membranes. These methods combine
the sampling methods of replica-exchange molecular
dynamics with novel generalized Born implicit solvent/
implicit membrane continuum electrostatic theories.
A key question these methods allow us to address
is the association of integral membrane proteins to form
oligomeric structures. Many important functional complexes of membrane proteins exist as oligomers, such as
the signal-transducing G protein–coupled receptors and
membrane-bound ion channels and transporters. Our
recent approach provides a way to predict the structures
of these key oligomeric states. Figure 1 shows the predicted oligomeric structures of glycophorin A (functionally a dimer), the tetrameric M2 transmembrane peptide
proton channel, and the phospholamban pentameric
194 MOLECULAR BIOLOGY
2006
THE SCRIPPS RESEARCH INSTITUTE
One recent advance came in exploring the structure
of the ribosome in complex with the SecY protein-conducting channel (PCC). The translocation of secreted
and membrane proteins across or into cell membranes
occurs through PCCs. Using an electron cryomicroscopy
reconstruction of the Escherichia coli PCC, which consisted of SecY complexed with the ribosome and a
nascent chain containing a signal anchor, we observed
the components of protein synthesis and translocation,
including mRNA, 3 tRNAs, the nascent chain, and
features of both a translocating PCC and a second,
nontranslocating PCC bound to mRNA hairpins (Fig. 2).
F i g . 1 . The predicted structure of dimeric glycophorin A, a dominant structural component of red blood cells, indicates the “classic” GVXXGV helical interface. For the M2 proton channel involved
in replication of the influenza virus, the structure of the functional
tetrameric proton-conducting channel is shown. In phospholamban,
which is localized in the membrane of the cardiac sarcoplasmic
reticulum and involved in phosphorylation-controlled regulation of
the cardiac calcium pump, the predicted pentameric structure selectively conducts calcium.
oligomer. Our calculations provide detailed predictions of
the protein-protein interfaces for these systems and may
be useful in elucidating the primary oligomerization
states. The predicted models shown in the figure are in
excellent agreement with existing structural models (from
experiments and other model building).
LARGE-SCALE FUNCTIONAL DYNAMICS IN
MOLECULAR ASSEMBLIES
Many naturally occurring machines, such as ribosomes, myosin, and viruses, require large-scale dynamical motions as a component of their normal functioning.
These motions involve the “mechanical” reorganization
of major parts of the structure of the machine in response
to binding of effectors or the addition of energy in the
form of thermal fluctuations or provided by chemical
catalysis. Exploring and understanding the character
and nature of such large-scale reorganization of biological machines are ongoing goals in our laboratory. Using
theoretical approaches derived from the treatment of
mechanoelastic materials, we developed new structure
refinement methods to model large-scale macromolecular
assemblies. The methods are based on atomic-level
structures of the component macromolecules (e.g.,
RNAs, DNAs, and proteins) or on single-particle or
tomographic images from electron microscopy. Using
these new methods, which we call normal mode flexible fitting, we have collaborated with several colleagues
in elucidating new structural models for functionally
important molecular assemblies.
F i g . 2 . Electron cryomicroscopy image of the ribosome with 2
bound PCCs obtained during the modeling of structural components
of the SecY dimer into the electron density for the nontranslocating
and translocating PCCs. The figure on the lower right illustrates the
structure of the SecY dimer fit into the experimental electron density map by using normal mode flexible fitting. NNMF indicates normal mode flexible fitting.
Normal mode flexible fitting of the SecYEb structure
into the PCC electron microscopy densities favors a
front-to-front arrangement of 2 SecYEG complexes in
the PCC and supports channel formation by the opening
of 2 linked SecY halves during polypeptide translocation. From the models elucidated by the combination
of electron cryomicroscopy and modeling based on
normal mode flexible fitting, we were able to develop
a model for cotranslational protein translocation.
PUBLICATIONS
Chen, J., Im, W., Brooks, C.L. III. Application of torsion angle molecular dynamics
for efficient sampling of protein conformations. J. Comput. Chem. 26:1565, 2005.
Chen, J., Im, W., Brooks, C.L. III. Balancing solvation and intramolecular interactions: toward a consistent generalized Born force field. J. Am. Chem. Soc.
128:3728, 2006.
Im, W., Chen, J., Brooks, C.L. III. Peptide and protein folding and conformational
equilibria: theoretical treatment of electrostatics and hydrogen bonding with
implicit solvent models. Adv. Protein Chem. 72:173, 1005.
Khandogin, J., Brooks, C.L. III. Constant pH molecular dynamics with proton tautomerism. Biophys. J. 89:141, 2005.
Khavrutskii, I.V., Byrd, R.H., Brooks, C.L. III. A line integral reaction path approximation for large systems via nonlinear constrained optimization: application to alanine dipeptide and the β-hairpin of protein G. J. Chem. Phys. 124:194903, 2006.
MOLECULAR BIOLOGY
2006
Konecny, R., Trylska, J., Tama, F., Zhang, D., Baker, N.A., Brooks, C.L. III,
McCammon, J.A. Electrostatic properties of cowpea chlorotic mottle virus and
cucumber mosaic virus capsids. Biopolymers 82:106, 2005.
Mitra, K., Schaffitzel, C., Shaikh, T., Tama, F., Jenni, S., Brooks, C.L. III, Ban,
N., Frank, J. Structure of the E. coli protein-conducting channel bound to a translating ribosome. Nature 438:318, 2005.
Natarajan, P., Lander, G.C., Shepherd, C.M., Reddy, V.S., Brooks, C.L. III, Johnson, J.E. Exploring icosahedral virus structures with VIPER. Nat. Rev. Microbiol.
3:809, 2005.
Patel, S., Brooks, C.L. III. Fluctuating charge force fields: recent developments
and applications from small molecules to macromolecular biological systems. Mol.
Simul. 32:231, 2006.
Patel, S., Brooks, C.L. III. Revisiting the hexane-water interface via molecular
dynamics simulations using nonadditive alkane-water potentials. J. Chem. Phys.
124:204706, 2006.
Patel, S., Brooks, C.L. III. Structure, thermodynamics, and liquid-vapor equilibrium of ethanol from molecular-dynamics simulations using nonadditive interactions. J. Chem. Phys. 123:164502, 2005.
Price, D.J., Brooks, C.L. III. Detailed considerations for a balanced and broadly
applicable force field: a study of substituted benzenes modeled with OPLS-AA. J.
Comput. Chem. 26:1529, 2005.
Tama, F., Brooks, C.L. III. Symmetry, form, and shape: guiding principles for
robustness in macromolecular machines. Annu. Rev. Biophys. Biomol. Struct.
35:115, 2006.
Tama, F., Brooks, C.L. III. Unveiling molecular mechanisms of biological functions
in large macromolecular assemblies using elastic network normal mode analysis.
In: Normal Mode Analysis: Theory and Applications to Biological and Chemical
Systems. Cui, Q., Bahar, I. (Eds.). Chapman & Hall/CRC Press, Boca Raton, FL,
2006, p. 111. Mathematical and Computational Biology Series.
Taufer, M., An, C., Kerstens, A., Brooks, C.L. III. Predictor@Home: a “protein
structure prediction supercomputer” based on global computing. IEEE Trans. Parallel Distributed Syst. 7:786, 2006.
Thorpe, I.F., Brooks, C.L. III. Conformational substates modulate hydride transfer
in dihydrofolate reductase. J. Am. Chem. Soc. 127:12997, 2005.
Trylska, J., McCammon, J.A., Brooks, C.L. III. Exploring assembly energetics of
the 30S ribosomal subunit using an implicit solvent approach. J. Am. Chem. Soc.
127:11125, 2005.
Yadav, M.K., Leman, L.J., Price, D.J., Brooks, C.L. III, Stout, C.D., Ghadiri, M.R.
Coiled coils at the edge of configurational heterogeneity: structural analyses of parallel and antiparallel homotetrameric coiled coils reveal configurational sensitivity to
a single solvent-exposed amino acid substitution. Biochemistry 45:4463, 2006.
Computation and Visualization
in Structural Biology
A.J. Olson, D.S. Goodsell, M.F. Sanner, S. Dallakyan,
A. Gillet, R. Harris, Y. Hu, R. Huey, J. Huntoon, S. Karnati,
W. Lindstrom, G.M. Morris, A. Omelchenko, M. Pique,
B. Norledge, R. Rosenstein, M. Utsintong, G. Vareille,
Q. Zhang, Y. Zhao
n the Molecular Graphics Laboratory, we develop
novel computational methods to analyze, understand,
and communicate the structure and interactions
of complex biomolecular systems. This past year, we
I
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195
showed the effectiveness of 3-dimensional molecular
models as a tangible human-computer interface in
educational and research settings. Within our component-based visualization environment, we continue to
develop methods for predicting biomolecular interactions,
analyzing biomolecular structure and function, and presenting the biomolecular world in education and outreach.
We have applied these methods to several important
systems in human health and welfare. In a novel distributed computing network, we continue the search for HIV
protease inhibitors to fight the growing problem of drug
resistance in HIV disease. We used AutoDock, a suite
of programs for predicting bound conformations and binding energies for biomolecular complexes, in the virtual
screening of large databases of compounds and ultimately
identified new compounds for use in the treatment of
cancer. We used methods for predicting protein interaction to probe the mechanism of blood coagulation.
TA N G I B L E I N T E R FA C E S I N S T R U C T U R A L B I O L O G Y
We have continued to develop autofabricated physical models (“solid printing”) of biological molecules and
the components and assemblies of the molecules; our
goal is to use the models in both research and education. We integrated computer graphics and computation
with these physical models by using augmented reality
to create custom interfaces to facilitate exploration and
computation of molecular interactions. We have begun
to use a self-assisted protein-folding model to teach elements of protein structure and assembly to our graduate
students. We are continuing to develop the software that
will enable the control of interactive computations
through manipulation of the tangible models.
In collaboration with T. Herman, Milwaukee School
of Engineering, Milwaukee, Wisconsin, we created a
model of the active site of acetylcholinesterase that can
be opened to show the buried active site and bound substrates (Fig. 1). This model was used, along with an
interactive Internet guide to the structure, as part of a
Waksman Challenge at Rutgers, the State University of
New Jersey. Groups of teachers and students were asked
to use the models and associated materials to explore
problems with insecticide resistance in compounds that
act on mosquito acetylcholinesterase.
Recently, we worked with Biomedical Graphics at
Scripps Research to establish a solid-model printing
service for researchers at Scripps and elsewhere. This
service is now in operation and has made a number of
molecular models for scientists working with molecular
structures. The users’ responses have been positive, and
196 MOLECULAR BIOLOGY
2006
F i g . 1 . A tangible model of the active site of acetylcholinesterase. The model separates into 4 pieces, allowing students to fit different substrates into the buried active site.
the research community is beginning to see how a solid
3-dimensional model can provide tangible, multimodal
feedback that mouse, keyboard, and image behind a
glass screen cannot provide.
A D VA N C E S I N C O M P U TAT I O N A L D O C K I N G
We have just completed developing and testing a
semiempirical free energy force field for use in AutoDock
and similar grid-based docking methods. The force field
is based on a comprehensive thermodynamic model that
allows incorporation of intramolecular energies into the
predicted free energy of binding. The model also incorporates a charge-based method for evaluating desolvation
designed to use a typical set of atom types. The method
was calibrated by using a set of 188 diverse proteinligand complexes of known structure and binding energy
and was tested by using a set of 100 complexes of
ligands with retroviral proteases. Compared with the
previous AutoDock force field, the new force field provides an improvement in redocking simulations.
AutoDock 4 has been modified to support more atom
types and to use an improved atom-typing mechanism.
Importantly, AutoDock 4 also now simulates the molecular system being docked in the unbound state, by generating and evaluating the extended conformation of the
ligand and moving side chains in the receptor before
the docking occurs. The unbound state is now considered in the calculation of the change in free energy upon
binding. AutoDock’s companion graphical user interface,
AutoDockTools, has been modified to support preparation of input files for AutoDock 4, in particular to allow
the definition of flexible side chains in macromolecules.
AutoDockTools has also been made easier to use by
simplifying the menus.
On the basis of the AutoDock force field, we developed a method for locating and characterizing the opti-
THE SCRIPPS RESEARCH INSTITUTE
mal binding site for ligands on the surface of a protein
of known structure. The method identifies the contiguous constant-volume region with the most favorable
binding affinity. The optimal binding sites identify regions
of primary binding affinity and regions of suboptimal
binding strength, which can be used to predict the function of proteins if the function is unknown or to identify target locations for the design of new inhibitors. We
showed the usefulness of the method in the design of
inhibitors for HIV type 1 protease, and we are applying
the method to a large database of protein structures.
C O M P O N E N T - B A S E D V I S U A L I Z AT I O N E N V I R O N M E N T S
To facilitate the integration and interoperation of
computational models and techniques from a wide variety of scientific disciplines, we continue to expand our
component-based software environment. The environment is centered on Python, a high-level, object-oriented,
interpretive programming language. This approach allows
the compartmentalization and reuse of software components. Python provides a powerful computation “glue”
for assembling computational components and, at the
same time, a flexible language for the interactive scripting of new applications.
We released version 1.4.1 of our software components in March 2006. This release contains substantial
enhancements, including a completely rewritten interface to the adaptive Poisson-Bolzman solver APBS,
making it easy to produce high-quality pictures of electrostatic potentials on molecular surfaces. A new control
panel provides a high-level interface for rapidly displaying molecular models in a variety of representations.
This new release is also distributed with installer programs for computers running the Windows and Macintosh OS X operating systems. We also added a parser
for macromolecular Crystallographic Information File
that allow users to read and write files in the macromolecular Crystallographic Information File format. This
addition helps overcome limitations in the Protein Data
Bank format such as maximum number of atoms or
chain IDs.
MODELING PROTEIN FLEXIBILITY IN DOCKING
We have developed a hierarchical and multiresolution
representation of the flexibility of biological macromolecules that can be used in computational simulations.
This treelike structure enables the computationally
tractable encoding of a small subset of a protein’s conformational subspace. After implementing the core infrastructure of the Flexibility Tree and developing intuitive
graphical interfaces for building such trees, we have
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2006
started exploring the use of this data structure in the context of automated docking. We reproduced a cross-docking experiment carried out earlier with AutoDock in which
20 inhibitors of HIV protease I where docked systematically into the 20 conformations of the receptor. We
showed that by adding receptor flexibility, we could
increase the rate of successful cross docking from
72% to 98%.
PROTEIN DOCKING
In collaboration with C. Bajaj, University of Texas,
Austin, we are investigating a novel fast Fourier transform–based method for predicting the association of protein in complexes. In parallel with this docking method,
we evaluated the effect on blurring molecular surfaces
on the shape complementarity at the interface between
proteins in a complex. We characterized the level of
distortion introduced by blurring atomic spheres by
using gaussian distributions and determined an optimal
blurring level for docking purposes. In addition, we
added software components for the calculating the curvature of meshes that are used in the docking procedure.
F I G H T I N G D R U G R E S I S TA N C E I N H I V D I S E A S E
As part of a program project, we continue our work
on inhibitors to fight drug resistance in the treatment of
AIDS. In collaboration with K.B. Sharpless and C.-H.
Wong, Department of Chemistry, we have designed and
optimized a series of inhibitors built around a triazole
formed in a click chemistry reaction. We are also exploring larger issues of resistance via docking experiments
with large chemical databases and large sets of mutant
protease structures. These massive docking experiments
are made possible by the resources available in the
FightAIDS@Home distributed computing system. FightAIDS@Home enlists the worldwide community in a
large computational effort to design effective therapeutic
agents to fight AIDS. Personal computers are used by
the program when the computers are not in use by their
owners, providing an enormous, and largely untapped,
computational resource. The current goal is to identify
inhibitors that are effective against the wild-type virus
and against common mutant forms of the virus.
In the past year, we moved FightAIDS@Home to the
IBM World Community Grid. This transition involved
working closely with the team at IBM to board our
automated docking software, AutoDock, to be able to
run on the Windows United Devices client and the Linux
and Macintosh OS X Berkeley Open Infrastructure for
Network Computing clients. This move has increased
the number of available processors to about 300,000
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and has enabled us to compute a complete scan of the
National Cancer Institute diversity set (2000 compounds)
against a panel of 200 mutant HIV proteases in a matter of 4 months. This computation required more than
2 quadrillion energy evaluations of ligand vs protein.
INTERACTIONS OF TISSUE FACTOR
We used our ligand-protein and protein-protein model
to study the interactions of tissue factor (TF) in the
initiation of blood coagulation and the related regulatory roles of the factor. TF plays a potential role in
metastasis, growth, and angiogenesis of tumor cells via
2 distinct mechanisms: interaction of the complex consisting of TF and factor VIIa with protease-activated
receptor 2 (PAR2) and interaction of the complex consisting of TF, factor VIIa, and factor Xa with PAR1 or
PAR2. However, no PAR structures are available for
studying these mechanisms. Because PAR2 is involved
in both pathways, we performed protein homology modeling studies of this receptor.
We found 8 unique PAR2 sequences. For each unique
sequence, we searched its homology sequences in the
Protein Data Bank and chose as the homology template
the sequence that has the highest-resolution x-ray crystal structure. Sequence alignment was then performed
between the PAR2 sequence and the template sequence.
The alignment was then input to MODELLER for building 10 homology structures, from which we chose the
structure with the best quality as the homology model.
The homology models have enabled us to use AutoDock
to perform docking studies of PAR2-activating peptides
and small molecules on PAR2. The discovered binding
modes are being confirmed by our collaborator, W. Ruf,
Department of Immunology.
STRUCTURE-BASED DRUG DESIGN IN GAUCHER
DISEASE
Gaucher disease is the most common lipid-storage
disorder caused by activity-compromising mutations in
glucosylceramidase and is the most common genetic
disease affecting Ashkenazi Jews. In addition to causing
great pain, anemia, and massive enlargement of the
liver and spleen, Gaucher disease can lead to neurologic
impairment or early demise. Among the most promising treatments, small-molecule chemical chaperones
can rescue the enzyme activity of the misfolded glucosylceramidase. The experimentally identified deoxynojirimycin-type inhibitors have a narrow concentration
range and cause a mild improvement in the activity of
the mutant enzyme.
We did a molecule fragment–based virtual screening with the National Cancer Institute diversity data set.
198 MOLECULAR BIOLOGY
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A total of 72 compounds identified as the best compounds of interest by virtual screening were tested in
the enzyme assays by our collaborator, J. Kelly, Department of Chemistry. A total of 13 compounds are insoluble in dimethyl sulfoxide at up to 10 mM; 25 precipitate
in the assay buffer. Among the remaining 34 compounds
examined by using in vitro enzyme assays, 16 show
significant inhibition of the enzyme. The top compounds
of interest have almost doubled the activities of the
mutant enzymes N370S and G202R in vivo.
P R O T E I N P H O S P H ATA S E 2 C I N H I B I T O R S
In collaboration with P. Greengard, Rockefeller University, New York, New York, we used AutoDock to screen
the National Cancer Institute diversity set against protein
phosphatase 2C (PP2C), an enzyme that must remain
active for tumor growth in breast cancer. Several compounds were identified as inhibitors of PP2C in the
computational screen. The compounds were ordered
from the National Cancer Institute and were assayed
experimentally. Several were inhibitory at micromolar
concentrations; the potency of the best was between
5 and 10 µM. The lead compounds discovered in this
study are the first nonphosphate-based PP2C inhibitors reported.
THE SCRIPPS RESEARCH INSTITUTE
to science education and outreach with a combination
of presentations, popular and professional illustrations
and animations, 3-dimensional tangible models, and a
presence on the Worldwide Web. In these projects, we
use the diverse visualization tools developed in the
Molecular Graphics Laboratory to disseminate results
that range from atomic structure to cellular function.
We also continued several regular features that
informally present molecular structure and function.
The “Molecule of the Month” at the Protein Data Bank
entered its seventh year of providing an accessible introduction to the central database of biomolecular structure. Each month, a new molecule is presented with
a description of the molecule’s structure, function, and
relevance to health and welfare (Fig. 2). Visitors are
M E C H A N I S T I C S T U D I E S O F B I O C ATA LY S T S I N
COCAINE ANTIBODIES
Currently, no effective treatment of cocaine addiction
approved by the Food and Drug Administration is available. One possible treatment based on receptor design
entails thorough investigation of cocaine hydrolysis by
catalytic antibodies. Between 2 possible reaction pathways (an oxyanion hole for carbonyl or an hydrogen-bond
trap for hydroxide ion formed by tyrosines at positions
H50 and L94), quantum mechanical, molecular docking, and molecular dynamics free-energy calculations
have shown no conclusive evidence for a dominant pathway between 2 possible ones. An explanation for the
low turnover rate of the antibody is the failure of the
antibody to promote any mechanism selectively because
of a homogeneous microenvironment generated by eliciting against a hapten with 2 equivalent phosphorusoxygen bonds. Computational modeling would help
improve hapten design and thus improve the efficiency
of the catalytic antibody.
V I S U A L M E T H O D S F R O M AT O M S T O C E L L S
Understanding structural molecular biology is essential to foster progress and critical decision making among
students, policy makers, and the general public. In the
past year, we continued our longstanding commitment
F i g . 2 . ATP synthase was presented as the Molecule of the
Month in 2005. The illustration of this complex molecular machine
was constructed from 4 separate entries in the Protein Data Bank:
1c17, 1e79, 2a7u, and 1l2p.
then given suggestions about to how to begin their own
exploration of the structures in the data bank. Other projects include “The Molecular Perspective,” articles in
the journal The Oncologist that present structures of
interest to clinical oncologists and provide a source of
continuing education for physicians; “Recognition in
Action,” a new series at the Journal of Molecular Recognition; and work with the Nanoscale Informal Science
Network supported by the National Science Foundation
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to develop new materials for presenting the science of
nanotechnology.
PUBLICATIONS
Beuscher, A., Olson, A.J., Goodsell, D.S. Identifying protein binding sites and optimal ligands. Lett. Drug Des. Discov. 2:483, 2005.
Cheng, T.-J., Goodsell, D.S., Kan, C.-C. Identification of sanguinarine as a novel
HIV protease inhibitor from high-throughput screening of 2,000 drugs and natural
products with a cell-based assay. Lett. Drug Des. Discov. 2:364, 2005.
Dickerson, T.J., Beuscher, A.E. IV, Rogers, C.J., Hixon, M.S., Yamamoto, N., Xu, Y.,
Olson, A.J., Janda, K.D. Discovery of acetylcholinesterase peripheral anionic site
ligands through computational refinement of a directed library. Biochemistry
44:14845, 2005.
Goodsell, D.S. Computational docking of biomolecular complexes with AutoDock.
In: Protein-Protein Interactions: A Molecular Cloning Manual, 2nd ed. Golemis, E.,
Adams, P. (Eds.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY,
2005, p. 885.
Goodsell, D.S. The molecular perspective: c-Abl tyrosine kinase. Oncologist
10:758, 2005; Stem Cells 24:209, 2006.
Goodsell, D.S. The molecular perspective: cisplatin. Oncologist 11:316, 2006;
Stem Cells 24:514, 2006.
Goodsell, D.S. The molecular perspective: double-stranded DNA breaks. Oncologist
10:361, 2005; Stem Cells 23:1021, 2005.
Goodsell, D.S. The molecular perspective: RAD51 and BRCA2. Oncologist
10:555, 2005; Stem Cells 23:1434, 2005.
Goodsell, D.S. The molecular perspective: tumor necrosis factor. Oncologist 11:83,
2006.
Goodsell, D.S. Recognition in action: DNA mimicry. J. Mol. Recognit. 18:427, 2005.
Goodsell, D.S. Representing structural information. In: Current Protocols in Bioinformatics. Baxeranis, A.D., Davison, D.B. (Eds.). Wiley & Sons, Hoboken, NJ,
2005, p. 5.4.1.
Huey, R., Morris, G.M., Olson, A.J., Goodsell, D.S. A semi-empirical free energy
force field with charge-based desolvation. J. Comput. Chem., in press.
Rogers, J.P., Beuscher, A.E. IV, Flajolet, M., McAvoy, T., Nairn, A.C., Olson, A.J.,
Greengard, P. Discovery of protein phosphatase 2C inhibitors by virtual screening.
J. Med. Chem. 49:1658, 2006.
Sanner, M., Stolz, M., Burkhard, P., Kong, X.-P., Min, G., Sun, T.-T., Driamov, S.,
Aebi, U., Stoffler, D. Nature at work from the nano to the macro scale.
Nanobiotechnology 1:7, 2005.
Predicting Protein Structure,
Association, and Inhibitors
R. Abagyan, J. An,* W. Bisson, A. Cheltsov, K. Hyun,
J. Kovacs, I. Kufareva, P. Lam,** G. Nicola, A. Saldanha
* Genome Sciences Centre, Vancouver, British Columbia
** Molsoft L.L.C., La Jolla, California
oday the Protein Data Bank contains more than
37,000 structures and is growing at a rate of
20 per day. These structures provide a unique
opportunity for functional studies and rational design
of therapeutic agents. We continue to focus on anno-
T
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tating and characterizing the protein structures in terms
of their interaction interfaces and flexibility; predicting
protein associations; modeling homologous structures
and membrane proteins; predicting conformational
rearrangements; and, finally, using ligand docking and
virtual screening to detect inhibitors of specific molecular targets. This past year, our efforts in the last area
led to new or improved inhibitors against the receptor
for epidermal growth factor (EGFR), anthrax lethal factor,
dynamin, α1-antitrypsin, and the androgen receptor.
B I O I N F O R M AT I C S A N D C H E M I N F O R M AT I C S
We helped G. Siuzdak and his group, Department
of Molecular Biology, build a cheminformatics system
for characterizing metabolites on the basis of liquid
chromatography–mass spectrometry data. The resulting software (XCMS) incorporates novel nonlinear
retention time alignment, matched filtration, peak
detection, and peak matching and is freely available
from http://metlin.scripps.edu/download/. The software
helps identify changes in specific endogenous metabolites, such as potential biomarkers.
We have proposed a method for sharing chemical
information in conjunction with data on experimental
compounds without revealing the identity of the compounds. Privacy of chemical structure is of paramount
importance in the industrial sector, and the proposed
solution opens a way to transfer a rich knowledge base
from the pharmaceutical industry to academia.
Finally, we collaborated with scientists at the
Structural Genomics Consortium, Oxford, England, to
improve the way the new structures are annotated,
distributed, and animated by using internal coordinates–based methods.
LIGAND DISCOVERY
Small-molecule therapeutic agents can be discovered by using docking and virtual chemical library
screening. The docking technology can also help in
understanding structural mechanisms of action of small
molecules and rational design of better molecules. However, modeling protein flexibility and ligand-induced conformational changes is a major challenge. We modeled
the induced receptor rearrangements at several levels,
including relevant normal modes combined with full
side-chain sampling and “minus-one” calculations. In
particular, we used the developed ligand-induced receptor simulation techniques to identify new antagonists
of the androgen receptor and the first small-molecule
inhibitors of α1-antitrypsin polymerization.
Our docking-based in silico chemical library screening
against the EGFR tyrosine kinase and the consequent
200 MOLECULAR BIOLOGY
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THE SCRIPPS RESEARCH INSTITUTE
experimental validation allowed identification of several
compounds with antiproliferative effects on cancer cells.
Among them, a C(4)-N(1)-substituted pyrazolo[3,4d]pyrimidine inhibits EGFR tyrosine kinase activity at
micromolar concentrations.
We screened a library of tyrphostins against the
GTPase activity of dynamin I and performed optimization of discovered compounds. The results yielded a
number of promising inhibitors that are effective at
micromolar concentrations.
Using a fragment-based approach, we developed
inhibitors of the lethal factor metalloproteinase of Bacillus anthracis. The discovered compounds are highly
potent and selective against lethal factor in in vitro
assays, including cell-based assays.
PEPTIDE DOCKING AND STRUCTURE PREDICTION
Predicting partial protein structure or molecular
association is a critical task of computational biology,
which remains a focus of our research. In particular,
we developed a method for ab initio prediction of peptide-MHC binding geometry for diverse class I MHC
allotypes. Such models are useful for predicting specific
ternary complexes with T-cell receptors and for designing
new molecules that interact with these complexes. The
surprisingly accurate prediction (0.75-Å backbone root
mean square deviation) that we achieved by using our
method for cross-docking of a highly flexible decapeptide,
dissimilar to the original bound peptide, and docking
predictions with homology models for 2 allotypes with
mean backbone root mean square deviations of less
than 1.0 Å illustrate the effectiveness of the method.
PREDICTING FUNCTIONAL SITES
Functional annotation of protein structures involves
identifying and characterizing protein-protein interfaces,
oligomerization states, and binding sites for small ligands.
We developed a method called protein interface recognition that can be used to predict interfaces on the basis
of an isolated protein structure and does not depend on
evolutionary information. The method was benchmarked
by using a diverse set of 748 protein interfaces. The
accuracy and efficiency make the method a suitable tool
for automated high-throughput annotation of protein
structures discovered in structural proteomics studies
(Fig. 1).
Some protein interfaces can safely be targeted for
drug discovery. We developed a systematic approach
to assessing the “druggability” of a protein interface.
The approach includes detecting a suitable ligand-binding pocket with maximal confidence in a functionally
F i g . 1 . Predicting protein oligomerization geometry by using
protein interface recognition.
sensitive location on the biomolecule and assessing the
reliability of the local structure. This approach was
applied to the Skp1-Cullin-F-box protein ubiquitin ligase
interface. It can be used before high-throughput or virtual library screening.
CD59 is a membrane glycoprotein with therapeutic
potential for treatment of inflammatory conditions. Using
scanning mutagenesis, refined nuclear magnetic resonance models, and additional site-specific mutations,
we identified a binding interface on CD59 that is much
broader than previously thought. We identified substitutions that decreased CD59 activity and a surprising
number of substitutions that enhanced it. On the basis
of these findings, we prepared clinically relevant soluble
mutant CD59-based proteins that had up to a 3-fold
increase in complement inhibitory activity.
PUBLICATIONS
Abagyan, R. Problems in computational structural genomics. In: Structural Proteomics. Sundstrom, M., Norin, M., Edwards, A. (Eds.). CRC Press, Boca Raton,
FL, 2006, p. 223.
Abagyan, R., Lee, W.H., Raush, E., Budagyan, L., Totrov, M., Sundstrom, M.,
Marsden, B.D. Disseminating structural genomics data to the public: from a data
dump to an animated story. Trends Biochem. Sci. 31:76, 2006.
Bordner, A., Abagyan, R.A. Ab initio prediction of peptide-MHC binding geometry
for diverse class I MHC allotypes. Proteins 63:512, 2006.
Cardozo, T., Abagyan, R. Druggability of SCF ubiquitin ligase-protein interfaces.
Methods Enzymol. 399:634, 2005.
Cavasotto, C.N., Orry, A.J.W., Abagyan, R. Receptor flexibility in ligand docking. In:
Handbook of Theoretical and Computational Nanotechnology. Rieth, M., Schommers,
W. (Eds.). American Scientific Publishers, Stevenson Ranch, CA, 2006, Vol.6, p. 217.
Cavasotto, C.N., Orry, A.J.W., Abagyan, R.A. The challenge of considering receptor flexibility in ligand docking and virtual screening. Curr. Comput. Aided Drug
Des. 1:423, 2005.
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Cavasotto, C.N., Ortiz, M.A., Abagyan, R.A., Piedrafita, F.J. In silico identification
of novel EGFR inhibitors with antiproliferative activity against cancer cells. Bioorg.
Med. Chem. Lett. 16:1969, 2006.
Forino, M., Johnson, S., Wong, T.Y., Rozanov, D.V., Savinov, A.Y., Li, W., Fattorusso, R., Becattini, B., Orry, A.J., Jung, D., Abagyan, R.A., Smith, J.W.,
Alibek, K., Liddington, R.C., Strongin, A.Y., Pellecchia, M. Efficient synthetic
inhibitors of anthrax lethal factor. Proc. Natl. Acad. Sci. U. S. A. 102:9499, 2005.
Hill, T., Odell, L.R., Edwards, J.K., Graham, M.E., McGeachie, A.B., Rusak, J.,
Quan, A., Abagyan, R., Scott, J.L., Robinson, P.J., McCluskey, A. Small molecule
inhibitors of dynamin I GTPase activity: development of dimeric tyrphostins. J.
Med. Chem. 48:7781, 2005.
Huang, Y., Smith, C.A., Song, H., Morgan, B.P., Abagyan, R., Tomlinson, S.
Insights into the human CD59 complement binding interface toward engineering
new therapeutics. J. Biol. Chem. 280:34073, 2005.
Kovacs, J.A., Cavasotto, C.N., Abagyan, R.A. Conformational sampling of protein
flexibility in generalized coordinates: application to ligand docking. J. Comput.
Theor. Nanosci. 2:354, 2005.
Kufareva, I., Budagyan, L., Raush, E., Totrov, M., Abagyan, R. PIER: protein
interface recognition for structural proteomics. Proteins, in press.
Orry, A.J., Abagyan, R.A., Cavasotto, C.N. Structure-based development of targetspecific compound libraries. Drug Discov. Today 11:261, 2006.
F i g . 1 . A novel nonlinear approach for correcting and analyzing
mass spectrometry data for characterization of metabolites.
V I R A L C H A R A C T E R I Z AT I O N
We have developed novel methods for characterizing
viruses that have applications to whole viruses, viral proteins, and viral metabolites. Our results have enabled us
to examine both local and overall viral structure, gaining insight into the dynamic changes of proteins on the
viral surface and the changes that occur during viral
infection (Fig. 2).
Smith, C.A., O’Maille, G., Want, E.J., Qin, C., Trauger, S.A., Brandon, T.R., Custodio, D.E., Abagyan, R., Siuzdak, G. METLIN: a metabolite mass spectral database. Ther. Drug Monit. 27:747, 2005.
Smith, C.A., Want, E.J., O’Maille, G., Abagyan, R., Siuzdak, G. XCMS: processing
mass spectrometry data for metabolite profiling using nonlinear peak alignment,
matching, and identification. Anal. Chem. 78:779, 2006.
Tetko, I.V., Abagyan, R., Oprea, T.I. Surrogate data: a secure way to share corporate data. J. Comput. Aided Mol. Des. 19:749, 2005.
Mass Spectrometry
F i g . 2 . A comprehensive approach for studying viral infection by
G. Siuzdak, J. Apon, H.P. Benton, E. Go, K. Harris, L. Hoang,
R. Lowe, A. Meyers, H. Morita, A. Nordstrom, T. Northen, G.
O’Maille, C. Qin, Z. Shen, C. Smith, M. Sonderegger,
S. Trauger, W. Uritboonthai, E. Want, W. Webb, W. Wikoff,
D. Wong
M E TA B O L I T E P R O F I L I N G
ndogenous small-molecule metabolites, ubiquitous
in biofluids, are crucial elements in understanding
living organisms whether in fundamental biochemistry, disease diagnosis, or drug toxicity. The inherent
advantage of monitoring small molecules rather than proteins is the relative ease of quantitative analysis of the
molecules with mass spectrometry. We are implementing
novel mass spectrometry and bioinformatics techniques
(Fig. 1) to investigate the profile of small-molecule metabolites. Our purposes are to correlate metabolite activity
with protein regulation and to develop metabolite analysis as a diagnostic method. Our ultimate goal is to create
analytical and chemical technologies and data management approaches to identify and structurally characterize
metabolites of physiologic importance.
E
using a combination of mass spectrometry techniques. Three different aspects of viral infection within an infected cell—the expression
kinetics of the viral proteins, changes in the expression levels of cellular proteins, and changes in cellular metabolites—were monitored.
These analyses reveal the complexity of the protein and metabolite
regulation involved in cellular transformations that occur during
viral infection.
MASS SPECTROMETRY IN SILICO
We are also developing ultra-high-sensitivity
approaches in mass spectrometry with a new strategy that involves pulsed laser desorption/ionization
from a silylated silicon surface. In desorption/ionization
on silicon, silicon is used to capture analytes and laser
radiation is used to vaporize and ionize these molecules. Using this technology, we can analyze a wide
range of molecules with unprecedented sensitivity, in
the yoctomole range.
PUBLICATIONS
Cohen, L., Go, E.P., Siuzdak, G. Small-molecule desorption/ionization mass analysis. In: A Practical Guide to MALDI MS: Instrumentation, Methods and Applications. Hillenkamp, F., Peter-Katalinic, J. (Eds.). Wiley & Sons, New York, in press.
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Lee, J.-C.,Wu, C.-Y., Apon, J.V., Siuzdak, G., Wong, C.-H. Reactivity-based onepot synthesis of tumor-associated antigen N3 minor octasaccharide for the development of a cleavable DIOS-MS sugar array. Angew. Chem. Int. Ed. 45:2753, 2006.
Lowe, R., Tong, G., Voelcker, N.H., Siuzdak, G. Monitoring EDTA and endogenous
metabolite from serum with mass spectrometry. Spectroscopy 19:137, 2005.
Luo, G., Chen, Y., Siuzdak, G., Vertes, A. Surface modification and laser pulse length
effects on internal energy transfer in DIOS. J. Phys. Chem. B. 109:24450, 2005.
Nordstrom, A., Apon, J.V., Uritboonthai, W., Go, E.P., Siuzdak, G. Surfactant
enhanced desorption/ionization on silicon mass spectrometry. Anal. Chem. 78:272,
2006.
Nordstrom, A., He, L., Siuzdak, G. Desorption/ionization on silicon (DIOS). In:
Hyphenation Methods. Niessen, W. (Ed.). Elsevier, St. Louis, in press. Encyclopedia of Mass Spectrometry, Vol 8. Gross, M.L., Caprioli, R.M. (Eds. in Chief).
Nordstrom, A., O’Maille, G., Qin, C., Siuzdak, G. Nonlinear data alignment for
UPLC-MS and HPLC-MS based metabolomics: quantitative analysis of endogenous
and exogenous metabolites in human serum. Anal. Chem. 78:3289, 2006.
Siuzdak, G. The Expanding Role of Mass Spectrometry in Biotechnology, 2nd ed.
MCC Press, San Diego, CA, 2006.
Smith, C.A., O’Maille, G., Want, E.J., Qin, C., Trauger, S.A., Brandon, T.R., Custodio, D.E., Abagyan, R., Siuzdak, G. METLIN: a metabolite mass spectral database. Ther. Drug Monit. 27:747, 2005.
Smith, C.A., Want, E.J., O’Maille, G., Abagyan, R., Siuzdak, G. XCMS: processing
mass spectrometry data for metabolite profiling using nonlinear peak alignment,
matching, and identification. Anal. Chem. 78:779, 2006.
Talkington, M.W., Siuzdak, G., Williamson, J.R. An assembly landscape for the
30S ribosomal subunit. Nature 438:628, 2005.
Want, E., Cravatt, B.F., Siuzdak, G. The expanding role of mass spectrometry in
metabolite profiling and characterization. Chembiochem 6:1941, 2005.
THE SCRIPPS RESEARCH INSTITUTE
We using a wide variety of biophysical techniques to
study the mechanism of assembly of the 30S ribosome in vitro.
The 30S ribosome can be reconstituted from purified components in vitro with extremely high efficiency,
a characteristic that has enabled detailed mechanistic
studies. The 30S ribosome is composed of a single
RNA chain of approximately 1500 nucleotides and 20
small proteins of 8–20 kD. Pioneering work by Nomura
more than 30 years ago led to the development of an
assembly map that outlines the basic order of protein
binding. However, the mechanistic basis for these early
observations was unknown. Using nuclear magnetic
resonance, x-ray crystallography, calorimetry, and fluorescence methods, we have studied the details of
assembly of small RNA-protein complexes derived from
the 30S subunit to elucidate these molecular events.
A guiding principle for ribosome assembly is that
each protein recognizes a small local region of the RNA
as its binding site. The protein cannot bind until the
RNA structure in its binding site is properly folded. We
showed that the assembly reaction can be considered
an alternating series of RNA conformational changes
and protein binding events (Fig. 1). RNA helices must
Want, E.J., O’Maille, G., Smith, C.A., Brandon, T.R., Uritboonthai, W., Qin, C.,
Trauger, S.A., Siuzdak, G. Solvent-dependent metabolite distribution, clustering,
and protein extraction for serum profiling with mass spectrometry. Anal. Chem.
78:743, 2006.
Assembly Landscape of the
30S Ribosome
J.R. Williamson, F. Agnelli, A. Beck, C. Beuck, A. Bunner,
A. Carmel, J. Chao, S. Edgcomb, M. Hennig, E. Johnson,
D. Kerkow, E. Kompfner, S. Kwan, P. Mikulecky, W. Ridgeway,
H. Schultheisz, L.G. Scott, E. Sperling, B. Szymczyna
he ribosome is a large molecular machine that
is responsible for synthesis of all proteins in the
cell. It is composed of 2 multicomponent subunits
that bind mRNA, tRNAs, and other factors to carry out
translation of the genetic code from RNA into protein
product. In bacteria, the large, or 50S, subunit is responsible for catalyzing the formation of peptide bonds,
whereas the small, or 30S, subunit is responsible for
reading out the genetic code. An elaborate process exists
for biogenesis of the ribosome machinery in cells to
assemble the ribosome from individual components.
T
F i g . 1 . A model for ribosome assembly. The RNA chain is
shown as cylinders representing helical regions of RNA structure.
In the first step, the RNA changes conformation, which creates a
protein-binding site for the protein S15. Next, a subsequent RNA
folding event occurs, which in turn creates a binding site for the
proteins S6 and S18. Assembly appears to proceed as an alternating series of folding and binding events.
be properly arranged to create the binding site for the
first protein, which is protein S15 in Figure 1. Binding
of S15 effectively consolidates the gains from RNA folding in the previous step. Furthermore, after S15 binding, the next RNA conformational change is facilitated;
this change sets up the binding site for the next proteins,
which are S6 and S18 in Figure 1.
Thus, each protein serves as a local reporter for
RNA folding in a specific region of the 30S subunit.
The overall assembly reaction can be schematically
illustrated as shown in Figure 2, where an unfolded
RNA chain is combined with 20 different proteins, a
change that after a complex series of RNA conforma-
MOLECULAR BIOLOGY
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THE SCRIPPS RESEARCH INSTITUTE
203
a number of parallel pathways exist by which the ribosome can assemble. In addition to the functional restrictions placed on the sequence of the RNA, most likely
the sequence is also selected under evolutionary pressure to fold efficiently under a variety of conditions
encountered by bacteria in the environment.
F i g . 2 . The 30S ribosome assembly reaction. The RNA chain is
represented as a thin line that is disordered at the beginning of the
experiment. The 20 small proteins that bind to the RNA are represented as circles. The final assembled subunit is composed of highly folded RNA with each protein bound at a specific location.
tional changes and protein-binding events results in the
structured 30S subunit. Our previous analyses involved
fragments of the overall structure, and we were interested in monitoring the kinetics of binding and assembly of the intact 30S subunits.
Monitoring the simultaneous binding of 20 different proteins to an RNA molecule is a serious technical
challenge. To surmount this challenge, we developed
an isotope-pulse chase assay in which mass spectrometry is used to indicate binding of the proteins to the
RNA. Assembly is initiated by using a pulse of a mixture
of the 20 15 N-labeled proteins; after a short assembly time, a mixture of 14 N-proteins is added as the
chase. The fully assembled subunits are isolated, and
the fraction of 15N for each protein is measured as a
function of the pulse time by using quantitative mass
spectrometry. In this way, the time course of binding
for each protein can be measured. The strength of the
method is that the binding rates can all be measured
simultaneously.
Using this method, we can perform mechanistic
experiments on 30S ribosome assembly by using the
kinetic tools of physical chemistry. We have varied the
protein concentration to show that the binding rates
correspond to a bimolecular association, not to a ratelimiting RNA conformational change. We have varied
the magnesium ion concentration to show that ions can
play 2 opposing roles during assembly. Some parts of the
30S subunit speed up at lower magnesium concentrations, and different parts slow down. Perhaps most
important, we have measured the rates as a function
of temperature and performed Arrhenius analysis of the
activation energies for binding.
The main conclusion from these studies is that 30S
assembly has no single global rate-limiting step. Rather,
PUBLICATIONS
Chao, J.A.., Lee, J.H., Chapados, B.R., Debler, E.W., Schneemann, A., Williamson,
J.R. Dual modes of RNA-silencing suppression by Flock House virus protein B2.
Nat. Struct. Mol. Biol. 12:952, 2005.
Davis, J.H., Tonelli, M., Scott, L.G., Jaeger, L., Williamson, J.R., Butcher, S.E.
RNA helical packing in solution: NMR structure of a 30 kDa GAAA tetraloop-receptor complex [published correction appears in J. Mol. Biol. 760:742, 2006]. J.
Mol. Biol. 351:371, 2005.
Hennig, M., Munzarova, M.L., Bermel, W., Scott, L.G., Sklenar, V., Williamson,
J.R. Measurement of long-range 1H-19F scalar coupling constants and their glycosidic torsion dependence in 5-fluoropyrimidine-substituted RNA. J. Am. Chem.
Soc. 128:5851, 2006.
Scott, L.G., Williamson, J.R. The binding interface between Bacillus stearothermophilus ribosomal protein S15 and its 5′-translational operator mRNA. J. Mol.
Biol. 351:280, 2005.
Talkington, M.T., Siuzdak, G., Williamson, J.R. An assembly landscape for the
30S ribosomal subunit. Nature 438:628, 2005.
Development of the Genetic
Code and Its Connection to
Human Disease
P. Schimmel, J. Bacher, K. Beebe, Z. Druzina, K. Ewalt,
M. Kapoor, E. Merriman, C. Motta, L. Nangle, F. Otero,
J. Reader, R. Reddy, M. Swairjo, K. Tamura, E. Tzima,
W. Waas, X.-L. Yang
he genetic code is thought to have developed in
the putative RNA world and thereby enabled the
transition to the modern world of proteins. The
early code was primitive and over many eons was
refined. This refinement came from the acquisition of
new activities by a group of proteins known as aminoacyl-tRNA synthetases. These proteins established the
rules of the code through aminoacylation reactions,
whereby each of the 20 amino acids is covalently joined
to its cognate tRNA. The tRNA harbors the genetic code
triplet associated with the specific amino acid that is
joined to the tRNA.
Each amino acid has a single tRNA synthetase. The
synthetases are thought to be among the earliest proteins, essential components of the translation apparatus that established the genetic code and that were
present in the last common ancestor of the universal
T
204 MOLECULAR BIOLOGY
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tree of life. As the tree developed and branched into
the 3 great kingdoms—archaebacteria, bacteria, and
eukaryotes—the enzymes were incorporated into every
cell type of every organism.
Detailed investigations of the structures and evolution of the aminoacyl-tRNA synthetases have provided
a picture of the development of the genetic code and
how the development was directed by the evolution of
the synthetases and tRNAs. In previous research, we
focused on the specifics of the molecular recognition
of tRNAs and how the enzymes distinguish one tRNA
from another to achieve accurate aminoacylation for a
precise genetic code. During these studies, examination
of a recent crystal structure of human tryptophanyl-tRNA
synthetase (TrpRS) in complex with the tRNA for tryptophan (tRNATrp) revealed 2 states of the enzyme-tRNA
complex (Fig. 1). In one state, the tRNA is entering the
THE SCRIPPS RESEARCH INSTITUTE
Recently, we showed how the error-correction activity
is essential for maintaining cell viability and how defects
in this activity can lead to disease. In collaboration with
S.L. Ackerman, Jackson Laboratories, Bar Harbor, Maine,
we found that a single point mutation in mice leads to
neurodegeneration (Fig. 2). In particular, Purkinje cells
F i g . 1 . Crystal structures of uncharged tRNA Trp associating with
TrpRS (A) and charged tRNATrp dissociating from the enzyme (B).
The location of the bound free amino acid (Trp) in the 2 active sites
of the homodimer is indicated. The tRNA binds across both subunits.
active site. In the other state, it has been charged (that
is, tryptophan has been joined to tRNATrp in the aminoacylation reaction) and is dissociating from the enzyme.
During the long evolutionary development of aminoacyl-tRNA synthetases and their populating of every cell
type, the enzymes adopted novel functions while keeping their canonical role as determinants of the genetic
code. Related to their central role, the enzymes acquired
novel domains enabling them to correct errors of aminoacylation and thereby ensure the stringent accuracy of
the code. Unrelated to the canonical activities of the
enzymes in translation, the expanded functions include
regulation of transcription and translation in bacteria,
RNA splicing in fungal organisms, and cytokine signaling in mammalian cells. These novel functions connect translation to other central pathways that control
growth, development, and regulation of all cell types.
F i g . 2 . Pathologic changes in sticky mutant mice (A). B-D, Cal-
bindin D-28 (Calb) immunohistochemistry of sagittal sections of
cerebella from 3-week-old (B), 6-week-old (C), and 12-month-old
(D) sti/sti mutant and 12-month-old wild-type (WT; E) mice. Cerebellar lobules are indicated by roman numerals. F-H, Hematoxylin
and eosin staining of Purkinje cells (arrowheads) in lobule II of
cerebella from 1-month-old (F) or 12-month-old (G) sti/sti mutant
and 12-month-old wild-type (H) mice. I-N, Cleaved caspase 3
(Casp3) immunohistochemistry (I-K) and TUNEL analysis (L-N) of
cerebella from 4-week-old mutant mice. Scale bars: For B-E, 500
µm; F-H, 50 µm; I-N, 10 µm.
in the brain deteriorate and ataxia develops. This simple,
heritable mutation is due to small amounts of misacylation of alanine-specific tRNA (tRNAAla) to generate, for
example, serine attached to the tRNA. In this instance,
small amounts of serine are incorporated in place of alanine in the polypeptides that are produced. Thus, a mild
MOLECULAR BIOLOGY
2006
editing defect can lead to heritable neurologic disorders.
A more severe defect in editing would doubtless be lethal
and not sustained in the population.
In other collaborative studies with R. Burgess, Jackson Laboratories, we established a connection between
glycyl-tRNA synthetase and Charcot-Marie-Tooth disease.
A single point mutation in the synthetase leads to the
disease. This mutation does not affect the aminoacylation
activity of glycyl-tRNA synthetase. Instead, the results
suggest that glycyl-tRNA synthetase has an additional
function, possibly in neurologic development. Several
examples of Charcot-Marie-Tooth disease due to mutations
in glycyl-tRNA synthetase in humans have been found.
To better understand the molecular origins of this
disease, we obtained crystals of human glycyl-tRNA
synthetase that diffract to about 3 Å. A structure is
being determined, and mutations found in humans
will be mapped on the structure. This information will
be used in conjunction with other assays and experiments to understand the connection between neurologic development, Charcot-Marie-Tooth disease, and
glycyl-tRNA synthetase.
These results with glycyl-tRNA synthetase support
the hypothesis that aminoacyl-tRNA synthetases in
mammals are not only components of the translation
apparatus but also a reservoir of cytokines with activities that are unmasked by specific activation events,
such as alternative splicing or generation of specific
fragments by proteolysis. Examples we are studying
include tyrosyl- and tryptophanyl-tRNA synthetases.
These are both procytokines that when split by alterative splicing or natural proteolysis, result in fragments
that are active in signal transduction pathways. For
example, a fragment of tryptophanyl-tRNA synthetase
is a potent angiostatic agent.
PUBLICATIONS
Lee, J.W., Beebe, K., Nangle, L.A., Jang, J., Longo-Guess, C.M., Cook, S.A.,
Davisson, M.T., Sundberg, J.P., Schimmel, P., Ackerman, S.L. Editing-defective
tRNA synthetase causes protein misfolding and neurodegeneration in the sticky
mouse. Nature 443:50, 2006.
Nangle, L.A., Motta, C.M., Schimmel, P. Global effects of mistranslation from an
editing defect in mammalian cells. Chem. Biol. 13:1091, 2006.
Reader, J.S., Ordoukhanian, P.T., Kim, J.-G., de Crécy-Lagard, V., Hwang, I.,
Farrand, S., Schimmel, P. Major biocontrol of plant tumors targets tRNA synthetase
[published correction appears in Science 310:54, 2005]. Science 309:1533, 2005.
Schimmel, P., Beebe, K. From the RNA world to the theatre of proteins. In: The
RNA World, 3rd ed. Gesteland, R.R., Cech, T.R. Atkins, J.F. (Eds.). Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, NY, 2005, p. 227.
Seburn, K.L., Nangle, L.A., Cox, G.A., Schimmel, P., Burgess, R.W. An active
dominant mutant of glycyl-tRNA synthetase causes neuropathy in Charcot-MarieTooth 2D mouse model. Neuron 51:715, 2006.
THE SCRIPPS RESEARCH INSTITUTE
205
Swairjo, M.A., Reddy, R.R., Lee, B., Van Lanen, S.G., Brown, S., de CrécyLagard, V., Iwata-Reuyl, D., Schimmel, P. Crystallization and preliminary x-ray
characterization of the nitrile reductase QueF: a queosine-biosynthesis enzyme.
Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 61(Pt. 10):945, 2005.
Tamura, K., Schimmel, P.R. Chiral-selective aminoacylation of an RNA minihelix:
mechanistic features and chiral suppression. Proc. Natl. Acad. Sci. U. S. A.
103:13750, 2006.
Tzima, E., Schimmel, P. Inhibition of tumor angiogenesis by a natural fragment of
a tRNA synthetase. Trends Biochem. Sci. 31:7, 2006.
Waas, W.F., de Crécy-Lagard, V., Schimmel, P. Discovery of a gene family critical
to wyosine base formation in a subset of phenylalanine-specific transfer RNAs. J.
Biol. Chem. 280:37616, 2005.
Yang, X.-L., Otero, F.J., Ewalt, K.L., Liu, J., Swairjo, M.A., Kohrer, C., RajBhandary, U.L., Skene, R.J., McRee, D., Schimmel, P. Two conformations of a crystalline human tRNA synthetase-RNA complex: implications for protein synthesis.
EMBO J. 25:2919, 2006.
Mechanisms of RNA Assembly
and Catalysis
M.J. Fedor, E.M. Calderon, J.W. Cottrell, C.P. Da Costa,
S. Daudenarde, J.W. Harger, Y.I. Kuzmin, E.M. Mahen,
M. Roychowdhury-Saha
ur goal is to generate basic insights into catalysis by RNA and RNA-protein enzymes, RNA
folding, and RNA interactions with small molecules. In addition to contributing basic knowledge of
RNA structure and function in normal growth and development, results of our studies provide a framework for
developing technical and therapeutic applications involving RNAs as targets and reagents.
Apart from the ribosome, which catalyzes peptidyl
transfer, the naturally occurring ribozymes catalyze transfer of phosphate groups. The small RNA enzymes that
we study catalyze reversible phosphodiester cleavage
reactions that generate 5′ hydroxyl and 2′,3′-cyclic
phosphate termini (Fig. 1). Possible strategies for catalysis of phosphoryl transfer reactions include aligning reactive groups in an optimal orientation for an in-line attack
mechanism, general acid-base catalysis of proton transfer
to activate nucleophilic oxygens or to stabilize oxyanionleaving groups, electrostatic stabilization of negative
charge that accumulates in the transition state, and
destabilizing the ground state. Our goal is to understand
which of these catalytic strategies RNA enzymes use.
In contrast to the chemical versatility of the amino
acid side chains that make up the active sites of protein enzymes, just 4 nucleotides are available for the
construction of ribozyme active sites. Nucleotides are
well suited to faithful storage and transmission of genetic
O
206 MOLECULAR BIOLOGY
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F i g . 1 . Chemical mechanism of RNA cleavage mediated by the
family of small catalytic RNAs that includes the hairpin ribozyme.
Cleavage of the phosphodiester bond occurs through an SN2-type
mechanism that involves in-line attack of the 2′ oxygen nucleophile
on the adjacent phosphorus to form a trigonal bipyramidal transition state. Breaking of the 5′ oxygen-phosphorus bond generates
products with 5′ hydroxyl and 2′,3′-cyclic phosphate termini.
information through complementary base pairing, but
they are not particularly adept at catalytic chemistry.
Protonation and deprotonation of nucleotides occur at
high or low pH extremes, a situation that would make
it difficult to mediate general acid or base catalysis at
neutral pH. No positively charged nucleotide functional
groups are expected to be available at neutral pH to
function as Lewis acids to activate a nucleophile or
stabilize an electronegative transition state or an oxyanion-leaving group.
Recent high-resolution structures of self-cleaving
RNAs lay the groundwork for experiments to probe fundamental questions about how RNA enzymes use their
functional groups for catalysis. Like all enzymes, hairpin
ribozymes combine several strategies to enhance catalytic rate. One important strategy, apparent from the
crystal structures, is the alignment of nucleophilic and
leaving-group oxygens in the optimal orientation for an
in-line SN2-type nucleophilic attack. The structure of the
hairpin ribozyme active site places guanine 8, adenine 9,
adenine 10, and adenine 38 nucleobases near the reactive phosphate. Guanine 8 and adenine 38 occupy positions reminiscent of 2 histidine residues in the active site
of ribonuclease A, a protein enzyme that catalyzes the
same reaction. Histidine residues perform general acidbase catalysis during ribonuclease A catalysis, so the
similarity between hairpin ribozyme and ribonuclease A
active sites raised the possibility that guanine 8 and
adenine 38 nucleobases might perform functions similar
to those of histidine residues.
Hairpin ribozyme activity increases with increasing
pH, consistent with the notion that activity depends on
the availability of the deprotonated form of guanine 8
to accept a proton from the 2′ hydroxyl nucleophile as
predicted by the general acid-base catalysis model. To
THE SCRIPPS RESEARCH INSTITUTE
test this model, we replaced guanine 8 with an abasic
residue, a substitution that eliminates the nucleobase but
leaves the phosphodiester backbone intact. However,
this abasic variant had the same pH dependence as
an unmodified ribozyme, arguing that the pH transition
does not involve guanine 8. Replacing adenine 38 with
an abasic residue, on the other hand, did eliminate the
pH-dependent transition in activity, implicating adenine
38 in a catalytically important deprotonation.
These and other results are consistent with 2 models
of the hairpin ribozyme catalytic mechanism in which
adenine 38 contributes either general acid-base catalysis (Fig. 2A) or electrostatic stabilization of negative
F i g . 2 . Two models of hairpin ribozyme catalysis. Results of mech-
anistic studies of the hairpin ribozyme are consistent with 2 models
in which the functional form of adenine 38 is either protonated or
unprotonated. In the first model (A), protonated adenine 38 would
act as a general acid by donating a proton to the 5′ oxygen, acting
in concert with hydroxide ion that activates the 2′ oxygen nucleophile
during cleavage, and unprotonated adenine 38 would act as a general base to activate the 5′ oxygen nucleophile during ligation. In the
second model (B), unprotonated adenine 38 accepts a hydrogen bond
from the 5′ hydroxyl nucleophile during ligation and accepts a hydrogen bond from a protonated bridging 5′ oxygen during cleavage, providing electrostatic stabilization to developing negative charge. In both
models, the amidine group of guanine 8, in its protonated form, donates
hydrogen bonds to the 2′ and phosphoryl oxygens that stabilize negative charge that develops in the transition state and positions reactive
groups in the orientation appropriate for an SN2 in-line nucleophilic
attack. Reproduced with permission from Fedor, M.J., Williamson, J.R.
The catalytic diversity of RNAs. Nat. Rev. Mol. Cell Biol. 6:399, 2005.
Copyright 2005 Nature Publishing Group/Macmillan Magazines Ltd.
charge that develops in the transition state as 5 electronegative oxygen atoms from transient bonds with
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2006
THE SCRIPPS RESEARCH INSTITUTE
207
phosphorus (Fig. 2B) and guanine 8 donates hydrogen
bonds to stabilize the transition state electrostatically.
Directed Evolution of Nucleic
Acid Enzymes
G.F. Joyce, S.E. Hamilton, D.P. Horning, T.A. Jackson,
G.C. Johns, B.J. Lam, B.M. Paegel, G.G. Springsteen,
S.B. Voytek
ll life known to exist on Earth today is based on
DNA genomes and protein enzymes, but most
likely it was preceded by a simpler form of life
based on RNA. This earlier era is referred to as the
“RNA world.” During that time, genetic information
resided in the sequence of RNA molecules and phenotype was derived from the catalytic behavior of RNA.
By studying the properties of RNA in the laboratory,
especially with regard to the evolution of catalytic
function, we can gain insight into the RNA world. In
addition, we can develop novel nucleic acid enzymes
that have applications in biology and medicine.
A
CONVERTING AN RNA ENZYME TO A DNA ENZYME
The transfer of sequence information between 2 different classes of nucleic acid–like molecules, for example
between RNA and DNA, is straightforward because it
relies on the 1-to-1 correspondence of Watson-Crick
pairing. Nearly 50 years ago, in articulating the central dogma of molecular biology, Francis Crick referred to
this property as “sequentialization.” Sequentialization
also applies to the transfer of information from RNA to
protein via the genetic code. The transfer of function,
however, is more difficult because function is an overall
property of a macromolecule and cannot be conveyed in
a sequential manner. There is no known example of an
RNA enzyme that retains catalytic activity when prepared
as the corresponding DNA molecule, and vice versa.
We used in vitro darwinian evolution to convert an
RNA enzyme to a DNA enzyme of the same function,
after the acquisition of a few critical mutations. The
starting RNA had the ability to join 2 RNA substrates
in a template-directed manner, with a catalytic rate of
0.14 min–1 (Fig. 1A). A corresponding DNA molecule
in which ribose was replaced by deoxyribose and uracil
was replaced by thymine had no detectable activity. The
DNA molecule was used as a starting point to generate
trillions of randomized variants, which were selected for
the ability to catalyze the RNA-joining reaction. After 10
F i g . 1 . Composition of an RNA enzyme (A) and a DNA enzyme
(B) related by evolutionary descent. Both enzymes contain about
50 nucleotides and catalyze the joining of 2 RNA substrates (S1
and S2). The evolved DNA enzyme contains 10 mutations relative
to the starting RNA enzyme (highlighted with black circles).
generations of evolution, we obtained a population of
DNA enzymes with the desired activity. A typical example
contains 10 mutations relative to the starting sequence
and has a catalytic rate of 0.052 min–1 (Fig. 1B). When
this DNA enzyme was prepared as the corresponding
RNA enzyme, it had no detectable activity. Thus, the
evolutionary transition from an RNA enzyme to a DNA
enzyme represents a switch in the chemical basis of
catalytic function.
Evolutionary pathways such as this one for conversion of an RNA enzyme to a DNA enzyme may exist
between other classes of nucleic acid–like molecules.
The RNA world may have been preceded by a simpler
“pre-RNA world” based on a nucleic acid–like molecule
that would have occurred more readily on the primitive
Earth. Our findings suggest that the catalytic function of
a pre-RNA molecule might have been transferred to a
corresponding RNA enzyme through darwinian evolution.
CONTINUOUS EVOLUTION OF RNA ENZYMES
Processes of darwinian evolution are fundamental
to understanding biological form and function but are
difficult to appreciate on the human timescale. During
the past decade, we have developed methods for evolving
molecules rapidly and under controlled laboratory conditions. One of the most powerful of these methods, and
the one that most closely resembles biological evolution,
is a system for the continuous in vitro evolution of RNA
enzymes. It involves a population of RNA enzymes that
catalyze an RNA-joining reaction. Any molecule in the
population that performs the reaction becomes amplified
to produce “progeny” molecules, which then have the
opportunity to perform the reaction again. The entire
process takes place within a common reaction mixture
and can be continued indefinitely, so long as an adequate supply of reaction materials is maintained.
208 MOLECULAR BIOLOGY
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Recently, we developed a novel approach for the
continuous evolution of RNA enzymes that uses microfluidic technology. With this approach, evolution can
be carried out in an automated fashion under computer
control, with continuous monitoring of the population
size and precise control over critical parameters such
as mutation frequency and selection pressure. We have
used the microfluidic device to conduct evolution experiments, beginning with a reaction mixture containing
about 1 billion RNA enzymes and carrying out repeated
rounds of RNA catalysis and selective amplification in
an automated fashion. The amount of RNA is monitored
continuously by using a confocal laser fluorescence
microscope. When a predetermined threshold concentration is reached, the computer initiates an automated
dilution and provides a fresh supply of reagents. This
process of selective amplification and dilution was carried out for 70 successive dilutions of 10-fold each during a period of 6.5 hours. The microfluidic system is
now being used to address fundamental questions of
macromolecular evolution, such as the role of genetic
diversity in escaping evolutionary bottlenecks and the
maximum frequency of mutation that can be tolerated
by an evolving population.
THE SCRIPPS RESEARCH INSTITUTE
Nature’s strategies to prepare novel molecules that perform specific functional tasks, such as regulating a gene,
destroying cancer, or catalyzing a reaction with enzymelike efficiency. We hope to apply these novel insights,
technologies, methods, and products to provide solutions
to human diseases, including cancer, HIV disease, and
genetic diseases.
D I R E C T I N G T H E E V O L U T I O N O F C ATA LY T I C F U N C T I O N
Using our concept of reactive immunization, we have
developed antibodies that catalyze aldol as well as
retro-aldol reactions of a wide variety of molecules. The
catalytic proficiency of the best of these antibodies
approaches 1014, a value 1000 times that of the best
catalytic antibodies reported to date and overall the
best of any synthetic protein catalyst. We have shown
the efficient asymmetric synthesis and resolution of a
variety of molecules, including tertiary and fluorinated
aldols, and have used these chiral synthons to synthesize
natural products (Fig. 1). The results highlight the poten-
PUBLICATIONS
Joyce, G.F., Orgel, L.E. Progress toward understanding the origin of the RNA
world. In: The RNA World, 3rd ed. Gesteland, R.F., Cech, T.R., Atkins, J.F. (Eds.).
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2006, p. 23.
Oberhuber, M., Joyce, G.F. A DNA-templated aldol reaction as a model for the formation of pentose sugars in the RNA world. Angew. Chem. Int. Ed. 44:7580, 2005.
Paul, N., Springsteen, G., Joyce, G.F. Conversion of a ribozyme to a deoxyribozyme through in vitro evolution. Chem. Biol. 13:329, 2006.
Studies at the Interface of
Molecular Biology, Chemistry,
and Medicine
C.F. Barbas III, M. Ahmad, K. Albertshofer,
L. Asawapornmongkul, N.S. Chowdari, S. Eberhardy,
R. Fuller, B. Gonzalez, R. Gordley, J. Guo, D.H. Kim,
R.L. Lerner, C. Lund, J. Mandell, S. Mitsumori, W. Nomura,
M. Popkov, D.B. Ramachary, S.S.V. Ramasastry,
L.J. Schwimmer, D. Shabat,* F. Silva, J. Suri, F. Tanaka,
U. Tschulena, N. Utsumi, Y. Ye, Y. Yuan, H. Zhang
* Tel Aviv University, Tel Aviv, Israel
W
e are concerned with problems in molecular
biology, chemistry, and medicine. Many of
our studies involve learning or improving on
F i g . 1 . A variety of compounds synthesized with the world’s
first commercially available catalytic antibody, 38C2, produced at
Scripps Research.
tial synthetic usefulness of catalytic antibodies as artificial
enzymes in addressing problems in organic chemistry
that are not solved by using natural enzymes or more
traditional synthetic methods.
Other advances in this area include the development
of the first peptide aldolase enzymes. By using both
design and selection, we have created small peptide
catalysts that recapitulate many of the kinetic features
of large enzyme catalysts. These smaller enzymes allow
us to address the relationship between the size of natural proteins and the proteins’ catalytic efficiency.
MOLECULAR BIOLOGY
2006
THE SCRIPPS RESEARCH INSTITUTE
209
O R G A N O C ATA LY S I S : A B I O O R G A N I C A P P R O A C H T O
C ATA LY T I C A S Y M M E T R I C S Y N T H E S I S
To further explore the principles of catalysis, we are
studying amine catalysis as a function of catalytic scaffold. Using insights garnered from our studies of aldolase antibodies, we determined the efficacy of simple
chiral amines and amino acids for catalysis of aldol and
related imine and enamine chemistries such as Michael,
Mannich, Knoevenagel, and Diels-Alder reactions.
Although aldolase antibodies are superior catalysts in
terms of the kinetic parameters, these more simple
catalysts are enabling us quantify the importance of
pocket sequestration in catalysis.
Furthermore, many these catalysts are cheap, environmentally friendly, and practical for large-scale synthesis. With this approach, we showed the scope and
usefulness of the first efficient amine catalysts of direct
asymmetric aldol, Mannich, Diels-Alder, and Michael
reactions. The organocatalyst approach is a direct outcome of our studies of catalytic antibodies and provides
an effective alternative to organometallic reactions that
use severe reaction conditions and often-toxic catalysts.
We think that our discovery that simple naturally
occurring amino acids such as L-proline and other amines
can effectively catalyze a variety of enantioselective intermolecular reactions will change the way many reactions
will be performed. As a testament to the mild nature of
this approach, we developed the first catalytic asymmetric Aldol, Mannich, Michael, and fluorination reactions involving aldehydes as nucleophiles. Previously,
such reactions were considered out of the reach of traditional synthetic methods.
In extensions of these concepts, we designed novel
amino acid derivatives that direct the stereochemical
outcome of reactions in ways not possible with proline
(Fig. 2). In other studies, we created the first asymmetric
small-molecule aldol catalysts that are highly effective
with water and seawater as solvent. We think that our
results are also relevant to the prebiotic synthesis of the
molecules of life. For example, we have shown that our
amino acid strategy can be used to synthesize carbohydrates directly, thereby providing a provocative prebiotic route to the sugars essential for life.
THERAPEUTIC ANTIBODIES, IN AND OUT OF CELLS
We developed the first human antibody phage display libraries and the first synthetic antibodies and
methods for the in vitro evolution of antibody affinity.
The ability to manipulate large libraries of human antibodies and to evolve such antibodies in the laboratory
provides tremendous opportunities to develop new medi-
F i g . 2 . Design of the new catalyst (3R,5R)-5-methyl-3-pyrrolidinecarboxylic acid (right) allows efficient access to anti-Mannich
products not accessible through proline catalysis (left).
cines. Laboratories and pharmaceutical companies
around the world now apply the phage display technology that we developed for antibody Fab fragments. In
our laboratory, we are targeting cancer and HIV disease.
One of our antibodies, IgG1-b12, protects animals
against primary challenge with HIV type 1 (HIV-1) and
has been further studied by many researchers. We
improved this antibody by developing in vitro evolution
strategies that enhanced its neutralization activity. By
coupling laboratory-evolved antibodies with potent toxins, we showed that immunotoxins can effectively kill
infected cells.
We are also developing genetic methods to halt HIV
by gene therapy. We created unique human antibodies
that can be expressed inside human cells to make the
cells resistant to HIV infection. In the future, these antibodies might be delivered to the stem cells of patients
infected with HIV-1, allowing the development of a
disease-free immune system that would obviate the
intense regimen of antiviral drugs now required to treat
HIV disease.
Using our increased understanding of antibody-antigen interactions, we extended our efforts in cancer therapy and developed rapid methods for creating human
antibodies from antibodies derived from other species.
We produced human antibodies that should enable us
to selectively starve a variety of cancers by inhibiting
angiogenesis and antibodies that will be used to deliver
radionuclides to colon cancers to destroy the tumors.
We hope that these antibodies will be used in clinical
trials done by our collaborators at the Sloan-Kettering
Cancer Center in New York City.
On the basis of our studies on HIV-1, we used intracellular expression of antibodies directed against angio-
210 MOLECULAR BIOLOGY
2006
THE SCRIPPS RESEARCH INSTITUTE
genic receptors to create a new gene-based approach
to cancer. Our studies indicate that this type of gene
therapy can be successfully applied to the treatment
of cancer.
our approach can become a key tool in selective chemotherapeutic strategies. To see a movie illustrating
this approach, visit http://www.scripps.edu/mb/barbas/
antibody/antibody.mov.
T H E R A P E U T I C A P P L I C AT I O N S O F C ATA LY T I C
ADAPTOR IMMUNOTHERAPY: THE ADVENT OF
ANTIBODIES
CHEMOBODIES
The development of highly efficient catalytic antibodies opens the door to many practical applications.
One of the most fascinating is the use of such antibodies
in human therapy. We think that use of this strategy can
improve chemotherapeutic approaches to diseases such
as cancer and AIDS. Chemotherapeutic regimens are
typically limited by nonspecific toxic effects. To address
this problem, we developed a novel and broadly applicable drug-masking chemistry that operates in conjunction with our unique broad-scope catalytic antibodies.
This masking chemistry is applicable to a wide range
of drugs because it is compatible with virtually any
heteroatom. We showed that generic drug-masking
groups can be selectively removed by sequential retroaldol–retro-Michael reactions catalyzed by antibody
38C2 (Fig. 3). This reaction cascade is not catalyzed
by any known natural enzyme.
We think that combining the chemical diversity of
small synthetic molecules with the immunologic characteristics of antibody molecules will lead to therapeutic
agents with superior properties. Therefore, we developed
a conceptually new device that equips small synthetic
molecules with both the immunologic effector functions
and the long serum half-life of a generic antibody molecule. For a prototype, we developed a targeting device
based on the formation of a covalent bond of defined
stoichiometry between (1) a 1,3-diketone derivative of
an arginine–glycine–aspartic acid peptidomimetic that
targets the integrins αvβ3 and αvβ5 and (2) the reactive
lysine of aldolase antibody 38C2 (Fig. 4). The resulting
F i g . 3 . Targeting cancer and HIV with prodrugs activated by catalytic antibodies. A bifunctional antibody is shown targeting a cancer
cell for destruction. A nontoxic analog of doxorubicin, prodoxorubicin,
is being activated by an aldolase antibody to the toxic form of the drug.
Application of this masking chemistry to the anticancer drugs doxorubicin, camptothecin, and etoposide
produced prodrugs with substantially reduced toxicity.
These prodrugs are selectively unmasked by the catalytic antibody when the antibody is applied at therapeutically relevant concentrations. The efficacy of this
approach has been shown in in vivo models of cancer.
Currently, we are developing more potent drugs and novel
antibodies that will allow us to target breast, colon, and
prostate cancer as well as cells infected with HIV-1.
On the basis of our preliminary findings, we think that
F i g . 4 . Designed small-molecule targeting agents (SCS-873 as
shown) program the specificity of the antibody 38C2 (A). The
resulting chemobodies (cp38C2, B) have characteristics that are
often superior to either those of either the small molecule or the
antibody alone.
complex spontaneously assembled in vitro and in vivo,
selectively retargeted antibody 38C2 to the surface of
cells expressing integrins αvβ3 and αvβ5, dramatically
increased the circulatory half-life of the peptidomimetic,
and effectively reduced tumor growth in animal models
of human Kaposi sarcoma, colon cancer, and melanoma.
ZINC FINGER GENE SWITCHES
The solution to many diseases might be simply turning genes on or off in a selective way. In order to pro-
MOLECULAR BIOLOGY
2006
duce switches that can turn genes on or off, we are
studying molecular recognition of DNA by zinc finger
proteins and methods of creating novel zinc finger DNAbinding proteins (Fig. 5). Because of their modularity
F i g . 5 . A designed polydactyl zinc finger binds 18 bp of DNA. A
single zinc finger domain is highlighted. With this approach, we
can now construct more than a billion gene switches and use them
to specifically turn genes on or off in multiple organisms. Further
elaboration of the approach allows every gene in the genome to be
either turned on or upregulated or downregulated, providing a new
approach to probe gene function across the genome.
and well-defined structural features, zinc finger proteins
are particularly well suited for use as DNA-binding proteins. Each finger forms an independently folded domain
that typically recognizes 3 nucleotides of DNA. We
showed that proteins can be selected or designed that
contain zinc fingers that recognize novel DNA sequences.
These studies are aiding the elucidation of rules for
sequence-specific recognition within this family of proteins. We selected and designed specific zinc finger
domains that will constitute an alphabet of 64 domains
that will allow any DNA sequence to be bound selectively. The prospects for this “second genetic code” are
fascinating and promise a major impact on basic and
applied biology.
We showed the potential of this approach in multiple mammalian and plant cell lines and in whole organisms. With the use of characterized modular zinc finger
domains, polydactyl proteins capable of recognizing an
18-nucleotide site can be rapidly constructed. Our results
suggest that zinc finger proteins might be useful as
genetic regulators for a variety of human aliments and
provide the basis for a new strategy of gene therapy.
Our goal is to develop this class of therapeutic proteins
to inhibit or enhance the synthesis of proteins, providing a direct strategy for fighting diseases of either somatic
or viral origin.
We are also developing proteins that will inhibit
the growth of tumors and others that will inhibit the
THE SCRIPPS RESEARCH INSTITUTE
211
expression of a protein known as CCR5, which is a key
to infection of human cells by HIV-1. We developed an
HIV-1–targeting transcription factor that strongly suppresses HIV-1 replication. Genetic diseases such as
sickle cell anemia are also being targeted with this
approach. Using a library of transcription factors, we
developed a strategy that effectively allows us to turn
on and off every gene in the genome. With this powerful new strategy, we can quickly regulate a target gene
or discover other genes that have a key role in disease.
In the future, we hope to use novel DNA-modifying
enzymes directed by zinc fingers to manipulate chromosomes themselves.
PUBLICATIONS
Alwin, S., Gere, M.B., Guhl, E., Effertz, K., Barbas, C.F. III, Segal, D.J., Weitzman, M.D., Cathomen, T. Custom zinc-finger nucleases for use in human cells.
Mol. Ther. 12:610, 2005.
Blancafort, P., Chen, E.I., Gonzalez, B., Bergquist, S., Zijlstra, A., Guthy, D.,
Brachat, A., Brakenhoff, R.H., Quigley, J.P., Erdmann, D., Barbas, C.F. III.
Genetic reprogramming of tumor cells by zinc finger transcription factors. Proc.
Natl. Acad. Sci. U. S. A. 102:11716, 2005.
Blau, C.A., Barbas, C.F. III, Bomhoff, A.L., Neades, R., Yan, J., Navas, P.A.,
Peterson, K.R. γ-Globin gene expression in chemical inducer of dimerization (CID)dependent multipotential cells established from human β-globin locus yeast artificial chromosome (β-YAC) transgenic mice. J. Biol. Chem. 280:36642, 2005.
Cheong, P.H.-Y., Zhang, H., Thayumanavan, R., Tanaka, F., Houk, K.N., Barbas,
C.F. III. Pipecolic acid-catalyzed direct asymmetric Mannich reactions. Org. Lett.
8:811, 2006.
Corte-Real, S., Collins, C., Aires da Silva, F., Simas, P., Barbas, C.F. III, Chang,
Y., Moore, P., Goncalves, J. Intrabodies targeting the Kaposi sarcoma-associated
herpesvirus latency antigen inhibit viral persistence in lymphoma cells. Blood
106:3797, 2005.
Dreier, B., Fuller, R.P., Segal, D.J., Lund, C.V., Blancafort, P., Huber, A., Koksch, B.,
Barbas, C.F. III. Development of zinc finger domains for recognition of the 5′-CNN3′ family DNA sequences and their use in the construction of artificial transcription
factors. J. Biol. Chem. 280:35588, 2005.
Eberhardy, S.R., Goncalves, J., Coelho, S., Segal, D.J., Berkhout, B., Barbas,
C.F. III. Inhibition of human immunodeficiency virus type 1 replication with artificial transcription factors targeting the highly conserved primer-binding site. J. Virol.
80:2873, 2006.
Lund, C.V., Popkov, M., Magnenat, L., Barbas, C.F. III. Zinc finger transcription
factors designed for bispecific coregulation of ErbB2 and ErbB3 receptors: insights
into ErbB receptor biology. Mol. Cell. Biol. 25:9082, 2005.
Mandell, J., Barbas, C.F. III. Zinc Finger Tools: custom DNA-binding domains for
transcription factors and nucleases. Nucleic Acids Res. 34(Web server
issue):W516, 2006.
Mase, N., Nakai, Y., Ohara, H., Yoda, H., Takabe, K., Tanaka, F., Barbas, C.F. III.
Organocatalytic direct asymmetric aldol reactions in water. J. Am. Chem. Soc.
128:734, 2006.
Mitsumori, S., Zhang, H., Cheong, P.H.-Y., Houk, K.N., Tanaka, F., Barbas, C.F. III.
Direct asymmetric anti-Mannich-type reactions catalyzed by a designed amino
acid. J. Am. Chem. Soc. 128:1040, 2006.
Nathan, S., Rader, C., Barbas, C.F. III. Neutralization of Burkholderia pseudomallei protease by Fabs generated through phage display. Biosci. Biotechnol.
Biochem. 69:2302, 2005.
Popkov, M., Rader, C., Gonzelez, B., Sinha, S.C., Barbas, C.F. III. Small molecule
drug activity in melanoma models may be dramatically enhanced with an antibody
effector. Int. J. Cancer 119:1194, 2006.
212 MOLECULAR BIOLOGY
2006
Suri, J.T., Mitsumori, S., Albertshofer, K., Tanaka, F., Barbas, C.F. III. Dihydroxyacetone variants in the organocatalytic construction of carbohydrates: mimicking
tagatose and fuculose aldolases. J. Org. Chem. 71:3822, 2006.
Suri, J.T., Steiner, D.D., Barbas, C.F. III. Organocatalytic enantioselective synthesis
of metabotropic glutamate receptor ligands. Org. Lett. 7:3885, 2005.
Swan, C.H., Buhler, B., Tschan, M.P., Barbas, C.F. III, Torbett, B.E. T-cell protection and enrichment through lentiviral CCR5 intrabody gene delivery. Gene Ther.
13:1408, 2006.
Tan, W., Dong, Z., Wilkinson, T.A., Barbas, C.F. III, Chow, S.A. Human immunodeficiency virus type 1 incorporated with fusion proteins consisting of integrase and
the designed polydactyl zinc finger protein E2C can bias integration of viral DNA
into a predetermined region in human cells. J. Virol. 80:1939, 2006.
Tanaka, F., Barbas, C.F. III. Enamine-based reactions using organocatalysts: from
aldolase antibodies to small amino acid and amine catalysts. J. Synth. Org. Chem.
Jpn. 63:27, 2005.
Tanaka, F., Fuller, R., Barbas, C.F. III. Development of small designer aldolase
enzymes: catalytic activity, folding, and substrate specificity. Biochemistry
44:7583, 2005.
Weinstain, R., Lerner, R.A., Barbas, C.F. III, Shabat, D. Antibody-catalyzed asymmetric intramolecular Michael addition of aldehydes and ketones to yield the disfavored cis-product. J. Am. Chem. Soc. 127:13104, 2005.
Synthetic Enzymes, Catalytic
Antibodies, Ozone Scavengers in
Asthma, Organometallic
Chemistry, and Biomolecular
Computing
E. Keinan, O. Reany, C.H. Lo, S. Bauer, N. Metanis,
E. Kossoy, M. Soreni, R. Piran, M. Sinha, I. Ben-Shir,
T. Ratner, T. Shekhter, T. Mejuch, E. Solel
e focus on synthetically modified enzymes,
antibody-catalyzed reactions, anticancer and
antiasthma agents, and biomolecular computation, as illustrated in the following examples.
W
SYNTHETIC ENZYMES
Efforts to generate new enzymatic activities from
existing protein scaffolds may not only provide biotechnologically useful catalysts but also lead to better
understanding of the natural process of evolution. We
profoundly changed the catalytic activity and mechanism of the enzyme 4-oxalocrotonate tautomerase by
means of rationally designed synthetic mutations. For
example, a single amino acid substitution that corresponds to a mutation in a single base pair led to a
dramatic change in the catalytic activity. Although the
wild-type enzyme catalyzes only the tautomerization of
4-oxalocrotonate, the mutant P1A catalyzes both the
THE SCRIPPS RESEARCH INSTITUTE
original tautomerization reaction via a general acid-base
mechanism and the decarboxylation of oxaloacetate via
a nucleophilic mechanism.
We also showed that the electrostatic manipulation
of an enzyme’s active site can alter the substrate specificity of the enzyme in a predictable way. We replaced
1, 2, or all 3 active-site arginine residues with citrulline
analogs to maintain the steric features of the active site
of 4-oxalocrotonate tautomerase while changing its electronic properties. These synthetic changes revealed that
the wild-type enzyme binds the natural substrate predominantly through electrostatic interactions. This and
other mechanistic insights led to the design of a modified enzyme that was specific for a new substrate that
had different electrostatic properties and that bound the
enzyme via hydrogen-bonding complementarity rather
than electrostatic interactions. This research on synthetic
enzymes is being done in collaboration with P.E. Dawson,
Department of Cell Biology.
C ATA LY T I C A N T I B O D I E S
Engineering herbicide resistance in crops facilitates
control of weed species, particularly weeds that are
genetically related to the crop, and may be useful in
selecting lines that have undergone multiple transformation events. We showed that herbicide-resistant plants
can be engineered by designing both a herbicide and
a catalytic antibody that destroys the herbicide within
the plants. First, we developed a carbamate herbicide
that can be catalytically destroyed by the aldolase antibody 38C2. Then we targeted the light chain and half
of the heavy chain (Fab) of the catalytic antibody to the
endoplasmic reticulum in 2 lines of Arabidopsis thaliana
transformants. Finally, we crossed the 2 transgenic plants
to produce a herbicide-resistant F1 hybrid (Fig. 1). Our
results suggest that in vivo expression of catalytic antibodies could become a general strategy to achieve
phenotype modifications not only in plants but also
in other organisms.
O Z O N E S C AV E N G E R S A N D A N T I A S T H M A A C T I V I T Y
A new hypothesis we proposed on the mechanism
of asthmatic inflammation has led to an ozone-scavenging compound that prevents bronchial obstruction in rats
with asthma. Previously, scientists at Scripps Research
discovered that ozone can be generated not only via the
antibody-mediated water oxidation pathway but also
by antibody-coated activated white blood cells during
inflammatory processes. This finding led us to speculate that the pulmonary inflammation in asthma might
be caused by ozone production by white blood cells in
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2006
F i g . 1 . Influence of herbicide (1) on the rooting and development
of seedlings of F1 hybrids and control A thaliana plants. The control
plants are shown in A and C; the hybrid plant lines (F1) expressing
both light and heavy chains of the catalytic antibody 38C2, in B
and D. Plantlets grown on medium without the herbicide are shown
in A and B; those grown with the herbicide are shown in C and D.
lungs and that inhalation of electron-rich olefins, which
are known ozone scavengers, might have antiasthmatic
effects. In experiments in rats, inhalation of such a
compound, limonene, caused a significant improvement
in signs of asthma. These results could have consequences in the management of asthma.
O R G A N O M E TA L L I C C H E M I S T R Y
Rhenium oxide, which is known primarily as a strong
oxidant, is a highly selective Lewis acid catalyst that
affects the heteroacylative dimerization of tetrahydrofuran at room temperature. This multicomponent reaction, which involves tetrahydrofuran, trifluoroacetic
anhydride, and a carboxylic acid, produces a nonsymmetrical diester (compound 3 in Fig. 2) in high yields.
The proposed catalytic cycle (Fig. 2) involves a multistep sequence of nucleophilic attacks, metal-oxygen
bond metathesis, and electrophilic cleavage by trifluoroacetic anhydride. This synthetically useful reaction
highlights the unique, frequently avoided Lewis acidity
of transition-metal oxides.
In study with the platinum complex TpPt(CO)CH3
(Tp = hydridotrispyrazolylborate), we found that the
proton exchange between water and the methyl group
involves the formation and deprotonation of a “sticky”
σ-methane ligand. The efficiency of this nontrivial process is attributed to the spatial orientation of functional
groups that operate in concert to achieve a multistep
proton walk. The key role played by the free pyrazolyl
nitrogen, acting as a proton carrier, is reminiscent of
the dual functionality of the histidine in the catalytic
triad of natural serine proteases.
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213
F i g . 2 . The catalytic cycle of the rhenium oxide–catalyzed heteroa-
cylative dimerization of tetrahydrofuran (THF), which is proposed on
the basis of isotope-labeling experiments, starts with an attack of a
rhenium oxo ligand on a coordinated tetrahydrofuran, then an attack
of the resultant alkoxide ligand on a second coordinated tetrahydrofuran, nucleophilic addition of the resultant alkoxide ligand to the
coordinated carboxylic acid, and finally, electrophilic cleavage of
the other coordinated alkoxide by trifluoroacetic anhydride (TFAA).
BIOMOLECULAR COMPUTING DEVICES
Previously, we described a programmable finite
automaton with 2 symbols and 2 states that computed
autonomously. All of the components of the device,
including hardware, software, input, and output, were
biomolecules mixed together in solution. The hardware
consisted of a restriction nuclease and a ligase; the
software (transition rules) and the input were doublestranded DNA oligomers. Computation was carried out
by processing the input molecule via repetitive cycles
of restriction, hybridization, and ligation reactions to
produce a final-state output in the form of a doublestranded DNA molecule.
More recently, we markedly increased the levels of
complexity and mathematical power of these automata
by the design of a 3-state–3-symbol automaton, thus
increasing the number of syntactically distinct programs
from 765 to 1 billion. We have further amplified the
applicability of this design by using surface-anchored
input molecules and surface plasmon resonance technology to monitor the computation steps in real time.
This technology allowed parallel computation with DNA
chips that carry multiple input molecules and can be
used as pixel arrays for image encryption.
PUBLICATIONS
Lo, H.C., Han, H., D‚Souza, L.J., Sinha, S.C., Keinan, E. Rhenium(VII) oxide-catalyzed heteroacylative ring-opening dimerization of tetrahydrofuran. J. Am. Chem.
Soc., in press.
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Lo, H.C., Iron, M.A. Martin, J.M.L., Keinan, E. Proton walk in the aqueous platinum complex TpPtMeCO via a sticky σ-methane ligand. Chem. Eur. J., in press.
Metanis, N., Keinan, E., Dawson, P.E. Synthetic seleno-glutaredoxin 3: highly reducing oxidoreductases with enhanced catalytic efficiency. J. Am. Chem. Soc., in press.
Tuttle, T., Keinan, E., Thiel, W. Understanding the enzymatic activity of 4-oxalocrotonate tautomerase and its mutant analogues: a computational study. J. Phys.
Chem. B Condens. Matter Mater. Surf. Interfaces Biophys. 110:19685, 2006.
Weiss, Y., Rubin, B., Shulman, A., Ben Shir, I., Keinan, E., Wolf, S. Determination of plant resistance to carbamate herbicidal compounds inhibiting cell division
and early growth by seed and plantlets bioassays. Nat. Protoc., in press.
Weiss, Y., Shulman, A., Ben Shir, I., Keinan, E., Wolf, S. Herbicide-resistance
conferred by expression of a catalytic antibody in Arabidopsis thaliana. Nat.
Biotechnol. 24:713, 2006.
Functional Characterization of
Proteases via Combinatorial
Libraries
J.L. Harris, J. Alves*
ith the complete sequencing of genomes
from multiple organisms, information on the
repertoire of genes can be readily established.
However, large gaps still remain in our knowledge of
the biological role of most genes. These gaps are mainly
due to the fact that most biological functions are regulated not at the gene or transcript level, but at the
posttranslational level. In contrast to the situation in
genomics, in which the changes in the content or amount
of cellular DNA or RNA can be readily examined, monitoring translational and posttranslational dynamics of
functional proteins on a genome-wide level is more
difficult. Progress in our current understanding of biological processes is limited by the available tools that
can be used to probe function at the posttranslational
level. We are developing and applying technologies
based on small-molecule protein modifiers to profile
the active state of enzymes.
In collaboration with N. Winssinger, Université
Louis Pasteur, Strasbourg, Germany, we have developed an encoding strategy that uses peptide nucleic
acid (PNA) sequences. Encoding combinatorial libraries
with PNA tags allows not only for the synthetic history
of the library to be captured in the resulting molecule
but also for spatial deconvolution of the molecules on
DNA microarrays.
Using this technology, we have created encoded
protease inhibitor and substrate libraries of thousands
of molecules. These libraries have been applied to var-
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THE SCRIPPS RESEARCH INSTITUTE
ious biological systems and have resulted in the identification and characterization of proteases within those
systems. For example, a cysteine protease from the
house dust mite Dermatophagoides pteronyssinus
was identified by using a 4000-member PNA-encoded
inhibitor library. The identified protease plays a key
role in allergic hypersensitivity through the selective
degradation of CD25 from T cells.
Another example of functional characterization of
protein activity is the profiling of the substrate specificity of proteases from Dengue virus, the etiologic agent
of dengue fever, dengue hemorrhagic fever, and dengue
shock syndrome. Using 2 substrate libraries of approximately 160,000 members, we characterized the structure-activity relationship of the NS3 protease from the
4 Dengue virus serotypes and facilitated the development of inhibitors of the virus.
PUBLICATIONS
Harris, J.L. Protease substrate profiling. In: Enzyme assays: high-throughput
screening, genetic selection and fingerprinting. Reymond, J.-L. (Ed.). Wiley-VCH,
New York, 2006, p. 303.
Harris, J.L., Winssinger, N. PNA encoding (PNA = peptide nucleic acid): from
solution-based libraries to organized microarrays. Chem. Eur. J. 11:6792, 2005.
Li, J., Lim, S.P., Beer, D., Patel, V., Wen, D., Tumanut, C., Tully, D.C., Williams,
J.A., Jiricek, J., Priestle, J.P., Harris, J.L., Vasudevan, S.G. Functional profiling of
recombinant NS3 proteases from all four serotypes of Dengue virus using tetrapeptide and octapeptide substrate libraries. J. Biol. Chem. 280:28766, 2005.
Petrassi, H.M., Williams, J.A., Li, J., Tumanut, C., Ek, J., Nakai, T., Masick, B.,
Backes, B.J., Harris, J.L. A strategy to profile prime and non-prime proteolytic
substrate specificity. Bioorg. Med. Chem. Lett. 15:3162, 2005.
Winssinger, N., Harris, J.L. Microarray-based functional protein profiling using
peptide nucleic acid-encoded libraries. Expert Rev. Proteomics 2:937, 2005.
Organic Synthesis and Selective
Drug Delivery
S.C. Sinha, R.A. Lerner, Z. Chen, S. De, S. Das, S. Abraham,
F. Guo
ur main research interests are synthesis of biologically important natural and nonnatural molecules,
synthetic methods, and antibody catalysis in
organic synthesis and selective drug delivery. During
the past year, we focused on 3 different classes of
compounds: the anticancer adjacent bis-tetrahydrofuran
annonaceous acetogenins, the antibacterial macrocyclic
lactones sorangiolides, and nonnatural small-molecule
drugs that target G protein–coupled receptors. In our
work on antibody catalysis, we developed a proadapter
approach for production of the chemically programmed
O
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2006
aldolase antibody 38C2 and new doxorubicin prodrugs
that are catalyzed by antibody 38C2 faster than the
previously reported drugs are.
S E L E C T I V E C H E M O T H E R A P Y W I T H C ATA LY T I C
ALDOLASE ANTIBODIES
For selective chemotherapy, we intend to develop
drug conjugates and prodrugs that will target cell-surface receptors, such as the glycoprotein integrins αvβ3
and α v β 5 . These integrins are directly implicated in
tumor angiogenesis; they are overexpressed in the vasculature of angiogenic tumors and in numerous cancer
cells but are less expressed on quiescent blood vessels.
Using antibody 38C2 and small-molecule antagonists
of αvβ3 and αvβ5 (or a targeting agent), we developed
antagonist-38C2 conjugates, also known as chemically
programmed 38C2 (Fig. 1).
F i g . 1 . Schematic drawings of the diketone and the proadapter
strategies used to produce chemically programmed antibody constructs that target cells expressing the integrins αvβ3 and αvβ5.
Abbreviations: Ab, antibody; TA, targeting agent.
The conjugation between the targeting agents and
the antibody takes place in the binding sites though
the diketone or vinyl ketone linkers. Because the vinyl
ketones are highly reactive, we used the corresponding
acetone adduct as the prolinker, which undergoes 38C2catalyzed reaction to produce the active linker before
the active linker reacts. This strategy has been termed
the proadapter approach. The conjugates prepared by
both approaches bound efficiently to cells expressing
αvβ3 and αvβ5, including human breast cancer cell
lines MDA-MB-435 and MDA-MB-231, and inhibited
the growth of both the primary tumors and secondary
metastasis in distant organs.
Development of these strategies for the formation
of antibody constructs can have a large effect on the
treatment of various diseases, including cancer.
In the alternative approach, we are developing prodrugs that can be efficiently activated by the aldolase
antibodies 38C2 and 93F3. These antibodies will be
targeted to tumor cells or the tumor vasculature by using
antagonists of αvβ3 and αvβ5. In the past year, we devel-
THE SCRIPPS RESEARCH INSTITUTE
215
oped new doxorubicin prodrugs that are not only more
stable than the previously reported analogous prodrugs
but also activated faster by using antibody 38C2. We
also produced 38C2-antagonist conjugates. The conjugates bound efficiently to MDA-MB-231 cells expressing
αvβ3 and αvβ5. We also found that the modified antibody can activate new doxorubicin prodrugs.
Therefore, we have all the tools to investigate prodrug therapy in animal models. The selective chemotherapy studies are carried out in collaboration with
C.F. Barbas, Department of Molecular Biology, and
B. Mueller, La Jolla Institute for Molecular Medicine,
San Diego, California.
S Y N T H E S I S O F N AT U R A L A N D N O N N AT U R A L S M A L L
MOLECULES
Total synthesis of naturally occurring and biologically
important compounds is important not only for confirming their structures but also for producing the compounds and their analogs for comprehensive biological
evaluations. To synthesize these compounds, we are
developing methods that involve both antibody catalysis and common synthetic routes. In the past year, in
addition to synthesizing sorangiolides, which are naturally occurring macrocyclic lactones, and the library of
bis-tetrahydrofuran annonaceous acetogenins, we focused
on small-molecule nonnatural ligands of G protein–coupled receptors. The studies on the synthesis of these
ligands are carried out in collaboration with E. Roberts,
Department of Chemistry.
Sorangiolides (Fig. 2) are weakly active antibacterial compounds. Our goal is to synthesize the highly
F i g . 2 . Structure of sorangiolides A and B (top) and a general
structure of bis-tetrahydrofuran annonaceous acetogenins (bottom).
active sorangiolide analogs. Thus, we have developed
synthetic routes that can provide the macrocyclic structure of sorangiolides. Using an intermediate, we will
synthesize both the natural and nonnatural molecules.
For other bis-tetrahydrofuran acetogenins, which are
among the most active cancer agents and are toxic to
several human cancer cell lines at much lower concen-
216 MOLECULAR BIOLOGY
2006
trations than doxorubicin is, we developed methods that
can provide all the stereoisomers of asimicin and bullatacin. The new methods involve a bidirectional approach.
Now, we are pursuing synthesis of the 64 stereoisomers of asimicin and bullatacin.
PUBLICATIONS
Das, S., Li, L.-S., Abraham, S., Chen, Z., Sinha, S.C. A bidirectional approach to
the synthesis of a complete library of adjacent-bis-THF annonaceous acetogenins.
J. Org. Chem. 70:5922, 2005.
Li, L.-S., Babendure, J.L., Sinha, S.C., Olefsky, J.M., Lerner, R.A. Synthesis and
evaluation of photolabile insulin prodrugs. Bioorg. Med. Chem. Lett. 15:3917, 2005.
Popkov, M., Rader, C., Gonzalez, B., Sinha, S.C., Barbas, C.F. III. Small molecule
drug activity in melanoma models may be dramatically enhanced with an antibody
effector. Int. J. Cancer 119:1194, 2006.
Structure, Function, and
Applications of Virus Particles
J.E. Johnson, M. Banerjee, A. Chatterji, Z. Chen, I. Gertsman,
R. Huang, R. Khayat, G. Lander, J. Lanman, K.K. Lee,
T. Matsui, P. Natarajan, A. Odegard, J. Speir
e investigate model virus systems that provide
insights for understanding assembly, maturation, entry, localization, and replication. We
have also developed viruses as reagents for applications
in nanomedicine, chemistry, and biology. We investigate viruses that infect bacteria, insects, plants, and
the extreme thermophile Sulfolobus. These viruses
have genomes of single-stranded RNA and doublestranded DNA.
We use a variety of physical methods to investigate
structure-function relationships, including single-crystal
and static and time-resolved solution x-ray diffraction,
electron cryomicroscopy and image reconstruction, mass
spectrometry, structure-based computational analyses,
and methods associated with thermodynamic characterization of virus particles and their transitions. Biological methods we use include genetic engineering of
viral genes and their expression in Escherichia coli,
mammalian cells, insect cells, and yeast and the characterization of these gene products by physical methods.
For cytologic studies of viral entry and infection, we
use fluorescence and electron microscopy and particles
assembled in heterologous expression systems. Our
studies depend on extensive consultations and collaborations with others at Scripps Research, including
groups led by C.L. Brooks, D.A. Case, B. Carragher,
M.G. Finn, M. Manchester, D.R. Millar, R.A. Milligan,
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THE SCRIPPS RESEARCH INSTITUTE
C. Potter, V. Reddy, A. Schneemann, G. Siuzdak, J.R.
Williamson, and M.J. Yeager, and a variety of groups
outside of Scripps.
DOUBLE-STRANDED DNA VIRUSES
HK97 is a double-stranded DNA virus similar to
phage λ. It undergoes a remarkable morphogenesis in
its assembly and maturation, and this process can be
recapitulated in vitro. We determined the atomic resolution structure of the 650-Å mature, head II particle and
discovered the mechanism used to concatenate the subunits of the particle into a chain-mail fabric similar to
that seen in armor of medieval knights. In the past year,
we focused on the dynamics of maturation.
Prohead II is a 500-Å metastable intermediate at
pH 7 that can be induced to begin maturation by lowering the pH to 4. Solution x-ray scattering and single-molecule fluorescence showed that the initial transition to
a particle of about 560 Å occurs as a highly cooperative, stochastic event with no detectable intermediates
that takes place in less than 1 second for an individual particle. A quorum of cross-links must form in this
particle to generate the second expansion intermediate
(about 650 Å), which also forms cooperatively with no
detectable intermediates. At pH 4, formation of crosslinks continues, with 360 formed per particle. Limited
pentamer dynamics (established from crystallography
and electron cryomicroscopy) prevents the last 60 crosslinks from forming, but pentamer trajectories extend
at pH 7, allowing these cross-links to form, completing maturation.
Bacteriophage P22 is the prototype of the Podoviridae, which are characterized by a T = 7 capsid with
a short tail structure incorporated into a unique 5-fold
vertex. We determined an asymmetric reconstruction
of this particle that revealed spooled DNA, the dodecameric portal, and the location of the 9 gene products
known to be in the particle.
Sulfolobus turreted icosahedral virus is an archaeal
virus isolated from Sulfolobus, which grows in the acidic
hot sulfur springs (pH 2–4, 72°C–92°C) in Yellowstone
National Park. An electron cryomicroscopy reconstruction
of the virus showed that the capsid has pseudo T = 31
quasi symmetry and is 1000 Å in diameter, including
the pentons. We solved the x-ray structure of the major
capsid protein of the virus, and it revealed a fold nearly
identical to the major capsid proteins of the eukaryotic
adenoviruses and PRD-1, a virus that infects bacteria.
These findings indicate a virus phylogeny that spans
the 3 domains of life. Difference electron density maps
in which the x-ray model is subtracted from the elec-
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2006
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217
tron cryomicroscopy density clearly shows an internal
membrane in which the capsid proteins are anchored.
Ochoa, W., Chatterji, A., Lin, T., Johnson, J.E. Generation and structural analysis of
reactive empty particles derived from an icosahedral virus. Chem. Biol. 13:771, 2006.
SINGLE-STRANDED RNA VIRUSES
Prasad, T., Turner, M., Falkner, J., Mittleman, D., Johnson, J.E., Lin, T., Colvin, V.
Nanostructured virus crystals for x-ray optics. IEEE Trans. Nanotechnol. 5:93, 2006.
Flock House virus is a T = 3, single-stranded RNA
virus that infects Drosophila. We are studying viral entry
and early expression and assembly of the capsid protein.
Recently, studies on viral entry indicated the presence
of an “eluted” particle early in infection that has initiated
its disassembly program but is then eluted back into the
medium. We did a phenotypic characterization of the
particles, and we are using electron cryomicroscopy
to study them. For studies on the expression and assembly of the capsid protein, we are using tags inserted
genetically in the capsid protein that allow the freshly
made proteins to be optically visualized with a fluorophore and in the electron microscope with photoconversion of the fluorophore. Recently, high-pressure freezing
of infected cells revealed exceptionally detailed features
of viral entry and regions of replication within the cell.
Refined atomic models of tetravirus structures and
structure-based mutagenesis combined with highly
sensitive assays for defining phenotypes have revealed
the electrostatic principals of maturation for the
T = 4 tetraviruses.
Cowpea mosaic virus is a 30-nM reagent that we
use for chemistry and nanomedicine. We found that
particles of the virus with doxorubicin bound internally
can be specifically targeted to tumor cells via peptides
on the viral surface that recognize receptors for vascularization signals that are highly expressed on tumor cells.
PUBLICATIONS
du Plessis, L., Hendry, D.A., Dorrington, R.A., Hanzlik, T.N., Johnson, J.E., Appel,
M. Revised RNA2 sequence of the tetravirus nudaurelia capensis ω virus (NωV):
annotated sequence record. Arch. Virol. 150:2397, 2005.
Khayat, R., Tang, L., Larson, E.T., Lawrence, C.M., Young., M., Johnson, J.E. Structure of an archaeal virus capsid protein reveals a common ancestry to eukaryotic and
bacterial viruses. Proc. Natl. Acad. Sci. U. S. A. 102:18944, 2005.
Lander, G.C., Tang, L., Casjens, S.R., Gilcrease, E.B., Prevelige, P., Poliakov, A.,
Potter, C.S., Carragher, B., Johnson, J.E. The structure of an infectious P22 virion
shows the signal for headful DNA packaging. Science 312:1791, 2006.
Lee, K.K., Tsuruta, H., Hendrix, R.W., Duda, R.L., Johnson, J.E. Cooperative reorganization of a 420 subunit virus capsid. J. Mol. Biol. 352:723, 2005.
Reddy, V.S., Johnson, J.E. Structure-derived insights into virus assembly. Adv.
Virus Res. 64:45, 2005.
Sapsford, K.E., Soto, C.M., Blum, A.S., Chatterji, A., Lin, T., Johnson, J.E.,
Ligler, F.S., Ratna, B.R. A cowpea mosaic virus nanoscaffold for multiplexed antibody conjugation: application as an immunoassay tracer. Biosens. Bioelectron.
21:1668, 2006.
Shepherd, C.M., Borelli, I.A., Lander, G., Natarajan, P., Siddavanahalli, V., Bajaj,
C., Johnson, J.E., Brooks, C.L. III, Reddy, V.S. VIPERdb: a relational database for
structural virology. Nucleic Acids Res. 34:386, 2006.
Soto, C.M., Blum, A.S., Vora, G.J., Lebedev, N., Meador, C.E., Won, A.P., Chatterji, A., Johnson, J.E., Ratna, B.R. Fluorescent signal amplification of carbocyanine
dyes using engineered viral nanoparticles. J. Am. Chem. Soc. 128:5184, 2006.
Speir, J.A., Bothner, B., Qu, C., Willits, D.A., Young, M.J., Johnson, J.E. Enhanced
local symmetry interactions globally stabilize a mutant virus capsid that maintains
infectivity and capsid dynamics J. Virol. 80:3582, 2006.
Tang, J., Johnson, J.M., Dryden, K.A., Young, M.J., Zlotnick, A., Johnson, J.E.
The role of subunit hinges and molecular “switches” in the control of viral capsid
polymorphism. J. Struct. Biol. 154:59, 2006.
Tang, L., Gilcrease, E.B., Casjens, S.R., Johnson, J.E. Highly discriminatory binding
of capsid-cementing proteins in bacteriophage L. Structure 14:837, 2006.
Taylor, D.J., Speir, J.A., Reddy, V., Cingolani, G., Pringle, F.M., Ball, L.A., Johnson,
J.E. Preliminary x-ray characterization of authentic providence virus and attempts to
express its coat protein gene in recombinant baculovirus. Arch. Virol. 151:155, 2006.
Walukiewicz, H.E., Johnson, J.E., Schneemann, A. Morphological changes in the
T = 3 capsid of Flock House virus during cell entry. J. Virol. 80:615, 2006.
Wikoff, W.R., Conway, J.F., Tang, J., Lee, K.K., Gan, L., Cheng, N., Duda, R.L.,
Hendrix, R.W., Steven, A.C., Johnson, J.E. Time-resolved molecular dynamics of
HK97 capsid maturation interpreted by electron cryo-microscopy and x-ray crystallography. J. Struct. Biol. 153:300, 2006.
Nanomanufacturing on an
Icosahedral Scaffold and
Neutralization of Avian H5N1
Influenza Viruses
T. Lin, J.E. Johnson, A. Censullo, A. Chatterji
MOLECULAR ELECTRONICS ON AN ICOSAHEDRAL
SCAFFOLD
Lin, T., Lomonossoff, G.P., Johnson, J.E. Structure-based engineering of an icosahedral virus for nanomedicine and nanotechnology. In: Nanotechnology in Biology
and Medicine: Methods, Devices, and Applications. Vo-Dinh. T. (Ed.). CRC Press,
Boca Raton, FL, in press.
Medintz, I.L., Sapsford, K.E., Konnert, J.H., Chatterji, A., Lin, T., Johnson, J.E.,
Mattoussi, H. Decoration of discretely immobilized cowpea mosaic virus with luminescent quantum dots. Langmuir 21:5501, 2005.
Natarajan, P., Lander, G.C., Shepherd, C.M., Reddy, V.S., Brooks, C.L. III, Johnson, J.E. Exploring icosahedral virus structures with VIPER. Nat. Rev. Microbiol.
3:809, 2005.
Molecular manufacturing, the essence of nanotechnology, involves the manipulation of molecules as the
self-assembling components at the nanometer scale to
build devices in mesoscale. Although small molecules
with novel electronic properties can be synthesized, making functional connectivity among the different components in designed patterns is generally difficult. In
contrast, biological macromolecules are more amenable
218 MOLECULAR BIOLOGY
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for self-assembly because of their versatility, programmability through genetic engineering, and propensity
to form arrays and can be used either directly as devices
or as scaffolds for patterning small molecules.
We have shown that cowpea mosaic virus (CPMV),
an icosahedral plant virus, can be used as the template
for nanochemistry by introducing unique cysteine residues
and exploiting the native lysine residues. In collaborative
studies with B.R. Ratna, Naval Research Laboratory,
Washington, D.C., the virus capsid was exploited as a
nano circuit board, and the reactive groups were used as
anchoring points for the assembly of the electronic molecules oligophenylene-vinylene and 1,4-C6H 4[trans-(4AcSC6H 4≡CPt(Pbu3)2≡C] 2. The establishment of the
molecular network was shown by measuring electronic
conductance with scanning tunnel microscopy.
THE SCRIPPS RESEARCH INSTITUTE
analysis of escape mutants in conjunction with the
vaccine development. Structural studies of antibody
interactions with the H5N1 viruses are also being carried out to shed light on the mechanism of neutralization of the viruses.
PUBLICATIONS
Medintz, I.L., Sapsford, K.E., Konnert, J.H., Chatterji, A., Lin, T., Johnson, J.E.,
Mattoussi, H. Decoration of discretely immobilized cowpea mosaic virus with luminescent quantum dots. Langmuir 21:5501, 2005.
Prasad, T., Turner, M., Falkner, J., Mittleman, D., Johnson, J.E., Lin, T., Colvin, V.
Nanostructured virus crystals for x-ray optics. IEEE Trans. Nanotechnol., in press.
Design and Informatics in
Structural Virology
N E U T R A L I Z AT I O N O F AV I A N H 5 N 1 I N F L U E N Z A
V.S. Reddy, S. Kumar, M. Tripp, P. Singh, R. Mannige,
VIRUSES
I. Borelli, J. Loo, C.L. Brooks III, J.E. Johnson,
Influenza is one of the most important viral diseases in humans. It has caused morbidity and mortality
in millions of people in frequent epidemics and pandemics throughout the centuries. Human influenza virus
is typically associated with 3 H subtypes: H1, H2, and
H3. In recent years, an avian H5 (H5N1) influenza
virus crossed the species barrier to infect humans with
high virulence. To date, the avian virus has not been
efficient in transmission from human to human, and
the disease has not spread in the human population.
However, the continuous circulation and spreading of
H5N1 viruses in avian species across the globe leads
to more human infections and increases the likelihood
that the virus will acquire the necessary characteristics for efficient human-to-human transmission through
genetic mutation or reassortment with a prevailing
human influenza A virus.
The possible emergence of an H5N1 virus highly
contagious to humans is a serious pandemic threat.
Therefore, producing effective vaccines to counter the
threat posed by the H5N1 viruses is important. CPMV
is an effective scaffold for the development of subunit
vaccines. We are developing a novel combinatorial strategy in which the CPMV system is used to identify vaccine candidates.
In another study in collaboration with scientists in
Hong Kong and Southern China, the epicenter of the
influenza outbreaks, we have produced more than 100
monoclonal antibodies against the avian influenza viruses
and have shown that many of these antibodies are neutralizing. These neutralizing antibodies are used in the
M. Manchester, G. Nemerow, A. Schneemann
e are interested in understanding the structural
underpinnings and requirements for formation
and function of viral capsids and in designing
novel protein shells that polyvalently display molecules
of interest. To this end, we use structural, computational, informatics, and genetic methods.
Viruses are highly evolved macromolecular machines
that perform a variety of functions during their life cycle,
including selective packaging of the genome, self-assembly into uniform capsids, binding to host cells, and delivery of the genome to the targeted cells. Simple viruses,
such as nonenveloped viruses, form closed protein shells
of uniform size and character by the self-association of
structural and functional components: proteins and the
nucleic acid genome. Hence, these viruses are useful for
structural and functional analyses.
In collaboration of with G.R. Nemerow, Department
of Immunology, we are using x-ray crystallographic
methods to determine the structure of the entire
human adenovirus particle, currently at about 9-Å resolution. We are continuing to collect diffraction data
at higher resolution. We continue to maintain and
expand the virus structure database, namely VIPERdb
(http://viperdb.scripps.edu), where the coordinates of the
characterized spherical capsid structures are stored and
organized in terms of viral taxonomy and capsid architecture. We are developing structural analysis tools to
“mine” the capsid structures in terms of protein-protein
interactions, contacting residue pairs, association ener-
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MOLECULAR BIOLOGY
2006
gies, contributions of individual residues, and surface
characteristics. VIPERdb is being developed as part
of the Multiscale Modeling Tools for Structural Biology,
a National Institutes of Health research resource headed
by C.L. Brooks, Department of Molecular Biology. In
addition, on the basis of the structural similarity that
occurs within a virus family, we are building homology
models for the uncharacterized members of virus families. These models will be useful for molecular virologists investigating structural and functional relationships
in viruses.
We are generating decoys of pathogenic molecules
on the surfaces of viral capsids that can be used as
vaccines against cytotoxins such as ricin. Currently,
tomato bushy stunt virus–like capsids are our display
platform of choice; the platform consists of multiple
copies of a 2-domain capsid protein subunit with the
C-terminal P-domain exposed on the surface. Such a
unique subunit structure is useful for attaching peptides
or proteins of interest at the end of the C terminus of
the capsid protein or for replacing the external P-domain
with the proteins of interest rather than inserting them
in a loop.
PUBLICATIONS
Hsu, C., Singh, P., Ochoa, W., Manayani, D.J., Manchester, M., Schneemann, A.,
Reddy, V.S. Characterization of polymorphism displayed by the coat protein
mutants of tomato bushy stunt virus. Virology 349:222, 2006.
Natarajan, P., Lander, G.C., Shepherd, C.M., Reddy, V.S., Brooks, C.L. III, Johnson, J.E. Exploring icosahedral virus structures with VIPER. Nat. Rev. Microbiol.
3:809, 2005.
Shepherd, C.M., Borelli, I.A., Lander, G., Natarajan, P., Siddavanahalli, V., Bajaj,
C., Johnson, J.E., Brooks, C.L. III, Reddy, V.S. VIPERdb: a relational database for
structural virology. Nucleic Acids Res. 34(Database Issue):D386, 2006.
Taylor, D.J., Speir, J.A., Reddy, V., Cingolani, G., Pringle, F.M., Ball, L.A., Johnson, J.E. Preliminary x-ray characterization of authentic providence virus and
attempts to express its coat protein gene in recombinant baculovirus. Arch. Virol.
151:155, 2006.
Biology and Applications of
Icosahedral Virus Capsids
A. Schneemann, B. Groschel, C. Hsu, J. Lee, D.J. Manayani,
D. Marshall, J.E. Petrillo, M.E. Siladi, P.A. Venter
oat proteins of nonenveloped, icosahedral viruses
perform multiple functions during the course of
viral infection, including capsid assembly, specific
encapsidation of the viral genome, binding to a cellular receptor, and uncoating. In some viruses, a single
type of protein is sufficient to carry out these functions;
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219
we are interested in the determinants that endow a
polypeptide chain with such versatility. We seek to
harness this versatility for novel applications of viruses
in biotechnology and nanotechnology.
We focus on a structurally and genetically well-characterized virus family, the T = 3 nodaviruses. Nodaviruses are composed of 180 copies of a single coat
protein and 2 strands of positive-sense RNA. Currently,
we are elucidating the mechanism by which the 2
genomic RNAs are packaged into a single virion. Our
long-term goal is to develop nodaviruses as RNA packaging and delivery vectors. Our data indicate that the
2 viral RNAs are recognized separately, but it is not yet
known whether packaging occurs sequentially and
whether one or more coat protein subunits are involved
in this process. Interestingly, we found that RNA
genome packaging is coupled to genome replication,
suggesting potential approaches for packaging of foreign RNAs.
In other studies, we are investigating the mechanism
by which nodaviral protein B2 suppresses RNA silencing
in infected cells. In collaboration with J.R. Williamson,
Department of Molecular Biology, we showed that B2
binds to double-stranded RNA in a sequence-independent manner and that it interferes with cleavage of
double-stranded RNA substrates by the cellular protein
Dicer. Moreover, in collaboration with J.L. Imler, University of Strasbourg, Strasbourg, France, we showed
that B2 is critical for nodaviral infection of Drosophila
and that Dicer plays an essential role in host defense
against nodaviruses in vivo.
We are also collaborating with several investigators
at Scripps Research, the Salk Institute, and Harvard
University to develop nodaviruses as platforms for delivery of anthrax antitoxins. To this end, we are using
particles to display the VWA domain of capillary morphogenesis protein 2, the cellular receptor for anthrax
toxin, in a multivalent fashion on the surface of the
virion. Two insertion sites yielding different patterns of
180 copies of the VWA domain were selected on the
basis of computational modeling of the high-resolution
crystal structure of the insect nodavirus Flock House
virus. The resulting chimeric viruslike particles functioned as a potent anthrax antitoxin in cell culture and
protected rats from challenge with lethal toxin. This
research is important because it shows that protein
domains containing more than 150 amino acids can be
displayed on Flock House virus in a biologically functional form, suggesting numerous additional applications.
220 MOLECULAR BIOLOGY
2006
Flock House virus particles are also good candidates
for novel materials in nanotechnology applications. The
particles are stable, easily manipulated, biocompatible, and nontoxic in vivo and can be produced easily
and in high quantities. The high-resolution x-ray structure of the virus revealed the potential for using chemical approaches to attach ligands to the surface of the
virus and for using genetic strategies to modify the
capsid. In collaboration with M. Manchester, Department of Cell Biology, and M. Ozkan, University of California, Riverside, we used conjugation chemistry to
couple inorganic nanotubes and quantum dots to Flock
House virus particles to produce an array of novel hybrid
structures. This approach may one day be used to fabricate unique materials for a variety of applications,
including biofilms with tunable pore size, 3-dimensional
scaffolds for production of nanoelectronic devices, and
drug delivery.
PUBLICATIONS
Chao, J.A., Lee, J.H., Chapados, B.R., Debler, E.W., Schneemann, A., Williamson,
J.R. Dual modes of RNA-silencing suppression by Flock House virus protein B2. Nat.
Struct. Mol. Biol. 12:952, 2005.
Destito, G., Schneemann, A., Manchester, M. Biomedical nanotechnology using
virus-based nanoparticles. Curr. Top. Microbiol. Immunol., in press.
Galiana-Arnoux, D., Dostert, C., Schneemann, A., Hoffmann, J.A., Imler, J.L.
Essential function in vivo for Dicer-2 in host defense against RNA viruses in
Drosophila. Nat. Immunol. 7:590, 2006.
Hsu, C., Singh, P., Ochoa, W., Manayani, D.J., Manchester, M., Schneemann, A.,
Reddy, V.S. Characterization of polymorphism displayed by the coat protein
mutants of tomato bushy stunt virus. Virology 349:222, 2006.
Schneemann, A. The structural and functional role of RNA in icosahedral virus
assembly. Annu. Rev. Microbiol. 60:51, 2006.
Singh, P., Destito, G., Schneemann, A., Manchester, M. Canine parvovirus-like
particles, a novel nanomaterial for tumor targeting. J. Nanobiotechnol. 4:2, 2006.
Walukiewicz, H.E., Johnson, J.E., Schneemann, A. Morphological changes in the
T = 3 capsid of Flock House virus during cell entry. J. Virol. 80:615, 2006.
Molecular Biology of
Retroviruses
J.H. Elder, A.P. de Parseval, Y.-C. Lin, S. de Rozieres, M.
Sundstrom, K. Tam, M. Giffin,* H. Heaslet,* C.D. Stout,
B.E. Torbett*
* Department of Molecular and Experimental Medicine, Scripps Research
ur research centers on the molecular characterization of retroviruses, with emphasis on feline
immunodeficiency virus (FIV) and development
of ways to interfere with the viral life cycle. FIV causes
O
THE SCRIPPS RESEARCH INSTITUTE
an AIDS-like syndrome in domestic cats and has structural and functional similarities to HIV, the causative
agent of AIDS in humans. Discovery of ways to interfere with FIV infection may ultimately result in development of treatments for infections in both cats and
humans. In recent studies, we continued to focus on
the molecular characterization of receptor interactions
and the molecular basis for the development of drug
resistance in the aspartic protease encoded by FIV.
RECEPTOR STUDIES
Like many strains of HIV, FIV uses the chemokine
receptor CXCR4 to enter the primary target cell, the
CD4 + T cell. However, unlike HIV, FIV does not use
the cell-surface protein CD4 as a primary binding
receptor. Rather, the feline lentivirus uses the activation antigen CD134 to initially bind to CD4+ T cells.
CD134 is expressed on activated CD4 + T cells, a
finding that explains why FIV can infect and kill CD4+
T cells, even though the virus does not bind CD4.
As reported last year, we showed that interaction
of the FIV surface glycoprotein gp95 with a soluble
version of CD134 allows productive infection of cells
that bear the entry receptor CXCR4 but lack cell-surface CD134. This finding is consistent with the notion
that binding of CD134 causes a conformational change
in gp95, which in turn increases the affinity of interaction with CXCR4 to facilitate infection of the target
cell. These effects are similar to the effects of binding
of soluble CD4 by gp120, the surface glycoprotein of
HIV and indicate that although different primary receptors are involved, the actual mechanism of infection
of FIV and HIV is strikingly similar. We speculate that
the benefit of this type of binding cascade is to limit
exposure of critical regions of the surface glycoproteins
to the immune system until the primary binding event
has already occurred, thus reducing the likelihood of
virus neutralization.
Using chimeric proteins consisting of feline and
human CD134 (the human homolog does not bind FIV
glycoprotein) and site-directed mutagenesis, we have
mapped regions of feline CD134 involved in interaction
with gp95. The results indicated that as few as 3 amino
acids in the C-terminal part of outer domain 1 of feline
CD134 are sufficient to impart FIV gp95 binding and
receptor function to human CD134. Studies are in progress to map the regions of gp95 that bind CD134.
Importantly, we have now a panel of antibodies
that bind and neutralize FIV only after CD134 is
bound; we have used peptides to map the region in
MOLECULAR BIOLOGY
2006
which these CD134-dependent neutralizing antibodies
react. These studies effectively map regions of the viral
glycoprotein critical for CD134 interaction. Cocrystallization studies are under way to determine the structure
of the region surrounding the antibody-binding site.
These experiments will contribute to our understanding of the nature of receptor binding and will define
targets for vaccine development.
P R O T E A S E D R U G R E S I S TA N C E
The aspartic protease of lentiviruses is responsible
for processing the viral Gag and Pol polyproteins into
the final gene products required for viral replication and
must function efficiently to generate infectious virus.
Drugs against HIV protease are keys to the success of
highly active antiretroviral therapy used to treat, but
not cure, patients infected with HIV. The substrate and
inhibitor specificities of FIV differ from those of HIV.
We investigated the nature of these differences to better understand the structural basis of development of
resistance to therapy, an ongoing problem with current
drugs used to treat HIV disease.
In certain instances, similarities exist between amino
acid positions that dictate differences in substrate specificity between FIV and HIV aspartic protease and those
that mutate in response to drug treatment. Mutations in
these sites increase the dissociation constant for a
given drug, but at a cost in catalytic efficiency for the
viral protease. Compensatory amino acid substitutions
can then occur that increase the catalytic efficiency of
the drug-resistant protease, thus increasing expression
of virus despite drug treatment.
We prepared mutants of FIV protease in which amino
acids found in drug-resistant HIV protease were placed in
the equivalent positions in the FIV enzyme. Then, using
cells transduced with gag/pol gene expression vectors
encoding HIV-FIV hybrid proteases, we tested the mutants
for relative drug sensitivity. We found that the Gag/Pol
polyproteins are processed by the hybrid proteases and
have drug sensitivity profiles similar to those of HIV
protease. However, the order of site cleavage, which is
critical to generation of infectious virus, is altered by
these specific changes. Studies are under way to establish a structural basis for this phenomenon. The findings
highlight yet another potential approach to interrupting
the viral life cycle.
PUBLICATIONS
Brik, A., Alexandratos, J., Lin, Y.-C., Elder, J.H., Olson, A.J., Wlodawer, A., Goodsell, D.S., Wong, C.-H. 1,2,3-Triazole as a peptide surrogate in the rapid synthesis
of HIV-1 protease inhibitors. Chembiochem 6:1167, 2005.
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221
de Parseval, A., Bobardt, M.D., Chatterji, A., Chatterji, U., Elder, J.H., David, G.,
Zolla-Pazner, S., Farzan, M.R., Lee, T.-H., Gallay, P.A. A highly conserved arginine
in gp120 governs HIV-1 binding to both syndecans and CCR5 via sulfated motifs.
J. Biol. Chem. 280:39493, 2005.
de Parseval, A., Grant, C.K., Sastry, K.J., Elder, J.H. Sequential CD134-CXCR4
interactions in feline immunodeficiency virus (FIV): soluble CD134 activates FIV
Env for CXCR4-dependent entry and reveals a cryptic neutralization epitope. J.
Virol. 80:3088, 2006.
Gonzalez-Lira, B., Rueda-Orozco, P.E., Galicia, O., Montes-Rodriguez, C.J., Guzman, K., Guevara-Martinez, M., Elder, J.H., Prospero-Garcia, O. Nicotine prevents
HIVgp120-caused electrophysiological and motor disturbances in rats. Neurosci.
Lett. 394:136, 2006.
Heaslet, H., Kutilek, V., Morris, G.M., Lin, Y.-C., Elder, J.H., Torbett, B.E., Stout
C.D. Structural insights into the mechanisms of drug resistance in HIV-1 protease
NL4-3. J. Mol. Biol. 356:967, 2006.
Liang, F.-S., Brik, A., Lin, Y.-C., Elder, J.H., Wong, C.-H. Epoxide in water and
screening in situ for rapid discovery of enzyme inhibitors in microtiter plates.
Bioorg. Med. Chem. 14:1058, 2006.
Whiting, M., Muldoon, J., Lin, Y.-C., Silverman, S.M., Lindstrom, W., Olson, A.,
Kolb, H.C., Finn, M.G., Sharpless, K.B., Elder, J.H., Fokin, V.V. Inhibitors of HIV-1
protease by using in situ click chemistry. Angew. Chem. Int. Ed. 45:1435, 2006.
Metalloenzyme Engineering
D.B. Goodin, C.D. Stout, S. Vetter, E.C. Glazer, R.F. Wilson,
A. Annalora, A.-M. Hays
ur goals are to understand the diverse reactivity
of heme enzymes and to use that information
to generate engineered forms with novel catalytic
properties. The primary hypothesis that has driven these
studies is that the chemical reactivity displayed by
these enzymes resides partially within the heme cofactor. One role of the protein is to limit or direct the access
of substrates to the active site in ways that result in
specific catalysis. In addition, many important examples exist in which the protein directly modulates the
activity of the heme. Thus, our goals are to delineate
the boundaries between these 2 roles for the protein
and then use this information to introduce sites where
nonnative substrates interact with the heme cofactor in
ways that will induce new catalytic reactions. We use
a number of techniques in structural biology and spectroscopy and strategies of rational protein redesign and
molecular evolution.
One area of emphasis is the basic physical, spectroscopic, and functional properties of heme enzymes.
For example, the FeIII/FeII and FeII/FeI redox couples
of inducible nitric oxide synthase have recently been
measured by using direct cyclic voltammetry in organic
films on graphite electrodes. These studies allow easy
measurement of electron transfer between the enzyme
and the electrode surface and have revealed the inter-
O
222 MOLECULAR BIOLOGY
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conversion of several coordination states of the heme.
The results will complement ongoing studies in which
the enzymes are directly and homogeneously coupled
to electrode surfaces by using molecular wires.
In other research, we used cavity complementation
to introduce small-molecule binding sites near the active
site of a protein environment. This approach has provided new ways to test ideas about the diversity of the
functions of heme enzymes and is a useful tool for testing predictions of protein-ligand interactions. For example, in a collaboration with B. Shoichet, University of
California, San Francisco, we completed a study in which
compounds in a database were docked into a buried
engineered cavity that has an unusual specificity for
charged ligands. Using x-ray crystallography, we verified the accuracy of the docking predictions for 15 of
the top 16 compounds.
In other studies, we used synthetic molecular wires,
substrate analogs linked to photochemical or redox-active
sensitizers, to bind at the active site of cytochrome
P450, peroxidases, and nitric oxide synthase. These
wires will be useful as reporters of the active-site environment and as triggers to study reaction mechanisms.
In collaboration with H.B. Gray, California Institute of
Technology, Pasadena, California, we recently solved
structures of cytochrome P450cam bound to 2 such wires.
Marked changes in the protein structure occurred near
2 helices that are similar to structural variations seen
in mammalian P450s, suggesting that the degree of
structural plasticity in prokaryotic P450s is similar to
that of mammalian forms.
In other research, we removed the proposed electron-transfer pathway from a peroxidase and replaced
it with a solvent-filled channel. We have designed surrogate molecular wires to replace the native pathway,
and we have shown that one of these binds the channel in a mode that is completely analogous to the native
structure. This technique will provide new ways to test
proposals about the specificity and structural requirements of this important structural element. Finally, we
are designing and synthesizing a class of cofactor-linked
ruthenium-diamine photosensitizers that are designed
to specifically target nitric oxide synthase at the pterinbinding site, allowing the role of the cofactor in catalysis to be probed by direct-charge injection-withdrawal
through the wire.
PUBLICATIONS
Brenk, R., Vetter, S., Boyce, S.E., Goodin, D.B., Shoichet, B. Probing molecular
docking in a charged model binding site. J. Mol. Biol. 357:1449, 2006.
THE SCRIPPS RESEARCH INSTITUTE
Control of Cell Division
S.I. Reed, C. Baskerville, L.-C. Chuang, S. Ekholm-Reed,
M. Henze, J. Keck, V. Liberal, K. Luo, B. Olson, S. Rudyak,
D. Tedesco, F. van Drogen, J. Wohlschlegel
iological processes of great complexity can be
approached by beginning with a systematic
genetic analysis in which the relevant components are first identified and the consequences of selectively eliminating the components via mutations are
investigated. We have used yeast, which is uniquely
tractable to this type of analysis, to investigate control
of cell division. In recent years, it has become apparent that the most central cellular processes throughout
the eukaryotic phylogeny are highly conserved in terms of
both the regulatory mechanisms used and the proteins
involved. Thus, it has been possible in many instances
to generalize from yeast cells to human cells.
B
CONTROL IN YEAST
In recent years, we have focused on the role and
regulation of the Cdc28 protein kinase (Cdk1). Initially
identified by means of a mutational analysis of the yeast
cell cycle, this protein kinase and its analogs are ubiquitous in eukaryotic cells and are central to a number
of aspects of control of cell-cycle progression.
One current area of interest is regulation of cellular
morphogenesis by Cdk1. The activity of Cdk1 driven by
mitotic cyclins modulates polarized growth in yeast cells.
Specifically, these activities depolarize growth by altering the actin cytoskeleton. We found that several proteins
that modulate actin structure are targeted by Cdk1, and
we are investigating whether these phosphorylation events
control actin depolarization.
A second major area of interest is in the regulation
of mitosis. A key aspect of mitotic regulation in yeast
is the accumulation of Cdc20, which triggers the transition from metaphase to anaphase. Cdc20 is an essential cofactor of the protein-ubiquitin ligase known as the
anaphase-promoting complex or APC/C. It is through the
ubiquitin-mediated proteolysis of a specific anaphase
inhibitor, securin (Pds1 in yeast), that anaphase is initiated. We found that cells are prevented from entering
mitosis when DNA replication is blocked by the drug
hydroxyurea, which causes the destabilization of Cdc20
and inhibition of Cdc20 translation.
While investigating mitosis, we found that a Cks1,
small Cdk1-associated protein, appears to regulate the
proteasome. Proteasomes are complex proteases that tar-
MOLECULAR BIOLOGY
2006
get ubiquitylated proteins, including important cell-cycle
regulatory proteins. Surprisingly, we found that Cks1 regulates a nonproteolytic function of proteasomes, the transcriptional activation of Cdc20. Specifically, Cks1 is
required to recruit proteasomes to the gene CDC20 for
efficient transcriptional elongation. Our investigations of
CDC20 have led to the conclusion that Cks1 is required
for recruitment of proteasomes to and transcriptional
elongation of many other genes as well. Currently, we are
elucidating the mechanism whereby Cks1 recruits proteasomes and facilitates transcriptional elongation. Our most
recent results suggest that Cks1 and proteasomes in conjunction with Cdk1 mediate remodeling of chromatin.
CONTROL IN MAMMALIAN CELLS
We showed previously that the human homologs
of the Cdc28 protein kinase are so highly conserved,
structurally and functionally, relative to the yeast protein kinase, that they can function and be regulated
properly in a yeast cell. Analyzing control of the cell
cycle in mammalian cells, we produced evidence for
the existence of regulatory schemes, similar to those
elucidated in yeast, that use networks of both positive
and negative regulators.
A principal research focus is the positive regulator
of Cdk2, cyclin E. Cyclin E is often overexpressed
and/or deregulated in human cancers. Using a tissue
culture model, we showed that deregulation of cyclin
E confers genomic instability, probably explaining the
link to carcinogenesis. The observation that deregulation of cyclin E confers genomic instability has led us
to hypothesize a mechanism of cyclin E–mediated carcinogenesis based on accelerated loss of heterozygosity
at tumor suppressor loci. We are testing this hypothesis in transgenic mouse models. We showed that a
cyclin E transgene expressed in the mammary epithelium markedly increases loss of heterozygosity at the
p53 locus, leading to enhanced mammary carcinogenesis. We are extending these investigations by using
mouse prostate, testis, and skin models.
In an attempt to understand cyclin E–mediated
genomic instability, we are investigating how deregulation of cyclin E affects both S phase and mitosis. Recent
data suggest that deregulation of cyclin E impairs DNA
replication by interfering with assembly of the prereplication complex. Cyclin E deregulation also impairs the
transition from metaphase to anaphase by promoting
the accumulation of inhibitors of anaphase.
Our interest in cyclin E deregulation in cancer led
us to investigate the pathway for turnover of cyclin E.
THE SCRIPPS RESEARCH INSTITUTE
223
We showed that phosphorylation-dependent proteolysis
of cyclin E depends on a protein-ubiquitin ligase known as
SCF hCdc4 . The F-box protein hCdc4 is the specificity
factor that targets phosphorylated cyclin E. We are
investigating how ubiquitylation of cyclin E is coordinated with other processes required for its degradation,
including prolyl isomerization. We are also investigating SCF hCdc4 ubiquitylation of other important cellular proteins.
Recently, we began determining the role of SCFhCdc4
in neurodegenerative disease. We found that parkin, a
protein often mutated in hereditary Parkinson’s disease,
regulates the stability of hCdc4, possibly leading to neuropathologic changes. Consistent with this idea, we
found that SCFhCdc4 targets peroxisome proliferator–activated receptor γ coactivator-1α which protects neurons
from oxidative damage. In addition, we showed that
SCF hCdc4 regulates the turnover of presenilins in the
brain, proteins strongly implicated in Alzheimer’s disease.
Another area of interest is the role of Cks proteins in
mammals, complementing our research in yeast. Mammals express 2 orthologs of yeast Cks1, known as Cks1
and Cks2. Experiments in mice lacking the gene for
Cks1 and Cks2 revealed that each ortholog has a specialized function. Cks1 is required as a cofactor for
Skp2-mediated ubiquitylation and turnover of inhibitors
p21, p27, and p130. Cks2 is required for the transition from metaphase to anaphase in both male and
female meiosis I. Nevertheless, mice nullizygous at the
individual loci are viable. However, doubly nullizygous
mice have not been observed because embryos die at
the morula stage, a finding consistent with an essential redundant function. We found that this function most
likely is involved in regulation of transcription and is
linked to chromatin remodeling, as in yeast.
PUBLICATIONS
Jackson, L.P., Reed, S.I., Haase, S.B. Distinct mechanisms control the stability of
the related S-phase cyclins Clb5 and Clb6. Mol. Cell. Biol. 26:2456, 2006.
Reed, S.I. Skp’n with Cks1: revelations from the Skp1-Skp2-Cks1-p27 structure.
Mol. Cell 20:1, 2005.
Reed, S.I. The ubiquitin-proteasome pathway in cell cycle control. Results Probl.
Cell Differ. 42:147, 2006.
Smith, A.P.L., Henze, M., Lee, J.A., Osborn, K.G., Keck, J., Tedesco, D., Bortner,
D.M., Rosenberg, M.P., Reed, S.I. Deregulated cyclin E promotes p53 loss of heterozygosity and tumorigenesis in the mouse mammary gland. Oncogene, in press.
Spruck, C., Sun, D., Fiegl, H., Marth C., Mueller-Holzner, E., Goebel, G., Widschwendter, M., Reed, S.I. Detection of low molecular weight derivatives of cyclin E1
is a function of cyclin E1 protein levels in breast cancer. Cancer Res. 66:7355, 2006.
van Drogen, F., Sangfelt, O., Malyukova, A., Matskova, L., Yeh, E., Means, A.R.,
Reed, S.I. Ubiquitylation of cyclin E requires the sequential function of SCF complexes containing distinct hCdc4 isoforms. Mol. Cell 23:37, 2006.
224 MOLECULAR BIOLOGY
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Wittenberg, C., Reed, S.I. Cell cycle-dependent transcription in yeast: promoters,
transcription factors, and transcriptomes. Oncogene 24:2746, 2005.
Wohlschlegel, J.A., Johnson, E.S., Reed, S.I., Yates, J.R. III. Improved identification of SUMO attachment sites using C-terminal SUMO mutants and tailored protease digestion strategies. J. Proteome Res. 5:761, 2006.
THE SCRIPPS RESEARCH INSTITUTE
expressed as an MBF target during late G1 phase, Nrm1
associates with MBF at target promoters and represses
expression as cells enter S phase (Fig. 1). Similarly, the
Nrm1 homolog, SpNrm1, in the fission yeast Schizosaccharomyces pombe regulates its only G1-specific transcription factor, MBF.
Transcriptional and Proteolytic
Control of Cell Proliferation
and Adaptation to
Environmental Stimuli
C. Wittenberg, M. Ashe, R. de Bruin, B.-K. Han. M. Guaderrama,
T. Kalashnikova
ellular decision making and coordination of cellular events often involves the differential regulation of the expression of genes. Recently, we
have focused on the mechanisms through which cells
exert control over gene expression to regulate cell proliferation and the response to changes in environmental conditions.
C
R E G U L AT I O N O F C E L L P R O L I F E R AT I O N
In most cells, commitment to a new round of cell
division during the G1 phase of the cell cycle is accompanied by the activation of a large family of genes that
encode activities involved in the duplication and segregation of cellular components. G1-specific genes also
encode regulatory factors that promote subsequent
cell-cycle events. In the budding yeast Saccharomyces
cerevisiae, G1-specific genes are regulated by 2 transcription factors: SBF and MBF. Using mass spectrometry–based multidimensional protein identification
technology, we have identified novel regulators of these
transcription factors.
SBF acts as a transcriptional activator and promotes
expression of its targets specifically during the G 1
interval. We established that promoter-bound SBF
associates with the Whi5 repressor during early G 1
phase and that Whi5 is inactivated via phosphorylation
by a G1-specific cyclin-dependent protein kinase, thereby
activating transcription (Fig. 1). This regulation is analogous to the regulation of E2F by the tumor suppressor
Rb in metazoans.
MBF, in conjunction with specific corepressors, acts
primarily as a transcriptional repressor and limits
transcription of target genes to the G1 phase. We identified Nrm1, a novel MBF-associated corepressor. When
F i g . 1 . Transcriptional circuitry regulating G1-specific gene expression. G1-specific transcription in S cerevisiae is regulated by 2 heterodimeric transcription factors: SBF and MBF. Both transcription factors are bound to promoters during G1 phase before commitment to a
new cell cycle. Commitment occurs when their target genes are activated by the action of the cyclin-dependent protein kinase Cln3/CDK1.
For SBF, Cln3/CDK1 activates transcription by phosphorylation and
inactivation of the SBF-specific repressor Whi5. Once activated, G1specific transcription leads to the accumulation of many proteins,
including those that promote repression of G1-specific transcription.
Nrm1 is an MBF-specific corepressor encoded by an MBF target
gene. Together, these regulatory proteins can explain the confinement of G1-specific transcription to the G1 phase.
The G1-specific transcriptional machinery is regulated by checkpoints that monitor the integrity of cellular structures and processes. When replication forks are
stalled during S phase in the fission yeast, repression
of MBF-regulated transcription is disrupted. We have
shown that that response, which requires the Rad3
(ATM) and Cds1 (Chk2) checkpoint protein kinases,
leads to the phosphorylation of SpNrm1 and dissociation from MBF-regulated promoters. Unexpectedly,
according to the literature, derepression of MBF target
genes also occurs via regulation of Nrm1 in response
to activation of the DNA replication checkpoint in budding yeast. Consequently, replication stress appears to
be associated with genomic instability in the absence
of Nrm1.
A D A P TAT I O N T O E N V I R O N M E N TA L S T I M U L I
Adaptation to environmental changes generally
involves remodeling of the gene expression program.
We have studied the regulation of the HXT genes, which
encode hexose permeases, in response to extracellular
glucose. Those genes are induced by glucose and are
repressed for most other carbon sources. Extracellular
glucose interacts with the Snf3 and Rgt2 receptors,
MOLECULAR BIOLOGY
2006
initiating a signaling cascade that culminates with the
activation of HXT gene transcription. We have shown
that signaling leads to the phosphorylation-dependent
destruction of a transcriptional corepressor, Mth1, by the
E3 ubiquitin ligase SCFGrr1. Destruction of Mth1 leads
to the phosphorylation of the transcriptional repressor
Rgt1 and its dissociation from HXT gene promoters. Conversely, repression of HXT gene expression requires Mth1
and is associated with Rgt1 dephosphorylation. We
recently identified a type 2A protein phosphatase complex
involved in Rgt1 dephosphorylation and are actively pursuing the protein kinase involved in Rgt1 phosphorylation.
Interestingly, SCFGrr1, the E3 ubiquitin ligase required
for HXT gene induction, is also important for destruction of phosphorylated G1 cyclins, critical regulators of
cell-cycle initiation. We are investigating the basis for
discrimination between targets by SCFGrr1. We found
that basic residues in the leucine-rich repeat and parts
of the carboxy terminus of the F-box protein Grr1 are
important for recognition of phosphorylated substrates.
Our recent identification of additional substrates and
additional characterization of Grr1 are facilitating
those studies.
PUBLICATIONS
de Bruin, R., Kalashnikova, T.I., Chawan, C., McDonald, W.H., Wohlschlegel, J.A.,
Yates, J. III, Russell, P., Wittenberg, C. Constraining G1-specific transcription to
late G1 phase: the MBF-associated corepressor Nrm1 acts via negative feedback.
Mol. Cell 23:483, 2006.
Cell-Cycle Checkpoints,
DNA Damage, and Oxidative
Stress Responses
P. Russell, C. Chahwan, C. Dovey, L.-L. Du, P.-H. Gaillard,
V. Martin, B.A. Moser, T.M. Nakamura,
M.A. Rodríguez-Gabriel, J. Williams, Y. Yamada
NA damage and oxidative stress elicit cellular
responses that are highly conserved throughout
eukaryotic evolution. Consequently, studies of
genetically tractable microorganisms such as the fission
yeast Schizosaccharomyces pombe can provide a useful framework for the design and interpretation of experiments with more complex multicellular organisms. We
use S pombe to study cell-cycle checkpoints, DNA repair,
and stress response mechanisms. Defects in these
mechanisms underlie a number of human diseases,
including cancer.
D
THE SCRIPPS RESEARCH INSTITUTE
225
CHECKPOINTS
The DNA replication and damage checkpoints prevent the onset of mitosis when DNA replication is
interrupted or when DNA is damaged. A single doublestrand break is sufficient to arrest the cell cycle. One
aim of our studies is to understand how cells detect
DNA damage and transmit a checkpoint signal that
arrests the cell cycle.
Chk1 is the effector kinase of the DNA damage
checkpoint. It regulates the activities of Cdc25 and
Mik1/Wee1 proteins, which modulate the inhibitory
phosphorylation of the cyclin-dependent kinase Cdc2.
Chk1 activation by Rad3 requires the adaptor protein
Crb2. Crb2 is rapidly recruited to double-strand breaks
in DNA. Rad3 and Tel1 (the ATM homolog in fission
yeast) stimulate Crb2 recruitment by phosphorylating
a serine residue near the C terminus of histone H2A in
the vicinity of double-strand breaks.
Our data indicate that tandem C-terminal BRCT
domains in Crb2 associate directly with phosphorylated
histone H2A. Crb2 recruitment to double-strand breaks
also requires the constitutive methylation of lysine at
position 20 in histone H4. This step most likely involves
a direct interaction with a Tudor motif in Crb2 that is
located to the N-terminal side of the BRCT motifs. We
recently found that these 2 histone modifications
cooperate in a nonredundant mechanism to promote
recruitment of Crb2 to double-strand breaks (Fig. 1).
Remarkably, neither histone modification is required for
recruitment of Crb2 to sustained double-strand breaks
that cannot be repaired by homologous recombination.
We recently discovered that the histone modification–
independent recruitment of Crb2 to double-strand breaks
involves association between phosphorylated threonine215 in Crb2 and another checkpoint protein known as
Cut5. In future studies, we will determine whether the
mechanisms that regulate Crb2 in fission yeast are
conserved for the analogous proteins in human cells.
DNA REPAIR
Bloom, Warner, and Rothmund-Thomson syndromes
in humans are typified by predisposition to cancer or
premature aging. These syndromes, which all result
from defects in DNA helicases, are characterized by
genomic instability arising from inappropriate homologous recombination. To better understand this process,
we used a 2-hybrid screen to identify novel proteins
that associate with Srs2 DNA helicase in fission yeast.
We discovered a previously uncharacterized protein
that promotes the formation of toxic recombination struc-
226 MOLECULAR BIOLOGY
2006
THE SCRIPPS RESEARCH INSTITUTE
current studies are aimed at providing a deeper biochemical and structural understanding of the SWS1XRCC2-RAD51D complex.
O X I D AT I V E S T R E S S R E S P O N S E
F i g . 1 . Parallel mechanisms of recruiting the DNA damage checkpoint protein Crb2 to sites of DNA damage. Top, A proposed mechanism of how Crb2 associates with double-strand breaks. One mode
of association involves interactions with modified histones. The
tandem C-terminal BRCT domains associate with the phosphory-
lated C-terminal region of histone H2A. The Tudor domain interacts
with the constitutive methylation of histone H4 on lysine at position 20. These modes of interaction are not redundant. A third mode
of interaction involves the phosphorylated threonine at position
215 (T215) region of Crb2 and the Cut5. Cut5 is proposed to specifically bind to the single-stranded DNA region near the end of the
double-strand break. This binding might involve the association of
Cut5 with other proteins that bind to single-stranded DNA. Bottom,
Fission yeast cells that express Crb2 were tagged with yellow fluorescent protein (YFP) and Cut5 tagged with cyan fluorescent protein (CFP). The cells were engineered to express the HO endonuclease and to contain a single HO cleavage site. The YFP-Crb2 and
Cut5-CFP foci indicate large-scale accumulation of these proteins
at the site of the double-strand break created by HO endonuclease.
tures in yeast mutants that lack one or more DNA helicases. This protein, which we christened Sws1 because
it has a SWIM-type zinc finger, is conserved from yeast
to humans. In collaborative mass spectrometry and proteomics studies with J.R. Yates, Department of Cell
Biology, we found that Sws1 forms a complex with 2
other proteins known as Rlp1 and Rdl1. Bioinformatic
analysis revealed that these 2 proteins are Rad51 paralogs (i.e., homologous sequences derived from gene
duplication) that promote the formation of the Rad51
nucleoprotein filament during homologous recombination. Rlp1 and Rdl1 are equivalent to human XRCC2
and RAD51D, proteins implicated in other human diseases characterized by genomic instability. In collaboration with C.H. McGowan, Department of Molecular
Biology, we found that using small interfering RNA to
silence the gene for human SWS1 reduced the occurrence of homologous recombination structures. Our
Oxidative stress caused by reactive oxygen species
can be highly toxic, causing damage to proteins, lipids,
and nucleic acids. Oxidative stress elicits a complex
gene expression response that is orchestrated in large
part by MAP kinase cascades. The fission yeast Spc1
MAP kinase pathway is homologous to the p38 pathway
in humans. We recently discovered Csx1, a protein that
collaborates with Spc1 to control gene expression in
response to oxidative stress. Csx1 is an RNA-binding
protein that mediates overall control of gene expression
in response to oxidative stress by binding and stabilizing mRNA that encodes Atf1, a transcription factor
that is also regulated by Spc1.
Most recently, we have focused on a newly discovered family of proteins that interact with Csx1. We have
named these proteins Cip1 and Cip2 (for Csx1-interacting proteins 1 and 2). Remarkably, elimination of
Cip1 or Cip2 results in substantial recovery of the
sensitivity of Csx1 mutant cells to oxidative stress,
suggesting that Cip1 and Cip2 are part of a mechanism that degrades Atf1 mRNA.
PUBLICATIONS
Cavero, S., Chahwan, C., Russell, P. Xlf1 is required for DNA repair by nonhomologous end-joining in Schizosaccharomyces pombe. Genetics, in press.
Coulon, S., Noguchi, E., Noguchi, C., Du, L.-L., Nakamura, T.M., Russell, P.
Rad22Rad52-dependent repair of ribosomal DNA repeats cleaved by Slx1-Slx4
endonuclease. Mol. Biol. Cell 17:2081, 2006.
de Bruin, R.A.M., Kalashnikova, T.I., Chahwan, C., McDonald, W.H.,
Wohlschlegel, J., Yates III, J.R., Russell, P., Wittenberg, C. Constraining G1-specific transcription to late G1-phase: The MBF-associated corepressor Nrm1 acts
via negative feedback. Mol. Cell. 23:483, 2006.
Du, L.-L., Nakamura, T.M., Russell, P. Histone modification-dependent and -independent pathways for recruitment of checkpoint protein Crb2 to double-strand
breaks. Genes Dev. 20:1583, 2006.
Martin, V., Chahwan, C., Gao, H., Blais, V., Wohlschlegel, J., Yates, J.R. III,
McGowan, C.H., Russell, P. Sws1 is a conserved regulator of homologous recombination in eukaryotic cells. EMBO J. 25:2564, 2006.
Martín, V., Rodríguez-Gabriel, M.A., McDonald, W.H., Watt, S., Yates, J.R. III,
Bähler, J., Russell, P. Cip1 and Cip2 are novel RNA-recognition-motif proteins that
counteract Csx1 function during oxidative stress. Mol. Biol. Cell 17:1176, 2006.
Matsumoto, S., Ogino, K., Noguchi, E., Russell, P., Masai, H. Hsk1-Dfp1/Him1,
the Cdc7-Dbf4 kinase in Schizosaccharomyces pombe, associates with Swi1, a component of the replication fork protection complex. J. Biol. Chem. 280:42536, 2005.
Nakamura, T.M., Moser, B.A., Du, L.-L., Russell, P. Cooperative control of Crb2 by
ATM family and Cdc2 kinases is essential for the DNA damage checkpoint in fission yeast. Mol. Cell. Biol. 25:10721, 2005.
Rodríguez-Gabriel, M.A., Russell, P. Distinct signaling pathways respond to arsenite and reactive oxygen species in Schizosaccharomyces pombe. Eukaryot. Cell
4:1396, 2005.
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2006
Rodriguez-Gabriel, M.A., Watt, S., Bähler, J., Russell, P. Upf1, an RNA helicase
required for nonsense-mediated mRNA decay, modulates the transcriptional
response to oxidative stress in fission yeast. Mol. Cell. Biol. 26:6347, 2006.
DNA Damage Responses in
Human Cells
C.H. McGowan, V. Blais, M. Duquette, E. Langley,
A. MacLaren, J. Scorah, D. Slavin, E. Taylor
omplex multicellular organisms, such as humans,
have large numbers of mitotically competent cells
that are capable of renewal, repair, and, to some
extent, regeneration. The advantages of being able to
replace damaged or aged cells are off set by the inherent susceptibility of mitotic cells to acquiring mutations
and becoming cancerous. DNA is inherently vulnerable
to many sorts of chemical and physical modification;
thus, as cells duplicate and divide, they can acquire
mutations. Both spontaneous and induced DNA damage must be repaired with minimal changes if growth,
renewal, and repair are to be successful. Our overall
objective is to understand how mammalian cells protect
themselves from DNA damage and thus from developing cancer.
Eukaryotic cells have evolved with a complex network of DNA repair processes and cell-cycle checkpoint
responses to ensure that damaged DNA is repaired
before it is replicated and becomes fixed in the genome.
These pathways are highly conserved throughout evolution, and much information about human responses
to DNA damage has been gained from studies of simple, genetically tractable organisms such as yeast. We
use a combination of molecular, cellular, and genetic
techniques to determine how these pathways operate
in human cells.
Checkpoints control the order and timing of events
in the cell cycle; they ensure that biochemically independent processes are coupled so that a delay in a
critical cell-cycle process will cause a delay in all other
aspects of progression of the cycle. In addition, checkpoints also coordinate repair with delays in progression
of the cell cycle and promote the use of the most appropriate repair pathway. We used genetic models to identify
2 checkpoint kinases in humans that limit progression
of the cell cycle when DNA is damaged. One of these
kinases, Chk2, is activated in response to DNA damage. Chk2 physically interacts with Mus81-Eme1, a
C
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227
conserved DNA repair protein that has homology to the
xeroderma pigmentosum F family of endonucleases.
Xeroderma pigmentosum is a cancer-prone disorder
that results from a failure to appropriately repair damaged DNA.
Biochemical analysis shows that Mus81-Eme1 has
associated endonuclease activity against structure-specific DNA substrates, including Holliday junctions. Enzymatic analysis, immunofluorescence studies, and the
use of RNA interference have all contributed to the conclusion that Mus81-Eme1 is required for recombination
repair in human cells. We are also using gene targeting
to study the function of the Mus81-Eme1 endonuclease
in mice. Inactivation of Mus81 in mice increases genomic instability and sensitivity to DNA damage but does
not promote tumorigenesis. In addition, we showed that
Mus81-Eme1 is specifically required for survival after
exposure to cisplatin, mitomycin C, and other commonly
used anticancer drugs. As a point of interaction between
checkpoint control and DNA repair, the relationship
between Mus8-Eme1 and Chk2 most likely provides
information critical to understanding the response to
DNA damage as a whole.
Anticancer therapy is largely based on the use of
genotoxic agents that damage DNA and thus kill dividing cells. Coordination of cell-cycle checkpoints and
DNA repair is especially important when unusually high
amounts of DNA damage occur after radiation or genotoxic chemotherapy. Hence, a detailed understanding of
cellular responses to DNA damage is essential in understanding both the development and the treatment of
disease in humans.
PUBLICATIONS
Martin, V., Chawan, C., Gao, H., Blais, V., Wohlschlegel, J., Yates, J.R. III,
McGowan, C.H., Russell, P. Sws1 is a conserved regulator of homologous recombination in eukaryotic cells. EMBO J. 25:2564, 2006.
DNA Repair and the
Maintenance of Genomic
Stability
M.N. Boddy, S. Pebernard, J. Prudden
NA repair pathways have evolved to protect
the genome from ever-present genotoxic agents.
Highlighting the importance of the pathways,
defects in DNA repair mechanisms strongly predispose
the host to cancer and to neurologic and developmental
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228 MOLECULAR BIOLOGY
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disorders. The DNA repair systems we study in fission
yeast are evolutionarily conserved, and therefore our
studies provide a valuable framework for understanding genome maintenance in human cells.
Although many DNA repair mechanisms have been
described, information on how they are coordinated with
necessary changes in chromatin structure is limited. We
are studying the essential structural maintenance of
chromosomes (SMC) complex Smc5-Smc6. The molecular
functions of Smc5-Smc6 are unknown, but the complex
is related to the SMC complexes that hold replicated sister chromatids together (cohesin) and condense chromatin before its segregation at mitosis (condensin).
In collaboration with J.R. Yates, Department of Cell
Biology, we purified the Smc5-Smc6 complex and determined the identity of the core components. The holocomplex consists of the Smc5-Smc6 heterodimer and 6
additional non-SMC elements, Nse1–Nse6. We expressed
and purified individual components of the complex and
determined the architecture of the holocomplex (Fig. 1).
THE SCRIPPS RESEARCH INSTITUTE
cation of target proteins with ubiquitin and the small
ubiquitin-like protein SUMO. Such protein modifications
play roles in DNA repair and chromatin remodeling.
We have carried out detailed genetic and biochemical analyses of the Nse5-Nse6 heterodimer. Nse5 and
Nse6 are not essential for growth; however, cells lacking either protein have high levels of spontaneous genome
damage and are hypersensitive to ultraviolet light and
other genotoxic agents. An important discovery is that
Nse5-Nse6 prevents the deleterious engagement of an
ordinarily beneficial DNA repair pathway called homologous recombination. Our studies indicate that Nse5Nse6, and by extension the Smc5-Smc6 complex, acts
either to prevent initiation of homologous recombination
or to separate physically linked chromosomes that arise
late in this process (Fig. 1B). Abrogating homologous
recombination by deleting a pivotal factor required for
the process (called Rad51) reduces the sensitivity of
Nse5-Nse6 mutant cells to DNA damage. The spontaneous DNA damage observed in Nse5-Nse6 mutant cells
is due to the attempted separation of chromosomes into
daughter cells while the chromosomes are still physically linked. Such defective chromosome separation in
humans could result in cancer and other diseases.
PUBLICATIONS
Pebernard, S., Wohlschlegel, J., McDonald, W.H., Yates, J.R. III, Boddy, M.N.
The Nse5-Nse6 dimer mediates DNA repair roles of the Smc5-Smc6 complex
[published correction appears in Mol. Cell. Biol. 26:3336, 2006]. Mol. Cell. Biol.
26:1617, 2006.
Raffa, G.D., Wohlschlegel, J., Yates, J.R. III, Boddy, M.N. SUMO-binding motifs
mediate the Rad60-dependent response to replicative stress and self-association. J.
Biol. Chem. 281:27973, 2006.
F i g . 1 . Architecture and function of the Smc5-Smc6 holocomplex.
A, Nse1, Nse3, and Nse4 form a stable heterotrimer that associates
with Smc5. Nse2 interacts directly with Smc5 in the absence of the
other Nse proteins. Smc6 interacts directly with Smc5 but with none
of the other components. Nse5 and Nse6 form a stable heterodimer
that also binds directly to Smc5. Double-headed arrows indicate
interactions between subcomplexes. Nse5-Nse6 may recruit the holocomplex to stalled replication forks and certain DNA damage sites.
B, Nse5-Nse6 might act to prevent the initiation of homologous
recombination catalyzed by a number of factors, including Rad51.
Nse5-Nse6 could be involved in the separation or “resolution” of physically linked chromosomes that can result from homologous recombination. Evidence suggests that Nse5-Nse6 and Smc5-Smc6 perform
such functions at replication forks and DNA double-strand breaks.
Nse1–Nse4 are essential for growth, and hypomorphic
mutants of these proteins cause cellular sensitivity to
genotoxic agents such as ultraviolet light and x-rays.
Notably, Nse1 and Nse2 contain certain zinc finger
domains that implicate these 2 elements in the modifi-
Delineating Oncogenic and
Tumor-Suppressing Signal
Transduction Pathways
P. Sun, Q. Deng, C. Kannemeier, R. Liao, A. Seit-Nebi,
N. Yoshizuka
evelopment of cancer is a result of multiple oncogenic genetic alterations, including activation
of oncogenes and inactivation of tumor suppressors. Despite the essential roles of these mutations in
tumor formation, normal cells usually respond to these
oncogenic changes by initiating tumor-suppressing
defense mechanisms such as premature senescence and
apoptosis. Our main interests are delineating the signal
transduction pathways that mediate these tumor-sup-
D
MOLECULAR BIOLOGY
2006
pressing responses and determining how oncogenes allow
a cell to evade the regulation by these cellular defense
mechanisms to cause cancer. Currently, we are focusing
on 2 well-known oncogenes: ras and mdm2.
The oncogene ras encodes a family of small GTPbinding proteins that transduce mitogenic signals from
extracellular growth factors. Constitutive activation of ras
is common in tumors and contributes to tumor development. In normal cells, however, the initial response to
ras activation is a stable growth arrest called premature
senescence. As a result, the senescence response triggered by ras must be evaded before transformation can
occur. We showed that ras induces senescence through
sequential activation of 2 MAP kinase pathways. Initially,
ras activates the MAP kinase kinase (MEK)–extracellular signal–regulated kinase (ERK) pathway. Sustained
activation of MEK-ERK turns on the stress-induced p38
pathway, which subsequently causes senescence.
These studies have revealed a novel, tumor-suppressing function of p38, in addition to its known roles in
inflammation and stress responses. In other studies,
we identified additional signaling components, either
upstream or downstream of p38, that mediate premature senescence. We found that 1 of the 4 isoforms of
p38 contributes to ras-induced senescence by activating the p53 tumor suppressor protein. In addition, a
serine/threonine protein kinase, which is a direct substrate of p38, also plays an essential role in ras-induced
senescence. Inactivation of this protein kinase disrupts
ras-induced senescence and promotes tumorigenesis
both in vitro and in vivo. Our results have confirmed
the tumor-suppressing function of the p38 pathway.
To determine how premature senescence is bypassed
in tumors, we dissected the functions of an adenovirusencoded oncoprotein, E1A, that can rescue ras-induced
senescence. Our results indicated that bypassing of
senescence requires binding of the cellular proteins Rb
and p300/CBP by E1A. Although interference with the
p16INK4A/Rb pathway or p300/CBP functions alone did
not result in bypassing of senescence, these 2 types of
genetic alterations cooperated to rescue cells from rasinduced senescence and lead to cellular transformation.
These results indicate that p300 and CBP are integral
components of the senescence pathway. Both p300 and
CBP have tumor-suppressing functions. The critical role
of p300 and CBP in the senescence response has provided a mechanistic basis for the tumor-suppressing function of these proteins.
Another focus of our research is mdm2, an oncogene that can mediate transformation primarily through
THE SCRIPPS RESEARCH INSTITUTE
229
inactivation of the tumor suppressor protein p53. However, we found that MDM2 confers resistance to a
growth-inhibitory cytokine, transforming growth factor β,
through a p53-independent mechanism. We are delineating this p53-independent activity of MDM2, which
may play an important role in tumorigenesis. We have
identified several MDM2 domains and activities that
are essential for the ability of MDM2 to mediate resistance to the growth factor.
In other research, we are systematically searching
for genetic alterations that contribute to specific tumorassociated phenotypes, such as drug resistance, cellular immortalization, and metastasis. For these studies,
we are using cDNA expression libraries or libraries of
short interfering RNAs.
PUBLICATIONS
Lin, S., Xiao, R., Sun, P., Xu, X., Fu, X.D. Dephosphorylation-dependent sorting of
SR splicing factors during mRNP maturation. Mol. Cell 20:413, 2005.
Hypocretins in Arousal, Feeding
Behavior, and Motivation
J.G. Sutcliffe, L. de Lecea
he 2 C terminally amidated hypocretin neuropeptides (also called orexins) are produced from a
precursor whose expression in rats is restricted
to a few thousand neurons of the lateral hypothalamus.
These neurons are active during wakefulness but are
quiescent during various phases of sleep. Two G protein–coupled hypocretin receptors have different distributions within the CNS.
The hypocretins are found in secretory vesicles at
synapses of fibers that project to areas within the posterior part of the hypothalamus that are implicated in
feeding behaviors and hormone secretion. Hypocretin
fibers also project to diverse targets in other brain regions
and the spinal cord, including several areas implicated
in cardiovascular function and sleep-wake regulation.
The peptides are excitatory when applied directly in
vivo. Most humans with narcolepsy have greatly reduced
levels of hypocretin peptides in their cerebral spinal fluid
and no or barely detectable hypocretin neurons in their
hypothalami, findings suggestive of autoimmune attack.
Hypocretin peptides excite noradrenergic neurons
in the locus coeruleus and serotonergic neurons in the
dorsal raphe to elevate muscle tone and histaminergic
tuberomammillary neurons to promote wakefulness.
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230 MOLECULAR BIOLOGY
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These components of the ascending reticular activating system, and the hypocretin neurons themselves,
project to and stimulate thalamic and basal forebrain
neurons, and all of these groups contribute to the depolarization of the cerebral cortex. Arousal-related signaling occurs through both hypocretin receptors.
These peptides have diverse effects on brain reward
and autonomic systems related to stress that increase
motivated behaviors, including feeding. The relation to
feeding is complex. Acute administration of hypocretin
peptides to sleeping rats increases food consumption.
However, patients and animals with impaired hypocretin signaling have an increased likelihood of being obese
despite reduced daily calorie intake.
PUBLICATIONS
de Lecea L., Sutcliffe, J.G. The hypocretins and sleep. FEBS J. 272:5675, 2005.
Desplats, P.A., Kass, K.E., Gilmartin, T., Stanwood, G.D., Woodward, E.L., Head,
S.R., Sutcliffe, J.G., Thomas, E.A. Selective deficits in the expression of striatalenriched mRNAs in Huntington’s disease. J. Neurochem. 96:743, 2006.
Hedlund, P.B., Huitrón-Reséndiz, S., Henriksen, S.J., Sutcliffe, J.G. 5-HT7 receptor inhibition and inactivation induce antidepressantlike behavior and sleep pattern.
Biol. Psychiatry 58:831, 2005.
Hedlund, P.B., Sutcliffe, J.G. 5-HT7 receptors as favorable pharmacological targets
for drug discovery. In: The Serotonin Receptors: From Molecular Pharmacology to
Human Therapeutics. Roth, B.L. (Ed.). Humana Press, Totowa, NJ, 2006, p. 517.
Hilbush, B.S., Morrison, J.H., Young, W.G., Sutcliffe, J.G., Bloom, F.E. New
prospects and strategies for drug target discovery in neurodegenerative disorders.
NeuroRx 2:627, 2005.
THE SCRIPPS RESEARCH INSTITUTE
nia of short, intermediate, and long duration and from
the prefrontal cortex of matched control subjects without schizophrenia. Among many genes and pathways
revealed as significantly altered in schizophrenia, we
are focusing on those related to glycosphingolipid metabolism and myelination. A total of 40 genes with altered
expression in patients with schizophrenia were related
to these systems.
To assess the effects of treatment with antipsychotic drugs on a subset of genes that encode structural components of myelin, we treated groups of mice
with haloperidol, a widely prescribed “typical” antipsychotic drug. Chronic haloperidol treatment caused significant decreases in the expression levels of at least 8
myelin-related genes in several white matter regions of
mouse brain as revealed by in situ hybridization analysis.
In other studies, we are investigating the molecular basis for heterogeneity in schizophrenia by identifying genes that have similar expression profiles in
subsets of patients with the disorder. Using weighted gene
coexpression network analyses, we identified distinct
subtypes in our schizophrenia cohort, most notably,
dramatic differences in the expression profiles between
patients with short versus long duration of illness. We
are exploring subtype-specific pathways associated with
these subgroups.
T R A N S C R I P T I O N A L D Y S R E G U L AT I O N I N
Sutcliffe, J.G. de Lecea, L. The hypocretin/orexin system. In: Handbook of Contemporary Neuropharmacology. Sibley, D.R., et al. (Eds.). Wiley-InterScience,
Hoboken, NJ, in press.
Sutcliffe, J.G., de Lecea, L. Hypocretins/orexins in brain function. In: Handbook of
Neurochemistry and Molecular Neurobiology: Neuroactive Proteins and Peptides, 3rd
ed. Lim, R. (Volume Ed.), Lajtha, A. (Series Ed.). Springer, New York, 2006, p. 499.
Molecular Neurobiology of
CNS Disorders
E.A. Thomas, J.G. Sutcliffe, P.A. Desplats, S. Narayan,
K.E. Kass, T. Gilmartin, L. Schaffer, S.R. Head
GENE PROFILING IN SCHIZOPHRENIA
chizophrenia is a life-long, heterogeneous mental illness with variable expression and unknown
etiology. We are interested in the molecular factors that influence the course of illness in schizophrenia and how treatment modifies these factors. Using
oligonucleotide microarrays, we generated gene expression profiles from tissue samples obtained at autopsy
from the prefrontal cortex of patients with schizophre-
S
H U N T I N G T O N ’ S D I S E A S E : S T R I ATA L S P E C I F I C I T Y
Much evidence supports a role for transcriptional
dysregulation in Huntington’s disease. Of particular
interest is how these disturbances may be specifically
manifested in the striatum, the primary region of neurodegeneration in Huntington’s disease. Using microarray analysis and a transgenic mouse of Huntington’s
disease, we identified a cluster of striatal-enriched genes
that was downregulated in the mice. The cluster included
the genes FoxP1, Bcl11b, and DRRF, which encode
zinc finger–containing transcription factors, and RARB
and RXRG, which encode nuclear receptors. Real-time
polymerase chain reaction validated 57% and 40%
reductions in levels of Bcl11b and FoxP1 mRNA, respectively, in the striatum of symptomatic transgenic mice
and a 73% decrease in the expression of FoxP1 in
human caudate from patients with Huntington’s disease.
Transcripts for both of these factors are expressed in
medium spiny projection neurons, which selectively
degenerate in Huntington’s disease. Further colocalization and coimmunoprecipitation studies have suggested
that Bcl11b and FoxP1 interact with polyglutamine-
MOLECULAR BIOLOGY
2006
expanded N-terminal huntingtin. Sequestration of these
factors into nuclear aggregates in Huntington’s disease
resulting in loss of function may contribute to specific
dysregulation of striatal gene expression. This mechanism may explain, in part, the specificity of the pathologic changes associated with Huntington’s disease.
PUBLICATIONS
Desplats, P.A., Kass, K.E., Gilmartin, T., Stanwood, G.D., Woodward, E.L., Head,
S.R., Sutcliffe, J.G. Thomas. E.A. Selective deficits in the expression of striatalenriched mRNAs in Huntington’s disease. J. Neurochem. 96:743, 2006.
Thomas, E.A. Apolipoprotein D and arachidonic acid interactions in the treatment
and pathology of schizophrenia. In: Fatty Acids and Oxidative Stress in Neuropsychiatric Disorders. Yao, J.K. (Ed.). Nova Science Publishers, Inc., Hauppauge, NY,
2006.
Narayan, S., Kass, K.E., Thomas, E.A. Chronic haloperidol treatment results in a
decrease in the expression of myelin/oligodendrocyte-related genes in the mouse
brain. J. Neurosci. Res., in press.
Thomas, E.A., Yao, J.K. Clozapine specifically alters the arachidonic acid pathway
in mice lacking apolipoprotein D. Schizophr. Res., in press.
Thomas, E.A. Molecular profiling of antipsychotic drug function: convergent mechanisms. In: The Pathology and Treatment of Psychiatric Disorders. Molecular Neurobiology, in press.
Thomas, E.A. Striatal specificity of gene expression dysregulation in Huntington's
disease. J. Neurosci. Res. 84:1151, 2006.
THE SCRIPPS RESEARCH INSTITUTE
231
at the 5-HT7 receptor. Thus, both blockade and inactivation of the 5-HT7 receptor yield the same result.
Sleep disturbances are common in depression.
Increased amounts of REM sleep are a frequent finding. Compared with mice that have the 5-HT7 receptor mice lacking the receptor spend less time in REM
sleep without alteration of other sleep parameters, further establishing the antidepressant-like profile of the
animals that lack the receptor.
Taken together our results suggest an important role
for the 5-HT7 receptor in depression, and antagonists
to this receptor should be evaluated as a treatment
for depression.
OBSESSIVE-COMPULSIVE DISORDER
Obsessive-compulsive disorder is related to depression, at least to the extent that antidepressants are
commonly used to treat both disorders. In an animal
model of obsessive-compulsive disorder (marble burying),
we showed that blockade or inactivation of the 5-HT7
receptor results in less compulsive behavior. Thus, the
5-HT7 receptor might be of interest as a putative target for treatment of obsessive-compulsive disorder.
SCHIZOPHRENIA
The 5-HT7 Receptor in
Neuropsychiatric Disorders
P.B. Hedlund, P.E. Danielson, S. Huitrón-Reséndiz,
S.J. Henriksen, S. Semenova, M.A. Geyer, A. Markou,
J.G. Sutcliffe
nterest in the serotonin 5-HT7 receptor as a putative
target in neuropsychiatric disorders has been growing continually. The interest was prompted by the
finding that several classes of drugs used to treat disorders such as depression and schizophrenia have high
affinity for the 5-HT 7 receptor. We have established
evidence that supports a role for this receptor in depression, obsessive-compulsive disorder, and schizophrenia.
I
DEPRESSION
The forced swim test and the tail suspension test
are animal models of behavioral despair that have high
value for predicting the antidepressant efficacy of drugs.
The tests can also be used to characterize animals in
which genes have been deleted. Using both of these
tests, we showed that mice lacking the 5-HT7 receptor
have a behavioral profile similar to that of mice treated
with antidepressants. We replicated these findings by
using a compound that acts as a selective antagonist
Prepulse inhibition (PPI) of the acoustic startle
reflex is a well-characterized animal model of schizophrenia. The model is especially relevant because
similar responses can be observed in patients with
schizophrenia. We showed that PPI per se is not altered
in mice lacking the 5-HT7 receptor, but that when PPI
is disrupted by phencyclidine, the mice are significantly
less affected than are mice that have the receptor.
Phencyclidine-induced disruption involves a glutamatergic
component of PPI that is relevant for the action of atypical antipsychotics such as clozapine. Clozapine is a
drug with relatively high affinity for the 5-HT7 receptor.
PUBLICATIONS
Hedlund, P.B., Huitrón-Reséndiz, S., Henriksen, S.J., Sutcliffe, J.G. 5-HT7 receptor inhibition and inactivation induce antidepressantlike behavior and sleep pattern.
Biol. Psychiatry 58:831, 2005.
Hedlund, P.B., Sutcliffe, J.G. 5-HT7 receptors as favorable pharmacological targets
for drug discovery. In: The Serotonin Receptors: From Molecular Pharmacology to
Human Therapeutics. Roth, B.L. (Ed.). Humana Press, Totowa, NJ, 2006, p. 517.
Hedlund, P.B., von Euler, G. Z-analysis: a new approach to analyze stimulation
curves with intrinsic basal stimulation. Biochem. Pharmacol. 70:170, 2005.
232 MOLECULAR BIOLOGY
2006
THE SCRIPPS RESEARCH INSTITUTE
Lysophospholipid Signaling and
Neural Aneuploidy
J. Chun, S. Appadurai, B. Almeida, B. Anliker, E. Birgbauer,
A. Dubin, S. Gardell, D. Herr, G. Kennedy, M. Kingsbury,
C.W. Lee, M. Lu, M. McCreight, C. Paczkowski, S. Peterson,
S. Rehen, R. Rivera, A.H. Yang, X.Q. Ye, Y. Yung, L. Zhu
n the past year, we gained significant new insights
into both lysophospholipid signaling and neural aneuploidy. First, we discovered that receptor-mediated
lysophosphatidic acid (LPA) signaling, mediated by the
cognate receptor known as LPA3, is essential for normal implantation of embryos in the uterine wall, a finding that may be relevant to the treatment of female
infertility. Second, we acquired new data that indicate
the potential function of genomically nonidentical brain
cells in normal brain in humans. In further studies in
mice, we found that aneuploid neurons can be integrated
into the normal circuitry of the brain, indicating that
the neurons are not simply dead or inert components
but rather have the potential to modify properties of
neural circuitry by virtue of their altered genomes.
I
F i g . 1 . Location of implantation sites in uteri at embryonic days
4.5 (E4.5) and 5.5 (E5.5). Bands indicate implantation sites. Mice
lacking the gene for LPA3 have delayed implantation and at later
times have reduced and abnormally spaced implantation (arrows).
LY S O P H O S P H O L I P I D S
Lysophospholipids such as LPA are simple phospholipids that act as extracellular signals that use cognate G
protein–coupled receptors to bring about myriad effects.
The 2 best studied lysophospholipids are LPA and sphingosine 1-phosphate (S1P). We continue to generate new
lines of mice that lack the genes for single and multiple
receptors and to characterize the mutant phenotypes. A
null mutation in LPA 3 resulted in a reduced-fertility
phenotype that was attributed to alterations in embryo
implantation (Fig. 1). We are elucidating the downstream
signaling effects of LPA3 in normal implantation.
NORMAL NEURAL ANEUPLOIDY
It is now clear that many cells in the brain have
nonidentical genomes by virtue of being aneuploid, that
is, the cells have gained and/or lost chromosomes. The
initial research on aneuploidy was done in mice, raising
the question of whether this phenomenon also existed in
humans. Use of double labeling with point probes, which
recognize a relatively discrete part of a chromosome,
and “paints,” which recognize much of a given chromosome, allowed the unambiguous identification of aneuploid neurons and glia in normal human brain (Fig. 2).
This finding led us to ask the additional question of
whether such cells were capable of normal function.
F i g . 2 . Nuclei isolated from the brains of different patients con-
taining 1 (E), 2 (F), 3 (G), or 4 (H) copies of chromosome 21. The
large, dark region indicates staining with DAPI (4′,6-diamidino-2phenylindole), whole-chromosome paint appears in light gray, and
the chromosome 21 point probes are indicated by arrows. A complete overlap between the paint and the point probe occurs, as
seen at higher magnification in the insets. Arrowheads indicate the
numbers of chromosome 21 per cell. Scale bar, 5 µm.
In mice, we found that indeed, aneuploid neurons
can have distant connections and physiologic activities,
suggesting that these genomically distinct cells can
function in normal neural circuitry. Currently, we are
determining the extent, forms, and roles of aneuploid
neural cells in normal and diseased mammalian brains.
MOLECULAR BIOLOGY
2006
PUBLICATIONS
Barbeito, L., Chun, J., Binder, L.I., Neto, V.M., Perry, G., Scazzochio, C., Violini, G.
The end of a Chilean institute. Science 308:792, 2005.
Chun, J. Lysophospholipids in the nervous system. Prostaglandins Other Lipid
Mediat. 77:46, 2005.
Gon, Y., Wood, M.R., Kiosses, W.B., Jo, E., Sanna, M.G., Chun, J., Rosen, H.
S1P3 receptor-induced reorganization of epithelial tight junctions compromises lung
barrier integrity and is potentiated by TNF. Proc. Natl. Acad. Sci. U. S. A.
102:9270, 2005.
Goparaju, S.K., Jolly, P.S., Watterson, K.R., Bektas, M., Alvarez, S., Sarkar, S.,
Mel, L., Ishii, I., Chun, J., Milstien, S., Spiegel, S. The S1P2 receptor negatively
regulates platelet-derived growth factor-induced motility and proliferation. Mol. Cell.
Biol. 25:4237, 2005.
Kingsbury, M.A., Friedman, B., McConnell, M.J., Rehen, S.K., Yang, A.H.,
Kaushal, D., Chun, J. Aneuploid neurons are functionally active and integrated into
brain circuitry. Proc. Natl. Acad. Sci. U. S. A. 102:6143, 2005.
Li, H., Ye, X., Mahanivong, C., Bian, D., Chun, J., Huang, S. Signaling mechanisms responsible for lysophosphatidic acid-induced urokinase plasminogen activator expression in ovarian cancer cells. J. Biol. Chem. 280:10564, 2005.
Rehen, S.K., Yung, Y.C., McCreight, M.P., Kaushal, D., Yang, A.H., Almeida,
B.S.V., Kingsbury, M.A., Cabral, K.M.S., McConnell, M.J., Anliker, B., Fontanoz,
M., Chun, J. Constitutional aneuploidy in the normal human brain. J. Neurosci.
25:2176, 2005.
Simon, M.F., Daviaud, D., Pradere, J.P., Grès, S., Guigné, C., Wabitsch, M.,
Chun, J., Valet, P., Saulnier-Blache, J.S. Lysophosphatidic acid inhibits adipocyte
differentiation via lysophosphatidic acid 1 receptor-dependent down-regulation of
peroxisome proliferator-activated receptor γ2 J. Biol. Chem. 280:1456, 2005.
Tölle, M., Levkau, B., Keul, P., Brinkmann, V., Giebing, G., Schönfelder, G.,
Schäfers, M., von Wnuck Lipinski, K., Jankowski, J., Jankowski, V., Chun, J., Zidek,
W., Van der Giet, M. Immunomodulator FTY720 induces eNOS-dependent arterial
vasodilation via the lysophospholipid receptor S1P3. Circ. Res. 96:913, 2005.
Ye, X., Hama, K., Contos, J.J., Anliker, B., Inoue, A., Skinner, M.K., Suzuki, H.,
Amano, T., Kennedy, G., Arai, H., Aoki, J., Chun, J. LPA3 lysophosphatidic acid
signalling in embryo implantation and spacing. Nature 435:104, 2005.
Chemical Glycobiology
J.C. Paulson, O. Blixt, L.K. Allin, H. Andersson-Sand,
O.V. Bohorov, B.E. Collins, S. Han, J. Hoffman, D. Lebus,
L. Liao, X. Liu, B. Ma, M. O’Reilly, N. Razi, P. Sobieszczuk,
L. Stewart, H. Tateno, H. Tian, D. Vasiliu, Y. Zeng
e investigate the roles of glycan-binding proteins that mediate cellular processes central
to immunoregulation and human disease. We
work at the interface of biology and chemistry to understand how the interaction of glycan-binding proteins
with their ligands mediates cell-cell interactions, endocytosis, and cell signaling. Our multidisciplinary approach
is complemented by a diverse group of chemists, biochemists, cell biologists, and molecular biologists.
W
BIOLOGICAL ROLES OF SIGLECS
The siglecs are a family of 11 sialic acid–binding
proteins that function as cell-signaling coreceptors.
They are expressed on glial cells and on a variety of
leukocytes that mediate acquired and innate immune
THE SCRIPPS RESEARCH INSTITUTE
233
functions, including B cells, eosinophils, macrophages,
dendritic cells, and natural killer cells. Siglecs are a
subfamily of the immunoglobulin superfamily that have
in common a unique N-terminal Ig domain that confers
the ability to bind to sialic acid–containing carbohydrate
groups (sialosides) of glycoproteins and glycolipids.
The cytoplasmic domains of most siglecs contain tyrosine-based inhibitory motifs characteristic of accessory proteins that regulate transmembrane signaling
and endocytosis of cell-surface receptor proteins. The
diverse specificity for their sialoside ligands and their
variable cytoplasmic regulatory elements provide siglecs
with attributes for unique roles in the cell-surface biology of each cell that expresses them.
The best understood siglec is CD22 (siglec-2), an
accessory molecule of the B-cell receptor (BCR) complex
that has both positive and negative effects on receptor
signaling. The carbohydrate ligand recognized by CD22 is
the sequence Siaα2-6Galbβ1-4GlcNAc found on glycoproteins of both B cells (cis ligands) and on cells that
interact with B cells (e.g., T cells, trans ligands). Interactions of CD22 with cis or trans ligands regulate aspects
of B-cell activation, proliferation, and development.
We found that CD22 is predominately associated
with clathrin-coated pits in resting B cells, whereas BCRs
are minimally associated with clathrin domains. Mice
deficient in the ligand for CD22 have greater colocalization of CD22 and the BCR in fused raft-clathrin domains
than do mice that have the ligand, accounting for the
immunosuppression in deficient mice. In wild-type mice,
after antigen activation, the BCR is endocytosed via
raft-clathrin domains, a logical site for the dampening
of B-cell signaling by CD22. In resting cells, CD22
undergoes constitutive endocytosis, which can result in
internalization of high-affinity ligands of CD22 (Fig. 1).
We also study siglec-F (murine) and siglec-8
(human), which are predominately expressed on eosinophils and recognize the sialoside Siaα2-3(6-SO4=)Galβ14GlcNAc and are targets for modulating eosinophil
activation. Another siglec being actively investigated is
myelin-associated glycoprotein (siglec-4). This siglec is
expressed on glial cells and recognizes the sialoside
Siaα2-3Galβ1-3(Siaα2-6)GalNAc-R found on O-linked
glycans of glycoproteins and glycolipids. Functionally,
myelin-associated glycoprotein stabilizes interactions
between glial cells and axons essential for normal organization of myelin and inhibits axonal regeneration,
which is currently a target for pharmaceutical intervention to promote nerve regeneration.
234 MOLECULAR BIOLOGY
2006
THE SCRIPPS RESEARCH INSTITUTE
F i g . 1 . Relationship between microdomain localization of the BCR
and CD22, a regulator of BCR signaling that binds glycan ligands.
A major barrier to studying the ligand-binding properties of siglecs and their role in siglec biology is the
difficulty in creating synthetic probes that compete with
endogenous (cis) ligands. Even highly multivalent polymers containing the natural glycan sequence recognized
by a siglec will not bind to cells unless cis ligands are
first destroyed. However, we found that high-affinity
analogs of the natural sialoside ligand of CD22 bind to
native B cells and are carried into the cell by receptormediated endocytosis. Similar constructs with the ligand
of siglec-F are also bound and endocytosed by eosinophils, but by a different endocytic mechanism. We have
also developed potent inhibitors of myelin-associated
glycoprotein that reverse its ability to block axon growth,
and in collaborative studies with R. Schnaar, Johns
Hopkins University, Baltimore, Maryland, we are investigating the potential of the inhibitors to promote nerve
growth in vivo.
With these successes, we have embarked on a major
effort to identify high-affinity analogs of each siglec to
produce ligand-based tools to investigate the biological
roles of the siglecs in innate and adaptive immunity.
S I A L O S I D E A N A L O G G LY C A N A R R AY S
We have developed a robotically printed glycan array
that displays sialoside analogs to assess the affinity of
siglecs for unnatural substituents at the C-9 and C-5
positions of sialic acids. Even in the initial experiments
with 65 acyl substituents at the C-9 position of sialic
acid, the method was a powerful one for identifying
substituents that increase the affinity of siglecs by
100-fold or more (Fig. 2). In collaboration with K.B.
Sharpless, Department of Chemistry, we have created
another 80 analogs by using by click chemistry to couple members of a library of alkynes to sialosides containing 9-azido-N-acetyl-neuraminic acid. Results from
the array can be rapidly assimilated into the synthesis
of high-affinity ligands and ligand-based probes of the
F i g . 2 . Sialoside analog glycan microarray reveals high-affinity
ligands for CD22. A, Sialoside ligands of CD22 with amino-terminated linkers are printed on N-hydroxyl succinimide (NHS)–activated glass slides, resulting in a covalent amide bond. B, The natural
ligand (3) with various substituents (1, 2, 4, 6) and a nonligand
control (5) are printed in 10 replicates at 10 two-fold diluted printing concentrations. Overlay with a fluorescence-labeled CD22-Ig
chimera reveals the increased binding to various substituents compared with the natural ligand.
corresponding siglec by using our flexible chemoenzymatic synthesis strategies.
BIOENGINEERING OF CELL-SURFACE SIALOSIDES
Sialic acids with substituents at the C-9 and C-5
positions are readily taken up by cells and incorporated
into cell-surface glycans of glycoproteins and glycolipids
by the natural glycosylation pathways. Taking advantage
of this concept, we developed a novel method for in
situ photoaffinity cross-linking of CD22 to its ligands on
the same cell (cis) or adjacent cell (trans) by using a
9-aryl-azide-sialic acid. When exposed to ultraviolet
light, CD22 is rapidly cross-linked to its cis ligands
through protein-glycan covalent bonds (Fig. 3). The
striking finding is that in addition to glycan structure,
microdomain localization of CD22 strongly influences
the glycoprotein ligands that CD22 interacts with. In
fact, the predominant cis ligands of CD22 were glycans of neighboring CD22 molecules, showing homomultimeric complexes of CD22 mediated by CD22’s
ligand-binding domain.
Another application is to incorporate sialic acid
analogs that increase or decrease the affinity of a siglec
for its natural ligand to perturb the dynamics of interactions of the siglec with its cis ligand. For example, a
9-biphenylcarboxyl substituent (Fig. 3) increases the
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2006
THE SCRIPPS RESEARCH INSTITUTE
235
influenza and related H1 avian influenza viruses and
the more recent avian influenza virus (H5N1) to identify mutations required to switch specificity from avian
receptors to human-type receptors.
PUBLICATIONS
Bochner, B.S., Alvarez, R.A., Mehta, P., Bovin, N.V., Blixt, O., White, J.R.,
Schnaar, R.L. Glycan array screening reveals a candidate ligand for siglec-8. J.
Biol. Chem. 280:4307, 2005.
Collins, B.E., Blixt, O., Han, S., Duong, B., Li, H., Nathan, J.K., Bovin, N., Paulson, J.C. High-affinity ligand probes of CD22 overcome the threshold set by cis
ligands to allow for binding, endocytosis, and killing of B cells. J. Immunol.
177:2994, 2006.
F i g . 3 . Bioengineering of cell-surface glycoproteins to carry substituents at the 9-position of N-acetyl–neuraminic acid that
increase affinity (biphenylcarboxyl) or allow in situ photoaffinity
cross-linking (9-aryl-azide) of CD22 to its ligands.
affinity for CD22 by 100-fold, resulting in the strong
CD22-mediated aggregation of B cells. These basic
approaches will be of general value in elucidating the
biology of other members of the siglec family.
C O N S O R T I U M F O R F U N C T I O N A L G LY C O M I C S
Members of our laboratory also staff 2 scientific
cores for the Consortium for Functional Glycomics,
organized to elucidate the mechanisms by which
glycan-binding proteins mediate cell communication
(http://www.functionalglycomics.org/). In the past
year, scientists in the Mouse Transgenics Core, led by
Peter Sobieszczuk, created 6 novel mouse strains
from C57Bl/6 embryonic stem cells that are deficient
in genes for key glycan-binding proteins that affect
immune function. Scientists in the Glycan Array Synthesis Core, led by Ola Blixt, have produced a library of
synthetic glycans by chemoenzymatic synthesis for
use in numerous applications. In addition, scientists
in the Scripps DNA Microarray Core, led by Steve
Head, designed and conduced investigator-initiated
analysis with a custom-based microarray with genes of
relevance for the consortium.
A major achievement by staff in the Glycan Array
Synthesis Core is the development of the world largest
glycan microarray, which currently has more than 300
unique structures, mostly synthetic glycans produced
by chemoenzymatic synthesis. Now produced in collaboration with the DNA Microarray Core, the microarray is widely used by investigators around the world
to assess the specificity of glycan-binding proteins that
mediate a broad scope of biological interactions. In an
exemplary collaboration with I.A. Wilson and J. Stevens,
Department of Molecular Biology, this array was used
to investigate the specificity of the 1918 pandemic
Collins, B.E., Smith, B.A., Bengtson, P., Paulson, J.C. Ablation of CD22 in liganddeficient mice restores B cell receptor signaling. Nat. Immunol. 7:199, 2006.
Comelli, E.M., Head, S.R., Gilmartin, T., Whisenant, T., Haslam, S.M., North,
S.J., Wong, N.K., Kudo, T., Narimatsu, H., Esko, J.D., Drickamer, K., Dell, A.,
Paulson, J.C. A focused microarray approach to functional glycomics: transcriptional regulation of the glycome. Glycobiology 16:117, 2006.
Comelli, E.M., Sutton-Smtih, M., Yan, Q., Amado, M., Panico, M., Gilmartin, T.,
Whisenant, T., Lanigan, C.M., Head, S.R., Goldberg, D., Morris, H., Dell, A.,
Paulson, J.C. Activation of murine CD4+ and CD8+ T lymphocytes leads to dramatic remodeling of N-linked glycans. J. Immunol. 177:2431, 2006.
Han, S., Collins, B.E., Paulson, J.C. Synthesis of 9-substituted sialic acids as
probes for CD22-ligand interactions on B. Oxford University Press, New York, in
press. ACS Symposium Series.
Leppanen, A., Stowell, S., Blixt, O., Cummings, R.D. Dimeric galectin-1 binds
with high affinity to α2,3-sialylated and non-sialylated terminal N-acetyllactosamine
units on surface-bound extended glycans. J. Biol. Chem. 280:5549, 2005.
Paulson, J.C., Blixt, O., Collins, B.E. Sweet spots in functional glycomics. Nat.
Chem. Biol. 2:238, 2006.
Raman, R., Raguram, S., Venkataraman, G., Paulson, J.C., Sasisekharan, R.
Glycomics: an integrated systems approach to structure-function relationships of
glycans. Nat. Methods 2:817, 2005.
Singh, T., Wu, J.H., Peumans, W.J., Rouge, P., Van Damme, E.J., Alvarez, R.A.,
Blixt, O., Wu, A.M. Carbohydrate specificity of an insecticidal lectin isolated from
the leaves of Glechoma hederacea (ground ivy) towards mammalian glycoconjugates. Biochem. J. 393:331, 2005.
Stevens, J., Blixt, O., Glaser, L., Taubenberger, J.K., Palese, P., Paulson, J.C.,
Wilson, I.A. Glycan microarray analysis of the hemagglutinins from modern and
pandemic influenza viruses reveals different receptor specificities. J. Mol. Biol.
355:1143, 2006.
Stevens, J., Blixt, O., Paulson, J.C., Wilson, I.A. Glycan microarray technologies: tools
to survey host specificity of influenza viruses. Nat. Rev. Microbiol. 4:857, 2006.
Stevens, J., Blixt, O., Tumpey, T.M., Taubenberger, J.K., Paulson, J.C., Wilson,
I.A. Structure and receptor specificity of the hemagglutinin from an H5N1 influenza
virus. Science 312:404, 2006.
Taniguchi, N., Nakamura, K., Narimatsu, H., von der Lieth, C.W., Paulson, J.C.
Human Disease Glycomics/Proteome Initiative workshop and the 4th HUPO Annual
Congress. Proteomics 6:12, 2006.
Tateno, H., Crocker, P.R., Paulson, J.C. Mouse siglec-F and human siglec-8 are
functionally convergent paralogs that are selectively expressed on eosinophils and
recognize 6′-sulfo-sialyl Lewis X as a preferred glycan ligand. Glycobiology
15:1125, 2005.
van Vliet, S.J., van Liempt, E., Saeland, E., Aarnoudse, C.A., Appelmelk, B.,
Irimura, T., Geijtenbeek, T.B., Blixt, O., Alvarez, R., van Die, I., van Kooyk, Y.
Carbohydrate profiling reveals a distinctive role for the C-type lectin MGL in the
recognition of helminth parasites and tumor antigens by dendritic cells. Int.
Immunol. 17:661, 2005.
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2006
Vasiliu, D., Razi, N., Zhang, Y., Jacobsen, N., Allin, K., Liu, X., Hoffmann, J.,
Bohorov, O., Blixt, O. Large-scale chemoenzymatic synthesis of blood group and
tumor-associated poly-N-acetyllactosamine antigens. Carbohydr. Res. 3451:1447,
2006.
Westerlind, U., Hagback, P., Tidback, B., Wiik, L., Blixt, O., Razi, N., Norberg, T.
Synthesis of deoxy and acylamino derivatives of lactose and use of these for probing the active site of Neisseria meningitidis N-acetylglucosaminyltransferase. Carbohydr. Res. 340:221, 2005.
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