News and Publications
DEPARTMENT OF MOLECULAR BIOLOGY
|Peter E. Wright, Ph.D. ||Member and Chairman, Cecil H. and Ida M. Green Investigator in Medical Research |
|William Balch, Ph.D.* ||Member |
|Carlos F. Barbas III, Ph.D. ||Associate Member |
|Donald E. Bashford, Ph.D. ||Associate Member |
|William H. Beers, Ph.D. ||Member, Senior Vice President, TSRI |
|Jeffrey D. Bleil, Ph.D. ||Assistant Member |
|Charles L. Brooks III, Ph.D. ||Member |
|Dennis R. Burton Ph.D.** ||Member |
|David A. Case, Ph.D. ||Member |
|Walter J. Chazin, Ph.D. ||Associate Member |
|Luis de Lecea, Ph.D. ||Assistant Member |
|Bruce S. Duncan, Ph.D. ||Assistant Member |
|H. Jane Dyson, Ph.D. ||Associate Member |
|John H. Elder, Ph.D. ||Member |
|Martha J. Fedor, Ph.D.*** ||Assistant Member |
|Terry M. Fieser, Ph.D. ||Adjunct Assistant Member |
|Christopher K. Garcia, Ph.D. ||Assistant Member |
|Larry Gerace, Ph.D.* ||Member |
|Dan D. Gerendasy, Ph.D. ||Assistant Member |
|Elizabeth D. Getzoff, Ph.D.** ||Associate Member |
|Adam Godzik, Ph.D. ||Assistant Member |
|David B. Goodin, Ph.D. ||Associate Member |
|David S. Goodsell, Jr., Ph.D. ||Assistant Member |
|Joel M. Gottesfeld, Ph.D. ||Member |
|Robert Hallewell, D.Phil. ||Adjunct Associate Member |
|Karl W. Hasel, Ph.D.**** ||Adjunct Assistant Member, Digital Gene Technologies, La Jolla, CA |
|Donald Hilvert, Ph.D.***** ||Member |
|Jonathan D. Hirst, Ph.D. ||Assistant Member |
|Richard A. Houghten, Ph.D. ||Adjunct Member |
|Kim D. Janda, Ph.D.***** ||Member |
|John E. Johnson, Ph.D. ||Member |
|Gerald F. Joyce, M.D., Ph.D ||Member |
|Angray S. Kang, Ph.D. ||Assistant Member |
|Peter A. Kast, Ph.D.***** ||Assistant Member |
|Ehud Keinan, Ph.D. ||Adjunct Member |
|Andrzej Kolinski, Ph.D. ||Associate Member |
|James LaClair, Ph.D. ||Assistant Member |
|Richard A. Lerner, M.D. ||Member, President, TSRI, Lita Annenberg Hazen Professor of Immunochemistry, Cecil H. and Ida M. Green Chair in Chemistry |
|Clare H. McGowan, Ph.D. ||Assistant Member |
|Duncan E. McRee, Ph.D. ||Assistant Member |
|David R. Milich, Ph.D. ||Associate Member |
|David P. Millar, Ph.D. ||Associate Member |
|Louis Noodleman, Ph.D. ||Associate Member |
|Arthur J. Olson, Ph.D. ||Member |
|James C. Paulson, Ph.D. ||Adjunct Member |
|Steven I. Reed, Ph.D. ||Member |
|Victoria A. Roberts, Ph.D. ||Assistant Member |
|Paul Russell, Ph.D. ||Associate Member |
|Arnold C. Satterthwait, Jr., Ph.D. ||Assistant Member |
|Paul R. Schimmel, Ph.D. ||Member |
|Sandra Schmid, Ph.D.* ||Associate Member |
|Anette Schneemann, Ph.D. ||Assistant Member |
|Charles G. Shevlin, Ph.D. ||Assistant Member |
|Subhash C. Sinha, Ph.D. ||Assistant Member |
|Gary E. Siuzdak, Ph.D. ||Assistant Member |
|Jeffrey Skolnick, Ph.D. ||Member |
|Robyn L. Stanfield, Ph.D. ||Assistant Member |
|Charles D. Stout, Ph.D. ||Associate Member |
|Enrico Stura, D.Phil. ||Assistant Member |
|J. Gregor Sutcliffe, Ph.D. ||Member |
|John A. Tainer, Ph.D. ||Member |
|Ian A. Wilson, D.Phil. ||Member |
|Peter M. Wirsching, Ph.D. ||Associate Member |
|Curt Wittenberg, Ph.D. ||Associate Member |
|Mark Yeager, M.D., Ph.D.* ||Associate Member |
|Todd O. Yeates, Ph.D. ||Adjunct Associate Member |
|John Chung, Ph.D. ||Manager, Nuclear Magnetic Resonance Facilities |
|Michael E. Pique ||Director, Graphics Development |
SENIOR RESEARCH ASSOCIATES
|Edelmira Cabezas, Ph.D. |
|Monica Carson, Ph.D. |
|Cindy L. Fisher, Ph.D.**** ||Structural Bioinformatics, Inc., San Diego, CA |
|Liliane A. Dickinson, Ph.D. |
|Flavio Grynszpan, Ph.D. |
|Jian Li, Ph.D. |
|Tianwei Lin, Ph.D. |
|Maria A. Martinez-Yamout, Ph.D. |
|Clifford Dean Mol, Ph.D. |
|Govinda Sridhar Prasad, Ph.D. |
|Vijay Sai Reddy, Ph.D. |
|Vicente M. Reyes, Ph.D. |
|Isabelle Rooney, Ph.D.****, ||La Jolla Institute for Allergy and Immunology, La Jolla, CA |
|Christoph B. Weber, Ph.D. |
|Douglas B. Williams, Ph.D. |
|Mason M. Yamashita, M.D., Ph.D. |
|Helena Almer, Ph.D. |
|Jennifer Andris-Widhopf, Ph.D. |
|Rebecca Alexander, Ph.D. |
|Carlos E. Alvarez, Ph.D. |
|Nicolaus Albert Bahr, Ph.D.****, ||University of Bern, Bern, Switzerland |
|Yawen Bai, Ph.D. |
|David Barondeau, Ph.D. |
|Roger R. Beerli, Ph.D. |
|Bonnie Bertolaet, Ph.D. |
|Robert Bjornestedt, Ph.D.**** ||Astra Biotech Lab, Stockholm, Sweden |
|Alessandra Blasina, Ph.D. |
|Michael N. Boddy, Ph.D. |
|Yves Bourne, Ph.D.**** ||Institut de Recherche Concertee 1, Marseille, France |
|Jean-Marc Brondello, Ph.D. |
|Ronald Brudler, Ph.D. |
|Christopher M. Bruns, Ph.D. |
|Mary A. Canady, Ph.D. |
|Yi Cao, Ph.D. |
|Danilo R. Casimiro, Ph.D.**** ||Merck Laboratories, West Point, PA |
|Silvia Cavagnero, Ph.D. |
|Daphne Chapman-Shimshoni, Ph.D.**** ||Weizman Institute of Science, Tel Aviv, Israel |
|Jonathan A. Chappel, Ph.D. || MRC Collaborative Centre, London, England |
|Udayan Chatterji, Ph.D. |
|Shu-Wen W. Chen, Ph.D. |
|Joe Chihade, Ph.D. |
|John Christodoulou, Ph.D. |
|Duncan J. Clarke, Ph.D. |
|Thomas Cleveland, Ph.D. |
|Brian R. Crane, Ph.D. |
|Orlando Crescenzi, Ph.D. |
|Aymeric de Parseval, Ph.D. |
|Massimo Degano, Ph.D. |
|Genevieve Degols, Ph.D.**** || Institut de Génétique Moléculaire, Montpellier, France |
|Eugene Demchuk, Ph.D. |
|Valerie Dillet, Ph.D. |
|Xiao Fan Dong, Ph.D. |
|David G. Donne, Ph.D. |
|Elena Dovalsantos, Ph.D. |
|Brendan Duggan, Ph.D. |
|David J. Eliezer, Ph.D. |
|Patricia A. Fagan, Ph.D. |
|Richard S. Fee, Ph.D. |
|Karin Maria Flick, Ph.D. |
|Katrina T. Forest, Ph.D. |
|Mark P. Foster, Ph.D. |
|Frederique Gaits, Ph.D. |
|Carlos Garcia, Ph.D. |
|Paul Geymayer, Ph.D.**** ||University of Bern, Bern, Switzerland |
|Jayant B. Ghiara, Ph.D. |
|Phalguni Ghosh, Ph.D.**** ||Hughes Institute, Roseville, MN |
|Svetlana I. Gramatikova, Ph.D. |
|Samantha Greasley, Ph.D. |
|Karl Gruber, Ph.D. |
|Yue Guan, Ph.D. |
|Zhuyan Guo, Ph.D. |
|Steven Blaine Haase, Ph.D. |
|Stephen G. Hall, Ph.D.**** ||GenMed Corporation, San Diego, CA |
|Seungil Han, Ph.D. |
|Joan Hanley-Hyde, Ph.D. |
|Matthew R. Haynes, Ph.D. |
|Peter B. Hedlund, Ph.D. |
|Andreas Heine, Ph.D. |
|Tamara Hendrickson, Ph.D. |
|Ludger Hengst, Ph.D. |
|Thomas Herzinger, M.D. |
|Torsten Hoffmann, Ph.D ||Hoffmann La Roche, Basel, Switzerland |
|Signe M.A. Holmbeck, Ph.D. |
|Yuchu G. Hsiung, Ph.D. |
|Wei-Ping Hu, Ph.D. ||National Chung-Cheng University, Chia-Wi, Taiwan |
|Mingdong Huang, Ph.D. |
|Michael J. Hunter, Ph.D. |
|Nathalie Jourdan, Ph.D. |
|Peter Kaiser, Ph.D. |
|Junko Kanoh, Ph.D. |
|Daniel Kaufmann, Ph.D.**** ||University of Bern, Bern, Switzerland |
|Arto Tapio Kesti, Ph.D. |
|Randal R. Ketchem, Ph.D. |
|Robert Konecny, Ph.D. |
|Xianjun Kong, Ph.D.**** || Molecular Simulations, Inc., San Diego, CA |
|Richard W. Kriwacki, Ph.D. |
|Gerard Johannes Kroon, M.D. |
|Gary S. Laco, Ph.D.**** ||University of Washington Medical School, Seattle, WA |
|John H. Laity, Ph.D. |
|Wai Chung Lam, Ph.D. |
|Stefan Lanker, Ph.D. |
|Teresa Larsen, Ph.D. |
|Janet Leatherwood, Ph.D.**** ||State University of New York, Stonybrook, NY |
|Brian M. Lee, Ph.D. |
|Glen Legge, Ph.D. |
|Ronald D. Lewis II, Ph.D. |
|Xiang Li, Ph.D.**** ||Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT |
|Ying-Chuan Lin, Ph.D. |
|Benjamin List, Ph.D. |
|Gang Liu, Ph.D. |
|Qiang Liu, Ph.D.**** ||Abgenix, Inc., Fremont, CA |
|Oded Livnah, Ph.D. |
|Terence Pui Kwan Lo, Ph.D. |
|John J. Long, Ph.D. |
|Antonia Lopez, Ph.D. |
|Guang Xiang Luo, Ph.D.+ |
|Thomas J. Macke, Ph.D. |
|Jens C. Madsen, Ph.D.**** ||Bruker Spectrospin, Taby, Sweden |
|Lena E. Maler, Ph.D. |
|Georges Mer, Ph.D. |
|Siobhan M. Miick, Ph.D.**** ||Nanogen, Inc., San Diego, CA |
|Debasisa Mohanty, Ph.D. |
|Vincent Moncollin, Ph.D. |
|Guillaume Mondesert, Ph.D. |
|Garrett M. Morris, D.Phil. |
|Rabi A. Musah, Ph.D. |
|Sangari Mylvaganam, Ph.D. |
|Padmaja Natarajan, Ph.D. |
|Tyzoon Nomanbhoy, Ph.D. |
|Jacek Nowakowski, Ph.D. |
|Eric H. Olender, Ph.D.**** ||San Jose State University, San Jose, CA |
|Phillip Ordoukhanian, Ph.D. |
|Angel Ramirez Ortiz, Ph.D. |
|Michael J. Osborne, Ph.D. |
|Michael R. Otto, Ph.D. |
|Jean-Luc Pellequer, Ph.D. |
|Gabriela Perez-Alvarado, Ph.D. |
|Jeroen A. Pikkemaat, Ph.D. |
|Stefan Prytulla, Ph.D.**** ||Institut für Pharmazeutische Chemie, Graz, Austria |
|Christoph Rader, Ph.D. |
|Ishwar Radhakrishnan, Ph.D. |
|Jennifer Lynn Radkiewicz, Ph.D. |
|Sun Ai Raillard-Yoon, Ph.D.**** ||Maxygen, Santa Clara, CA |
|Mohan Srinivasa Rao, Ph.D. |
|Boris Alexander Reva, Ph.D. |
|Nicholas Rhind, Ph.D. |
|Lluis Ribas De Pouplana, Ph.D. |
|Olaf Ritzeler, Ph.D.**** ||University of Bern, Bern, Switzerland |
|Jeffrey Keith Rogers, Ph.D. |
|Christopher Rosin, Ph.D. |
|Franziska Ruchti, Ph.D. |
|Leszek Rychlewski, Ph.D. |
|Kandasamy Sakthivel, Ph.D. |
|Michel F. Sanner, Ph.D. |
|Niranjan Sardesai, Ph.D. |
|Mallika Sastry, Ph.D. |
|Kevin Y. Sato, Ph.D. |
|Sergio D.B. Scrofani, Ph.D. |
|David Segal, Ph.D. |
|Marisa Segal, Ph.D. |
|Brent W. Segelke, Ph.D.**** ||Lawrence Livermore National Laboratory, Livermore, CA |
|Doron Yacov Shabatt, Ph.D. |
|Joan-Emma Shea, Ph.D. |
|Terry Lee Sheppard, Ph.D. |
|Kazuhiro Shiozaki, Ph.D. |
|William Arthur Shirley, Ph.D. |
|Anjana Sinha, Ph.D.**** ||University of California, San Diego, CA |
|Doree F. Sitkoff, Ph.D. |
|Monique F.M. Smeets, Ph.D. |
|Velin Slatkov Spassov, Ph.D. |
|Jeffrey A. Speir, Ph.D. |
|Charles H. Spruck III, Ph.D. |
|Jayashree Srinivasan, Ph.D. |
|Brian Steer, Ph.D. |
|Peter Steinberger, Ph.D. |
|Heimo Strohmaier, Ph.D. |
|David T. Stuart, Ph.D. |
|Ying Su, Ph.D. |
|Kwang-Ai Won Szymanski, Ph.D. |
|Fujie Tanaka, Ph.D. |
|John Tate, Ph.D. |
|Maria Julie Thayer, Ph.D. |
|Elizabeth A. Thomas, Ph.D. |
|Robert Turner, Ph.D. |
|Maria Henar Valdivieso, Ph.D.**** ||University of Salamanca, Salamanca, Spain |
|Sara Venturini, Ph.D. |
|Michal Vieth, Ph.D. |
|John Howard Viles, Ph.D. |
|Keisuke Wakasugi, Ph.D. |
|Chien-Chia Wang, Ph.D. |
|Mark Howard Watson, Ph.D. |
|Douglas B. Williams, Ph.D. |
|Pamela A. Williams, Ph.D. |
|Christer Wingren, Ph.D. |
|Martin Charles Wright, Ph.D.**** ||Phylos, Boston, MA |
|Bin Xia, Ph.D. |
|Jian Xu, Ph.D. |
|Jing Xu, Ph.D. |
|Jian Yao, Ph.D. |
|William S. Young, Ph.D.**** ||Molecular Simulations, Inc., San Diego, CA |
|Ke Zeng, Ph.D. |
|Baohong Zhang, Ph.D. |
|Hongyu Zhang, Ph.D.**** ||Center for Advanced Research in Biotechnology, Rockville, MD |
|Li Zhang, Ph.D. |
|Mingzhu Zhang, Ph.D.**** ||Vanderbilt University, Nashville, TN |
|Ouwen Zhang, Ph.D. |
|Ruoheng Zhang, Ph.D. |
|Xu-Yang Zhao, Ph.D. |
|Guo-Fu Zhong, Ph.D. |
|Zhongxiang Zhou, Ph.D.**** ||Alanex Corporation, San Diego, CA |
|Leiming Zhu, Ph.D. |
|Andrew S. Arvai |
|Diane Marie Kubitz |
|Stephen J. Benkovic, Ph.D. ||The Pennsylvania State University, University Park, PA |
|Sture Forsen, Ph.D. ||University of Lund, Lund, Sweden |
|Arne Holmgren, M.D., Ph.D. ||Karolinska Institute, Stockholm, Sweden |
|Tai-huang Huang, Ph.D. ||Institute of Biomedical Sciences, Academica Sinica, Taipei, Taiwan |
|Kenneth Kustin, Ph.D. ||Brandeis University, Waltham, MA |
|Johannes Langedijk, Ph.D. ||Institute for Animal Sciences and Health, Lelystad, the Netherlands |
|Robert D. Rosenstein, Ph.D. ||Lawrence Berkeley Laboratory, Berkeley, CA |
|Michael Taussig, Ph.D. ||Babraham Research Institute, Cambridge, England |
| * Joint appointment in Department of Cell Biology |
| ** Joint appointment in Department of Immunology |
| *** Joint appointment in The Skaggs Institute for Chemical Biology |
| **** Appointment completed; new location shown |
|***** Joint appointment in Department of Chemistry |
| + Appointment completed |
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Peter E. Wright, Ph.D.
Research in the Department of Molecular Biology encompasses a wide range of disciplines, extending from structural biology at one extreme to molecular genetics at the other. During the past year, our scientists have continued to make exciting progress toward understanding the fundamental molecular events that underlie the processes of life. These accomplishments have been reported on the pages of the most influential journals and have been rewarded by a higher than ever level of grant support. The faculty members of this department have every reason to be proud of these achievements, as do the exceptionally talented postdoctoral fellows, graduate students, technologists, and support personnel, who are absolutely vital to the success of each and every research program.
Within the confines of this brief overview, I cannot do justice to the many exciting research programs within the department. These are described in detail on the following pages, and a few of the highlights are mentioned briefly here.
A collaboration between Joel Gottesfeld of this department and Peter Dervan at the California Institute of Technology led to the development of small synthetic molecules that bind in the minor groove of DNA and can regulate gene expression. Gottesfeld, Dervan, and their colleagues showed that these compounds, pyrrole-imidazole polyamides, can be designed to target specific DNA sequences and interfere with the transcription of specific genes. Polyamides directed against DNA target sequences within the HIV enhancer and promoter are highly effective inhibitors of viral replication in isolated human blood peripheral lymphocytes. These compounds offer the potential for design of small molecules that can regulate the transcriptional activity of selected target genes in living cells. These results are extremely exciting and could eventually lead to a new class of therapeutic agents directed against a broad spectrum of viral and other diseases.
A different approach to targeted gene regulation is being pursued by Carlos Barbas and his colleagues. Proteins containing multiple zinc-finger domains are being engineered to recognize specific binding sites containing as many as 18 nucleotides. Such proteins can target a unique locus in the human genome and have considerable potential as genetic regulators in a variety of human diseases.
In the area of structural biology, a number of spectacular new advances were made during the past year. Ian Wilson and his coworkers determined the three-dimensional structure of murine CD1, a protein that is distantly related to MHC molecules and that presents antigens to T cells. The overall structure is similar to that of an MHC class I molecule, but CD1 contains a deep, almost entirely hydrophobic, binding cavity. The structure reinforces the view that the role of CD1 is to display lipid and glycolipid antigens.
In another major advance, Elizabeth Getzoff and coworkers determined the atomic resolution structure of a signaling intermediate in the light cycle of a photoreceptor protein by using millisecond time-resolved x-ray crystallography. This achievement is impressive, because the lifetime of the photocycle intermediate is less than 1 second, and the findings provide the first detailed insights into the structure of a light-activated intermediate. The structural changes observed, relative to the ground state, suggest a mechanism for signal transduction and provide a general framework for understanding the structural mechanisms of protein photocycles.
Other important structures determined during the past year include catalytic antibodies; animal and plant viruses; calcium signal transduction proteins; the first structure of a complex between a transcriptional activation sequence and a domain of the coactivator CREB-binding protein; and the structure of a human DNA-repair enzyme, uracil-DNA glycolase. This last structure is of interest for the insights it provides into the mechanism of recognition of damaged bases, which are flipped out from the double helix and into the active site of the enzyme, where they are excised.
Gerald Joyce and Martin Wright developed a new method to evolve catalytic RNA molecules continuously in vitro. This method is a major advance in the evolution of novel RNA catalysts. In addition, continuous in vitro evolution provides a more realistic model of biological evolution and should provide new insights into evolutionary mechanisms. Other work in the Joyce laboratory led to the development of a small and highly efficient DNA enzyme that can be tailored to recognize and cleave different RNA target sequences. Such DNA enzymes have potential applications both as laboratory reagents and in medicine.
In the area of cell-cycle regulation, Curt Wittenberg and his colleagues published several key articles describing the phosphorylation-dependent degradation of cyclins. Cyclins regulate cell-cycle transitions in eukaryotes through association with cyclin-dependent protein kinases. Wittenberg and coworkers showed that phosphorylation of a G1 cyclin, by the kinase that the cyclin activates, provides a signal that promotes rapid degradation of the cyclin by a ubiquitin-dependent pathway and thereby makes activation self-limiting. This work is providing important new insights into the complex molecular mechanisms that govern the major cell-cycle transitions.
Finally, I am delighted to report that we have recruited several outstanding scientists to full or joint appointments in the department. Gary Siuzdak, director of the Mass Spectrometry Facility, joined our faculty during the past year, and Paul Schimmel, Jamie Williamson, and Martha Fedor will soon bring new strengths to our programs in the molecular and structural biology of RNA. In addition, Jonathan Hirst, Chris Garcia, Robyn Stanfield, and Luis de Lecea have recently joined us and strengthen our research in the areas of theory, x-ray crystallography, and molecular neurobiology.
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Crystallographic Analyses of Viruses and Macromolecular Assemblies
J. Johnson, A. Schneemann, V. Reddy, T. Lin, W. Wikoff, A. Kumar, P. Natarajan, S. Hall, J. Tate, F. Dong, M. Canady, H. Giesing, B. Sheehan, H. Langedijk*
* Institute for Animal Science and Health, Lelystad, the Netherlands
Our group investigates the structure and function of viruses to elucidate molecular mechanisms of infection. We use this information to determine potential targets for antiviral agents and to use viruses as reagents for understanding and exploiting the biology of the cell. We have used a variety of virus families for our investigations; each family has novel properties appropriate for specific lines of investigation.
For research on assembly, we use virus groups readily studied in vitro and in vivo. An area of specific interest is the formation of quasi-equivalent viral capsids in which the same gene product participates in polymorphic interactions to generate both hexamers and pentamers in the formation of icosahedral shells. These structures are the biological equivalent of Buckminster Fuller's geodesic domes. They provide particles large enough to package and protect the genome and have targeting properties to deliver the genome to host cells for viral replication.
Knowledge of the structure of cowpea chlorotic mottle virus has enabled us to determine the parts of the subunit that control different aspects of assembly. In vitro assembly with structure-based mutations has been studied in collaboration with M. Young at Montana State University, who has developed an Escherichia coli expression system for the cowpea chlorotic mottle virus subunit and can purify, refold, and assemble the protein into viruslike particles. These studies confirm the postulated functional roles, based on crystallographic studies, of different regions of the subunit.
To further understand assembly polymorphism, we examined the different patterns of assembly of the coat protein of alfalfa mosaic virus. In nature, this multipartite RNA virus exists as four bacilliform particles in which capsid protein forms a particle proportional to the size of the RNA genome segment packaged. Purified viral protein can assemble in vitro into an icosahedral particle in the absence of RNA. We determined the structure of this assembly product at 4.0-Å resolution. Using the coordinates of the subunit structure, we created a model of the bacilliform particles that agrees in detail with the hexagonal lattice observed in electron microscopy studies.
In collaboration with A. Schneemann, we are exploring structure-function relationships in the nodavirus and tetravirus families of animal viruses. Our studies have shown that capsid polymorphism is controlled by a protein switch and/or RNA, that the maturation cleavage required for infection is autocatalytic and depends on assembly and the binding of calcium ions, that the release of RNA probably depends on a specific protein-RNA interaction that occurs only once in the context of the symmetric capsid, and that membrane translocation of RNA is probably mediated by a pentameric helical bundle that is rendered covalently independent from the subunit by the maturation cleavage.
Although the quaternary structures and subunits of nodaviruses and tetraviruses differ dramatically in size, our studies have shown an evolutionary relationship between the two families. Using heterologous protein expression systems in which the capsid protein spontaneously assembles to form particles, we crystallized and solved the structures of particles specifically mutated to reveal the atomic details of the phenomena described earlier. Recently, we used computational chemistry methods to explore assembly trajectories and their dependence on intersubunit stabilities.
We are investigating the use of plant viral particles as carriers for genetically inserted, heterologous polypeptides up to 40 amino acids long. These polypeptides have been used to generate neutralizing antibodies to HIV and to present peptide antagonists that trigger cell-surface phenomena. Structures of these "chimeric viruses" have enabled us to design presentations that increase the efficacy of inserted peptides and to better understand factors that affect peptide folding and viral assembly.
We are continuing the first crystallographic study of the capsid of HK97, a -like double-stranded DNA bacteriophage. The T = 7, 620-Å diameter particles were assembled by expressing the gene for the capsid protein in E. coli. These particles were crystallized, and x-ray diffraction patterns beyond 3.5-Å resolution were obtained. A data set has been collected at the Cornell High Energy Synchrotron Source, processed to 7.0-Å resolution, and merged with ultralow resolution data (200- to 16-Å resolution) collected at the Stanford Synchrotron Radiation Laboratory. Phases between 200- and 50-Å resolution were computed with the particle model determined by electron cryo-microscopy and image reconstruction at 35-Å resolution. Phases for the higher resolution data were determined by using extension procedures and the 60-fold noncrystallographic symmetry. The structure shows a classical T = 7 lattice with the subunits formed predominantly of -helices. On the basis of the x-ray structure and a electron cryo-microscopy reconstruction of the 450-Å diameter procapsid, we have proposed a detailed mechanism for particle maturation.
Baker, T.S., Johnson, J.E. Low resolution meets high: Towards a resolution continuum from cells to atoms. Curr. Opin. Struct. Biol. 6:585, 1996.
Baker, T.S., Johnson, J.E. Principles of virus structure determination. In: Structural Biology of Viruses. Chiu, W., Burnett, R.M., Garcea, R. (Eds.). Oxford University Press, New York, 1997, p. 38.
Bothner, B., Dong, X., Bibbs, L., Johnson, J., Siuzdak, G. Evidence of viral capsid dynamics using limited proteolysis and mass spectrometry. J. Biol. Chem., in press.
Chandrasekar, V., Johnson, J. The structure of tobacco ringspot virus: A link in the evolution of icosahderal capsids in the picornavirus superfamily. Structure, in press.
Chandrasekar, V., Munshi, S., Johnson, J.E. Crystallization and preliminary x-ray analysis of tobacco ringspot virus. Acta Crystallogr. D53:125, 1997.
Flasinski, S., Dzianott, A., Speir, J.A., Johnson, J.E., Bujarski, J.J. Structure-based rationale for the rescue of systemic movement of brome mosaic virus by spontaneous second-site mutations in the coat protein gene. J. Virol. 71:2500, 1997.
Johnson, J.E., Lin, T., Lomonossoff, G.P. Presentation of heterologous peptides on plant viruses: Genetics, structure and function. Annu. Rev. Phytopathol. 35:67, 1997.
Johnson, J.E., Rueckert, R.R. Packaging and release of the viral genome. In: Structural Biology of Viruses. Chiu, W., Burnett, R.M., Garcea, R. (Eds.). Oxford University Press, New York, 1997, p. 269.
Johnson, J.E., Schneemann, A. Nodavirus endopeptidase. In: Handbook of Proteolytic Enzymes. Barret, A.J., Rawlings, N.D.,Woessner, J.F. (Eds.). Academic Press, San Diego, in press.
Johnson, J.E., Speir, J.A. Quasi-equivalent viruses: A paradigm for protein assemblies. J. Mol. Biol. 269:665, 1997.
Kumar, A., Chipman, P., Yusibov, V., Fita, I., Hatta, Y., Loesch-Fries, S., Baker, T.S., Rossmann, M.G., Johnson, J.E. The structure of alfalfa mosaic virus capsid protein assembled as a T = 1 icosahedral particle at 4.0-Å resolution. J. Virol. 71:7911, 1997.
Porta, C., Lin, T., Johnson, J., Lomonossoff, G. The development of cowpea mosaic virus as a potential source of novel vaccines. Intervirology 39:79, 1996.
Reddy, V.S., Giesing, H., Kumar, A., Morton, R., Post, C.B., Brooks, C., Johnson, J.E. Energetics of quasi-equivalence: Computational analysis of protein-protein interactions in icosahedral viruses. Biophys. J., in press.
Schneemann, A., Reddy, V., Johnson, J.E. The structure and function of nodavirus particles: A paradigm for understanding chemical biology. Adv. Virus Res., in press.
Spall, V.E., Porta, C., Taylor, K.M., Lin, T., Johnson, J.E., Lomonossoff, G.P. Antigen expression on the surface of a plant virus for vaccine production. In: Engineering Crops for Industrial End Uses. Shewry, P.R., Napier, J.A., Davis, P. (Eds.). Portland Press, London, in press.
Wikoff, W., Tsai, C.J., Wang, G., Baker, T.S., Johnson, J.E. Crystallographic analysis and cryoelectron microscopy reconstruction of cucumber mosaic virus. Virology 232:91, 1997.
Zlotnick, A., Natarajan, P., Munshi, S., Johnson, J.E. Resolution of space group ambiguity and the structure determination of nodamura virus to 3.3 Å resolution from pseudo R32 (monoclinic) crystals. Acta Crystallogr., in press.
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Assembly and Uncoating of Icosahedral Viruses
A. Schneemann, D. Marshall, V. Reddy, F. Dong, J. Johnson
Coat proteins of nonenveloped, icosahedral animal viruses have multiple functions during the course of viral infection and spread, including assembly of the viral capsid, specific encapsidation of the viral genome, binding to a cellular receptor, and uncoating. We are interested in the chemical and structural determinants that endow a single polypeptide chain with such versatility. Our investigations focus on a structurally and genetically well-characterized model system, the T = 3 nodaviruses.
Nodaviruses are assembled from 180 copies of a single coat protein and two strands of messenger sense RNA. To acquire infectivity, assembled particles must undergo a maturation step in which subunits of the coat protein autocatalytically cleave into two polypeptides. We are investigating the precise pathway of viral assembly and the mechanism of the associated cleavage reaction. Our studies are guided by the high-resolution structures of several nodaviruses that have been determined in the laboratory of J. Johnson, Department of Molecular Biology. Analysis of the atomic structures of these viruses has enabled us to detect regions in the coat protein that appear to be critical in regulating viral assembly, stability, and maturation. For example, genetic, biochemical, and biophysical analyses have shown that the N-terminal part of the coat protein regulates the shape of viral particles, whereas the C-terminal part controls specific encapsidation of the viral genome.
A second area of research focuses on viral uncoating. Uncoating and delivery of viral genomes into the cytosol of susceptible cells are poorly understood processes. The viral nucleic acid must cross a cellular membrane at some point during entry, but the mechanistic details of this step remain unknown. The nodaviruses contain a preformed helical bundle just below the surface of the shell that may form a channel within the endosomal membrane after receptor-mediated endocytosis of the virion. This channel might be used for translocation of genomic RNA across the membrane into the cytosol. We have obtained preliminary evidence that supports this model.
The third project in our laboratory is determining and isolating the viral receptor protein. We have generated monoclonal antibodies that inhibit nodavirus infection of cultured cells; these antibodies will be used to isolate the receptor protein. Availability of the viral receptor will enable us to study the initial interactions of the viral particle with the cell surface and will help us to elucidate the mechanism of viral uncoating in detail.
Johnson, J.E., Schneemann, A. Nodavirus endopeptidase. In: Handbook of Proteolytic Enzymes. Barret, A.J., Rawlings, N.D., Woessner, J.F. (Eds.). Academic Press, San Diego, in press.
Schneemann, A., Reddy, V., Johnson, J.E. The structure and function of nodavirus particles: A paradigm for understanding chemical biology. Annu. Rev. Virus Res., in press.
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X-Ray Crystallographic Studies of Immunologically Important Macromolecules, Growth Factors, Receptors, and Enzymes Involved in Human Disease
I.A. Wilson, E.A. Stura, R.L. Stanfield, K.C. Garcia, T.A. Cross, W.L. Densley, M. Degano, S.E. Greasley, K. Gruber, J.B. Ghiara, M.R. Haynes, A. Heine, T. Horton, K. Hotta, M. Huang, D.A. Jewell, H.-M. Li, O. Livnah, G. Luo, E.E. Ollmann, K.A. Renner, V.M. Reyes, C.E.A. Scott, B.W. Segelke, J.A. Speir, R.S. Stefanko, Y. Su, M.J. Taussig,* D.B. Williams, J. Xu, M.M. Yamashita, K. Zeng
* Babraham Institute, Cambridge, England
The main goal of our research program is to understand macromolecular recognition, especially in the immune system. Current projects include x-ray crystallographic studies of antibody-antigen complexes; complexes that consist of T-cell receptors (TCRs), MHC molecules, and peptide antigens; classical and nonclassical MHC molecules and their antigens; human growth factors and their receptors; and enzyme targets for chemotherapy.
The TCR is a heterodimeric glycoprotein expressed on the surface of T lymphocytes that plays a central role in the recognition of peptide antigens in the context of the MHC. The ability to discriminate between self and foreign antigens depends on the fine specificity of the interaction between TCRs and MHC molecules. We have determined the three-dimensional structure of the murine class I TCR 2C to a resolution of 2.5 Å. Overall, the structure generally resembles an Fab fragment of an antibody. However, significant differences are found in the pairing of the variable and the constant domains and in the unconventional fold of the constant domain of the -chain. The hypervariable loops form a flat surface with a deep central pocket that is involved in the recognition of the peptide antigen bound to the MHC molecule.
To define the molecular basis of the TCR-MHC interaction, we have crystallized and are determining the structures of 2C-MHC complexes with both self and foreign peptide antigens bound to the MHC. The peptide residues contribute about 25% of the surface buried in the formation of the complex. The third complementarity-determining regions of the - and ß-chains of the TCR interact with the central residue of the peptide bound to the MHC. Specific recognition of the MHC molecule is achieved through contacts of complementarity-determining regions 1 and 2 of the - and the ß-chain of the TCR to the helices of the peptide-binding domain.
In the MHC class II system, our focus has been on the production and crystallization of TCRs specific for the murine class II MHC molecule IAd in complex with ovalbumin, hemagglutinin, and insulin peptides. Structural studies of class II TCR-MHC complexes would address the important question of whether TCR recognition of class I MHC molecules differs substantially from TCR recognition of class II molecules. The TCR-MHC studies are being done in collaboration with L. Teyton, Department of Immunology.
CLASS I AND CLASS II MHC MOLECULES
Several cell-surface molecules involved in T-cell activation have been expressed in soluble forms in collaboration with Dr. Teyton and A. Brunmark, R.W. Johnson Pharmaceutical Research Institute. These include murine MHC class I molecules H-2Ld, H-2Kb mutants and murine class II molecule IAd, each complexed with peptides. Evaluation of a number of protein engineering strategies was necessary to produce milligram quantities of IAd for structure-function studies.
H-2Ld is alloreactive with the H-2Kb--specific TCR 2C, and therefore a structural comparison between H-2Ld and H-2Kb is of great value. The structure of H-2Ld has been determined with a mixture of peptides (Fig. 1).
The results show that the allogeneic reaction appears to be due mainly to the C-terminal residues of specific peptides that provide the optimal shape and chemistry for 2C reactivity normally present in the syngeneic H-2Kb interaction. The class II molecule IAd is biologically well characterized and is a factor in murine models of autoimmunity. Two crystal structures of IAd with different peptides have been determined at 2.4- and 2.6-Å resolution and reveal novel elements of this particular class II peptide binding.
Other unusual interactions between MHC class I molecules and small hapten antigens were examined by determining the structure of H-2Kb complexed with a glycopeptide. The disaccharide of the ligand is visible on the outer boundaries of the binding groove, where interactions with TCRs would predominate. These studies have furnished detailed new insights on antigen selection and presentation to TCRs and have advanced our understanding of these complex interactions that are critical to cellular immunity.
The structure of 13G5, an antibody to metallocene that catalyzes the disfavored exo Diels-Alder reaction, has been determined at 1.95-Å resolution for the unbound form and for the antibody in complex with a ferrocene derivative (Fig. 2).
Interactions between antibody side chains and the inhibitor provide the basis for understanding the chemical transformation. Modeling studies with transition-state analogs and substrates suggest hypotheses for the preference for the exo vs the endo pathway.
The structure of 33F12, an aldolase antibody that was generated by reactive immunization, has been determined at 2.15-Å resolution. The particular sequence of residues in the binding pocket and the hydrophobic environment of the pocket can account for the unusual pKa (~6.0) and reactivity of a lysine in the active site. Determination of the structure of a similar natural aldolase enzyme is in progress.
Antibody 5C8 catalyzes the disfavored 6-endo-tet ring closure reaction of trans-epoxy alcohols (violating "Baldwin's rules"). The crystal structures of the Fab fragment of 5C8, unbound (to 2.5 Å) and in complex with the transition-state analog (to 2.0 Å), have been determined (Fig. 3). Further crystallographic and modeling studies are being done to establish the catalytic mechanism and explain the observed regiospecificity and stereospecificity.
The Fab fragments of two antibodies that catalyze another Diels-Alder reaction and a decarboxylation reaction have been crystallized both in their native forms and in complex with their corresponding transition-state analogs. The x-ray data for these two Fabs in both native and complex forms were collected at 2.1 and 2.2 Å, respectively, for each antibody. Refinements of these four structures are in progress. The catalytic antibody projects are being done in collaboration with R. Lerner of the Departments of Chemistry and Molecular Biology; with C. Barbas and C. Shevlin of the Department of Molecular Biology; and with K. Janda, D. Hilvert, and C.-H. Wong of the Department of Chemistry.
NEUTRALIZING ANTIBODIES TO HIV TYPE I
We are studying several antibodies that neutralize HIV type 1. Four of these antibodies were generated by Repligen Corporation (Needham, MA) against a peptide of 40 amino acids that corresponds to the third hypervariable (V3) loop of the HIV type 1 surface glycoprotein gp120. These four antibodies differ in their viral-strain specificity and have overlapping but nonidentical epitopes on the V3 loop. We have determined the structures for Fab fragments of antibody 50.1 in the unliganded and peptide-bound forms, of antibody 59.1 in two peptide-bound forms, and of antibody 58.2 in several peptide-bound forms.
The structures of the peptides bound to the Fabs of 50.1 and 59.1 show that the V3 loop adopts an extended structure, followed by a type II ß-turn around residues Gly-Pro-Gly-Arg. The type II turn is followed by a turn of 310 helix, resulting in a double turn at the tip of this loop. We replaced an alanine residue in the double-turn region of the loop with an -aminoisobutyric acid residue to constrain the secondary structure in this part of the peptide. The crystal structure of Fab 59.1 in complex with the designed peptide containing the -aminoisobutyric acid residue showed no difference, as expected, from the original complex. However, nuclear magnetic resonance studies by H. Dyson, Department of Molecular Biology, of the original and the designed peptides showed that the -aminoisobutyric acid residue does indeed confer additional structure on the free peptide in solution.
The structures of the Fab of 58.2 in complex with linear and cyclic peptides and with peptides containing -aminoisobutyric acid show that the V3 peptide can adopt a conformation that differs from that of the V3 peptide bound to Fabs of 50.1 and 59.1. In the 58.2 complexes, the peptide differs around residues Gly-Pro-Gly-Arg, adopting a type I turn, followed again by another unclassified turn to form a different type of double turn. The linear and cyclic peptides and the peptides containing -aminoisobutyric acid all adopt this alternative conformation. These results indicate that the V3 loop can adopt at least two conformations on the viral surface.
Antibodies b12 and 3B3 are neutralizing antibodies that recognize the CD4 binding site of gp120. These antibodies were developed in the laboratories of C. Barbas and D. Burton, Department of Molecular Biology. Phage display technology was used to obtain the antibodies from a library of antibodies derived from sera from patients with long-term asymptomatic HIV infection. These antibodies potently neutralize more than 75% of American HIV isolates and 50% of foreign isolates tested and strongly neutralize primary viral isolates. Studies to obtain crystals of the Fab fragments of these antibodies in complex with gp120 are under way. In a collaboration with Progenics (Tarrytown, NY), we are also studying complexes of various CD4 constructs with gp120.
Refinement is in progress for a complex of testosterone with an Fab. A molecular replacement solution has also been obtained for the free Fab. The refinement of the Fab to estrone-3-glucuronide is nearing completion.
The blood coagulation protease cascade initiated by tissue factor (TF) can be greatly inhibited in vivo by 5G9, a potent monoclonal antibody to human TF. This antibody binds the carboxyl module of the extracellular domain of TF at a nanomolar binding constant and inhibits formation of the ternary complex consisting of TF, factor VIIa, and factor X that is essential for the initiation of the protease cascade in blood coagulation.
To study recognition of TF by the antibody, we determined the crystal structures of the extracellular modules of human TF, an Fab fragment of 5G9, and a complex consisting of TF and the Fab at 2.4, 2.5, and 3.0 Å, respectively, and measured the binding constants of a panel of TF mutants with 5G9 in collaboration with T. Edgington and W. Ruf, Department of Immunology. The structure of the Fab complexed with TF (Fig. 4)
explains and confirms the mutagenesis results, illustrates the molecular mechanism of antibody-antigen recognition, and provides insights into the mechanism by which 5G9 can inhibit formation of the ternary complex.
IL-2 is one of the most important regulators of the immune response. It produces its effects by binding to its receptor, which is composed of three distinct chains, , ß, and . We are involved in crystallographic studies of this heterotrimeric IL-2 receptor system in a collaboration with T. Ciardelli at Dartmouth College.
The structure of the extracellular domain of the receptor for erythropoietin and an antagonist peptide has been determined to the resolution of 2.8 Å. The antagonist peptide was derived from a set of agonist peptides, which were discovered via phage-display techniques in collaboration with R.W. Johnson Pharmaceutical Research Institute and Affymax, Palo Alto, California. A single amino acid modification (tyrosine to 3,5-dibromotyrosine) reversed the activation properties of the peptide. The structure reveals a 2:2 assembly in which two peptides bind two receptor molecules and generate an asymmetric receptor dimerization, a condition that differs from the perfect twofold symmetry observed for the complex containing the peptide agonist.
Symmetric dimerization is a common feature of other erythropoietin receptor--peptide complexes that have agonistic properties. It is well established that in many systems receptor dimerization is the initial key event in signaling, but in this instance, we showed that a nonproductive mode of dimerization occurs. Such a difference in dimer formation can have a great impact on the understanding of signaling in both cytokine and other receptor systems. We are currently determining structures of the agonist peptide complexes with the erythropoietin receptor in collaboration with L. Jolliffe, R.W. Johnson Pharmaceutical Research Institute.
Glycinamide ribonucleotide transformylase and aminoimidazole carboxamide ribonucleotide transformylase are folate-dependent enzymes involved in the de novo biosynthesis of purine. These enzymes are potential targets for anticancer and antiinflammatory drugs. Crystal structures are in progress for a number of folate-derived inhibitors synthesized in D. Boger's laboratory, Department of Chemistry, and cocrystallized with wild-type Escherichia coli glycinamide ribonucleotide transformylase. These x-ray structures will guide future development of novel nonfolate analogs specific for glycinamide ribonucleotide transformylase, thereby reducing nonspecific interaction with other folate-dependent enzymes in the cell.
Recently, a novel mutant form of E. coli glycinamide ribonucleotide transformylase that lacks the ability to form dimers was developed by S. Benkovic, Pennsylvania State University, in conjunction with P. Jenning's laboratory, University of California, San Diego. We are determining the x-ray structures of this mutant and of additional nonactive mutant structures of glycinamide ribonucleotide transformylase in a number of complexed forms to provide evidence for the mechanism of transformylation.
GUANINE NUCLEOTIDE DISSOCIATION INHIBITOR
The guanine nucleotide dissociation inhibitor (GDI) regulates the retrieval of Rabs in the vesicular traffic while inhibiting the dissociation of GDP from Rabs, small GTP-binding proteins related to the Ras superfamily. The crystal structure of the bovine -isoform of GDI expressed from E. coli has been determined to 1.8-Å resolution. Mutagenesis studies have shown that a substructure in domain I is possibly involved in Rab association. The structure of the GDI-Rab complex has also been investigated. Meanwhile, a more active form of GDI expressed from insect cells has been crystallized, and x-ray data have been collected at 1.7-Å resolution. The high-resolution structure of this GDI will reveal any posttranslational modifications that this molecule undergoes in higher eukaryotic systems.
CD1 is an MHC-like antigen-presenting molecule. Recent studies have shown that CD1 may present mycobacterial cell wall lipids or phage display--derived peptides as antigens to T cells. Attempts to elute a natural ligand directly from CD1 molecules have been unsuccessful. The crystal structure of mouse CD1d1 has been determined to 2.8-Å resolution in two different space groups. Determination of the structure revealed a molecule markedly similar to MHC class I molecules but with a larger, mostly hydrophobic, ligand-binding groove (Fig. 5).
A second structure determination was undertaken to elucidate the nature of the interaction between CD1 and a large hydrophobic peptide presented to T cells by mouse CD1d1. Several features of the molecule are now apparent that were poorly ordered in the original crystal structure, including several carbohydrate moieties. Surprisingly, a long unbranched ligand occupying the binding groove is also apparent. The density for this ligand is consistent with density seen in the binding groove from the original structure determination and is not consistent with peptide. Attempts to crystallize the CD1d1-peptide complex are continuing. The studies on mouse CD1d1 have been done in collaboration with P. Peterson's group and others at R.W. Johnson Pharmaceutical Research Institute; those on human CD1, with M. Kronenberg, La Jolla Institute of Allergy and Immunology.
We are also attempting to crystallize the human CD1b isoform. Earlier reports stated that this molecule presents mycolic acid derived from mycobacterial extracts to T lymphocytes. Recent studies detected a series of natural lipoglycans that are ligands for human CD1b and that elicit T-cell responses. The structure of the complex of CD1b with a lipid would provide insights into the presentation of nonpeptide antigens by MHC-like molecules to T cells. The studies on CD1b have been done in collaboration with R. Modlin, University of California, Los Angeles, with ligands provided by M. Brenner and S. Porcelli, Harvard Medical School.
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Methods for the Crystallization of Macromolecules
E.A. Stura, S. Ghosh, A. Muhlberg, K. Hotta, P. Shim
The development of methods and techniques for the rapid determination of crystallization conditions with minimal amounts of proteins and nucleic acids has been the focus of the research this current year.
Reverse screening, a method based on the principle that a precipitant or buffer should play no role other than that of inducing supersaturation or maintaining the pH, has been applied to proteins that have a binding site for phosphate moieties. In collaboration with J. Elder and C. Stout, Department of Molecular Biology, we used sodium citrate instead of ammonium sulfate, which is a phosphate mimic, to obtain complex crystals of dUTPase with deoxynucleotide diphosphates and triphosphates. These complexes are not compatible with the 1.8-Å diffracting crystals obtained by using cacodylate, another phosphate mimic, which can accommodate only deoxynucleotide monophosphates.
Reverse screening has been also applied to the crystallization of the humanized catalytic antibody Hu21D8. Crystals of the native antibody were obtained by slightly changing the conditions used for the crystallization of Fab fragments of catalytic antibodies obtained in the P.G. Schultz laboratory at the University of California, Berkeley, by using a similar expression and purification system. In collaboration with C. Stout and W. Chazin, Department of Molecular Biology, we are applying the principles of reverse screening to crystallization of RNA and RNA-DNA enzymes with good success.
Dynamic light scattering can be used to determine the aggregation state and polydispersity of macromolecules. Polydispersity can be considered a measure of heterogeneity and, to a certain extent, of the likelihood of crystallization. This technique has been used to differentiate the dimeric and monomeric states of the erythropoietin receptor in the presence of different ligands and to confirm the trimeric state of dUTPase of feline immunodeficiency virus. In collaboration with S. Schmid, Department of Molecular Biology, we have determined conditions in which dynamin remains relatively monodisperse in its tetrameric form.
Initial crystals of gap junctions have been obtained in collaboration with B. Gilula, Department of Cell Biology.
Cabezas, E., Stanfield, R.L., Wilson, I.A., Satterthwait, A.C. Defining conformational requirements for the principle neutralizing determinant on HIV-1. In: Peptides: Chemistry, Structure and Biology. Proceedings of the 14th American Peptide Symposium. Kaumaya, P. (Ed.). ESCOM, Leiden, the Netherlands, 1996, p. 800.
Desmet, J., Wilson, I.A., Joniau, M., De Maeyer, M., Lasters, I. Computation of the binding of fully flexible peptides to proteins with flexible side chains. FASEB J. 11:164, 1997.
Garcia, K.C., Scott, C.A., Brunmark, A., Carbone, F.R., Peterson, P.A., Wilson, I.A., Teyton, L. CD8 enhances formation of stable T-cell receptor/MHC class I molecule complexes. Nature 384:577, 1996.
Ghiara, J.B., Ferguson, D.C., Satterthwait, A.C., Dyson, H.J., Wilson, I.A. Structure-based design of a constrained peptide mimic of the HIV-1 V3 loop neutralization site. J. Mol. Biol. 266:31, 1997.
Haynes, M.R., Heine, A., Wilson, I.A. Catalytic antibody structures: An early assessment. Isr. J. Chem. 36:133, 1996.
Haynes, M.R., Lenz, M., Taussig, M.J., Wilson, I.A., Hilvert, D. Sequence similarity and cross-reactivity of a Diels-Alder catalyst and an anti-progesterone antibody. Isr. J. Chem. 36:151, 1996.
Stura, E.A., Ruf, W., Wilson, I.A. Crystallization and preliminary crystallographic data for a ternary complex between tissue factor, factor VIIa, and a BPTI-derived inhibitor. J. Cryst. Growth 168:260, 1996.
White J.M., Hoffman, L.R., Arevalo, J.H., Wilson, I.A. Attachment and entry of influenza virus into host cells: Pivotal roles of the hemagglutinin. In: Structural Biology of Viruses. Chiu, W., Burnett, R.M., Garcea, R. (Eds.). Oxford Press, New York, 1997, p. 80.
Wu, S.K., Zeng, K., Wilson, I.A., Balch, W.E. Structural insights into the function of the Rab GDI superfamily. Trends Biochem. Sci. 21:472, 1996.
Zeng, Z.H., Castano, A.R., Segelke, B.W., Stura, E.A., Peterson, P.A., Wilson,I.A. Crystal structure of mouse CD1: An MHC-like fold with a large hydrophobic binding groove. Science, in press.
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Principles of Protein Structure for Recognition, Interaction, and Function
E.D. Getzoff, A.S. Arvai, S.L. Bernstein, I.L. Canestrelli, B.R. Crane, T. Cross, C.L. Fisher, K.T. Forest, U.K. Genick, S. Hartsock, F. Henderson, C.K. Koike, T.P.K. Lo, S.E. Mylvaganam, C.D. Mol, E.H. Olender, J.L. Pellequer, M.E. Pique, M.M. Thayer
We determine the structural basis for protein recognition, function, and interaction by using x-ray crystallography and molecular biology, coupled with new computational and computer graphics approaches and tested by protein design. We focus on crystallographic studies for five proteins that undergo functionally important conformational and spectroscopic changes mediated by protein-cofactor interactions: photoactive yellow protein, to characterize a protein photocycle; sulfite reductase hemoprotein, to define the structural basis for the six-electron reduction of sulfite to sulfide; Root-effect hemoglobin, to study extreme pH effects on allostery and oxygen binding; Cu,Zn superoxide dismutase (SOD) metalloproteins, to understand their unusual stability and efficiency in regulating reactive oxygen; and, most recently, nitric oxide synthase, to understand the mechanism by which it oxidizes arginine to produce nitric oxide for biological signaling and defense.
To understand how a chromophore and protein interact to undergo a light cycle, we are studying photoactive yellow protein, a bacterial blue-light photosensor. Sequence homologies suggest that the fold in this protein is the structural prototype for the superfamily of Per-Arnt-Sim domains found in diverse biological sensors and clock proteins. We are extending our structure of dark-state photoactive yellow protein to 0.82-Å resolution, where individual atoms appear as spheres (Fig. 1).
We used millisecond time-resolved Laue crystallography and simultaneous optical spectroscopy to determine the first atomic structure for a protein photocycle intermediate. The structures of the two different states of photoactive yellow protein reveal the synergistic interactions between the chromophore and the protein that tune the spectral and kinetic properties of the light cycle for efficient protein-mediated signal transduction. We made photoactive yellow proteins with site-directed mutations and modified chromophores by recombinant expression and chemical attachment of the chromophore to experimentally test hypotheses for the light-cycle mechanism.
Sulfite and nitrite reductases catalyze fundamental chemical transformations for biogeochemical cycling of sulfur and nitrogen. We determined the 1.6-Å crystallographic structure of sulfite reductase hemoprotein, which catalyzes the concerted six-electron reductions of sulfite to sulfide and nitrite to ammonia, by using multiwavelength anomalous diffraction of the native siroheme and Fe4S4 cluster cofactors, multiple isomorphous replacement, and selenomethionine sequence markers. A distinctive three-domain /ß fold controls assembly and reactivity of the cofactor and contains a sulfite or nitrite reductase repeat common to a redox-enzyme superfamily. Coupled spectroscopy and crystallography of the enzyme in three oxidation states showed that heme activation occurs via reduction-mediated ligand exchange. Examination of refined crystallographic complexes with substrates, inhibitors, intermediates, and products showed how the active site facilitates the reaction and accommodates the varied reaction intermediates without release (Fig. 2).
The fish Root-effect hemoglobins act as acid-controlled molecular oxygen pumps, delivering oxygen against high oxygen pressures to the swim bladder for neutral buoyancy and to the retina for visual acuity. Our 2-Å crystal structures of the ligand-bound hemoglobin from the fish Leiostomus xanthurus show that key residues of the hemoglobin recruit conserved residues to strategically assemble positive-charge clusters that promote the extremely pH-dependent allosteric RT switch with concomitant release of oxygen.
For SOD metalloenzymes, we solved structures of bacterial, bovine, and human mutant enzymes to characterize the structural basis of the enzymes' activity and stability. Structures of the oxidized and reduced states of bovine SOD and active-site mutants of human SOD provide information on the enzymes' mechanism. Our bacterial SOD structure is representative of the class of SODs from bacterial pathogens and provides the potential for drug design. Although the bacterial SOD subunit fold and active-site geometry match those of human SOD, the elements recruited to form the dimer interface, the active-site channel, and the disulfide bond are strikingly different. Our SOD structural results suggest a hypothesis for the mechanism by which single-site mutations in human SOD cause the fatal degenerative disease amyotrophic lateral sclerosis.
Antibody interactions are a final focus for characterizing protein recognition, function, and interaction. Structures of the E8 antibody in the free state and bound to cytochrome c provided information on conformational changes that occur on binding, electrostatic interactions, and the key role of water molecules at the antibody-antigen interface. With V. Roberts, Department of Molecular Biology, and S. Benkovic, Pennsylvania State University, we designed, constructed, and characterized metalloantibodies and catalytic antibodies that exploit the versatility of the sequence-variable but structurally conserved antibody scaffold.
Boissinot, M., Karnas, S., Lepock, J.R., Cabelli, D.E., Tainer, J.A., Getzoff, E.D., Hallewell, R.A. Function of the Greek key connection analysed using circular permutants of superoxide dismutase. EMBO J. 16:2171, 1997.
Bourne, Y., Redford, S.M., Steinman, H.M., Lepock, J.R., Tainer, J.A., Getzoff, E.D. Novel dimeric interface and electrostatic recognition. Proc. Natl. Acad. Sci. U.S.A. 93:12774, 1996.
Crane, B., Siegel, L., Getzoff, E.D. Probing the catalytic mechanism of sulfite reductase by x-ray crystallography: Structure of the E. coli hemoprotein in complex with substrates, inhibitors, intermediates and products. Biochemistry, in press.
Crane, B., Siegel, L., Getzoff, E.D. Structures of the siroheme and Fe4S4-containing active center of sulfite reductase in different states of oxidation: Heme activation via reduction-gated exogenous ligand exchange. Biochemistry, in press.
Crane, B.R., Bellamy, H., Getzoff, E.D. Multiwavelength anomalous diffraction of sulfite reductase hemoprotein: Making the most of MAD data. Acta Crystallogr. D53:8, 1997.
Crane, B.R., Getzoff, E.D. Determining phases and anomalous scattering models from the multiwavelength anomalous diffraction of native protein metal clusters: Improved MAD phases error estimates and anomalous scatterer positions. Acta Crystallogr. D53:23, 1997.
Crane, B.R., Getzoff, E.D. The relationship between structure and function for the sulfite reductases. Curr. Opin. Struct. Biol. 6:744, 1996.
Devanathan, S., Genick, U.K., Getzoff, E.D., Meyer, T.E., Cusanovich, M.A., Tolin, G. Preparation and properties of a 3,4-dihydroxycinnamic acid chromophore variant of the photoactive yellow protein. Arch. Biochem. Biophys. 340:83, 1997.
Fisher, C.L., Cabelli, D.E., Hallewell, R.A., Beroza, P., Lo, T.P., Getzoff, E.D., Tainer, J.A. Computational, pulse-radiolytic, and structural investigations of lysine-136 and its role in the electrostatic triad of human Cu,Zn superoxide dismutase. Proteins, in press.
Genick, U.K., Borgstahl, G.E.O., Ng, K., Ren, Z., Pradervand, C., Burke, P.M., Srajer, V., Teng, T.-T., Schildkamp, W., McRee, D.E., Moffat, K., Getzoff, E.D. Structure of a protein photocycle intermediate by millisecond time-resolved crystallography. Science 275:1471, 1997.
Genick, U.K., Devanathan, S., Meyer, T.E., Canestrelli, I.L., Williams, E., Cusanovich, M.A., Tollin, G., Getzoff, E.D. Active site mutants implicate key residues for control of color and light cycle kinetics of photoactive yellow protein. Biochemistry 36:8, 1997.
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Structural Molecular Biology and Protein Design
J.A. Tainer, A.S. Arvai, D. Barondeau, G.E.O. Borgstahl, S.L. Bernstein, Y. Bourne, C. Bruns, B. Crane, T. Cross, D. Daniels, C.L. Fisher, K. Forest, Y. Guan, R.A. Hallewell, S. Han, F. Henderson, M.J. Hickey, D. Hosfield, C. Koike, T. Lo, T. Macke, C. Mol, S. Parikh, J.L. Pellequer, M.E. Pique, C. Putnam, S.M. Redford, M.M. Thayer
We focus on four classes of proteins at the interface of structural biology and cellular chemistry: (1) enzymes that regulate reactive oxygen and xenotoxins (superoxide dismutases [SODs], catalase, and glutathione transferase), (2) enzymes that control DNA repair and evolution enzymes, (3) pilus fiber motility and adherence proteins of bacterial pathogens, and (4) proteins that control the cell cycle. Current structural and design results are providing an understanding of these systems that should contribute to new treatments for infectious disease, degenerative diseases, and cancer.
We seek to improve our understanding of the many bacterial pathogens that use long fibers called type IV pili for attachment and mobility and to develop new treatments for infections caused by these microorganisms. We solved the crystallographic structure of pilin, the protein that forms the fiber. The fiber is essential to the virulence of these pathogens, which include Neisseria meningitidis, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Dichlobacter nodosus, Moraxella bovis, Vibrio cholerae, and enterotoxogenic Escherichia coli.
The pilin subunit is ladle-shaped with an extended -helix spine wrapped at one end by a ß-sheet. Interactions between a few key residues allow the remaining part of a hypervariable region to undergo extreme antigenic variation to escape the host's immune response. The assembled fiber shows extreme sequence variation plus glycosylation and phosphorylation at the surface. However, we have defined two conserved regions recognized by human, mouse, and rabbit antibodies. Current efforts include the redesign of pilin to develop potential vaccines by replacing the hypervariable region with epitopes from other essential target proteins.
REACTIVE OXYGEN AND XENOBIOTIC CONTROL ENZYMES
Atomic structures of human cytoplasmic copper-zinc SODs, the mitochondrial manganese SODs, and schistosomal glutathione transferases are improving our understanding of the control of reactive oxygen and xenotoxins within cells. SODs are master regulators for reactive oxygen species involved in injury, pathogenesis, aging, and degenerative diseases.
For copper-zinc SODs, we defined the structural chemistry of the active site responsible for the rapid reaction. We are now examining how single-site mutations cause degenerative disease such as Lou Gehrig disease or familial amyotrophic lateral sclerosis. For manganese SOD, we found that single-site mutations can destabilize the tetramer and also reduce stability and activity in ways that may cause degenerative diseases.
Structures of glutathione-S-transferase, which is an essential detoxification enzyme in all organisms, showed how the leading antischistosomal drug praziquantel binds to this enzyme. This information may enable us to design new drugs to overcome the growing resistance to current antischistosomal drugs. Protein design of glutathione-S-transferases includes using random libraries for the loop regions surrounding the active site, thereby developing glubodies as a new class of binding proteins in biotechnology.
DNA REPAIR AND GENETIC EVOLUTION
Cells must balance DNA repair to preserve fidelity with DNA variation that allows evolutionary changes. Because more than 10,000 DNA bases per day are repaired in each human cell, DNA excision-repair enzymes are essential to cell survival and to protection against cancer-causing mutations. Surprisingly, DNA-repair inhibitors may improve current radiation therapy and chemotherapy for cancer by specifically killing cancer cells. Unlike normal cells, cancer cells often undergo DNA synthesis and cell division with unrepaired DNA, resulting in the death of the cells.
Our structures of DNA-repair enzymes show in atomic detail how damaged DNA bases are recognized and removed. These enzymes repair DNA by flipping the DNA nucleotides out from the double helix and into specific binding pockets (Fig. 1),
which are ideal for the design of inhibitors for anticancer therapies. We confirmed our understanding of these binding pockets by deliberately altering the specificity of the DNA-repair enzyme uracil-DNA glycosylase. We made mutants that remove cytosine or thymine from normal DNA, resulting in mutator phenotypes in vivo.
We found that the endonuclease III structure is representative of a superfamily of DNA-repair enzymes and a key HhH motif that recognizes DNA backbone. Our new structure of the major DNA-repair abasic-site endonuclease, which cuts DNA at sites where bases are missing, defines the active site of the enzyme and indicates its mechanism for recognizing missing bases.
Human dUTP pyrophosphatase (dUTPase) catalyzes the breakdown of uracil nucleotide triphosphates to keep the RNA base uracil out of DNA and to provide material for the biosynthesis of the DNA building block dTTP. These dUTPase functions prevent cycles of uracil misincorporation and removal that would generate multiple breaks in DNA strands and eventual cell death, a process called thymine-less cell death. Atomic structures of dUTPase with bound nucleotides show that uracil binds within a groove that is then capped when the flexible tail region closes over the bound dUTP substrate. These structures establish how dUTPase recognizes its substrate with exquisite specificity and provide a basis for the design of inhibitors as future anticancer drugs.
CONTROL OF THE CELL CYCLE
Together with S. Reed's group, Department of Molecular Biology, we are working to define the structural basis for control of the cell cycle. Structures of the Cks or suc1 proteins, which are essential to the progression of the cell cycle, provide clues for new mechanisms for regulation of the cycle via a conformational switch that controls two distinct Cks folds and assemblies. A straight ß-hinge conformation of Cks, which forms a dimer of swapped ß-strands, blocks binding to the cell-cycle kinase Cdk2. Formation of a closed, bent ß-hinge conformation creates a single domain fold that promotes Cdk2 binding (Fig. 2).
Preliminary experiments by Dr. Reed's group show that blocking expression of Cks results in cell death for several types of cancer cells. This finding suggests that Cks is a useful target for the development of anticancer drugs.
Boissinot, M., Karnas, S., Lepock, J.R., Cabelli, D.E., Tainer, J.A., Getzoff, E.D., Hallewell, R.A. Function of the Greek key connection analysed using circular permutants of superoxide dismutase. EMBO J., in press.
Bourne, Y., Redford, S.M., Steinman H.M., Lepock, J.R., Tainer, J.A., Getzoff, E.D. Novel dimeric interface and electrostatic recognition in bacterial Cu,Zn superoxide dismutase. Proc. Natl. Acad. Sci. U.S.A. 93:12774, 1996.
Crane, B.R., Arvai, A.S., Gachhui, R., Wu, C., Ghosh, D.K. The structure of NO synthase oxygenase domain and inhibitor complexes. Science, in press.
Fisher, C.L., Cabelli, D.E., Hallewell, R.A., Beroza, P., Lo, T.P., Getzoff, E.D., Tainer, J.A. Computational, pulse-radiolytic and structural investigations of lysine 136 and its role in the electrostatic triad of human of Cu,Zn superoxide dismutase. Proteins, in press.
Forest, K.T., Tainer, J.A. Type IV pilin structure, assembly, and immunodominance: Applications to vaccine design. In: Vaccines. Brown, F., et al. (Eds.). Cold Spring Harbor Press, Cold Spring Harbor, NY, 1997, p. 167.
Forest, K.T., Tainer, J.A. Type IV pilus structure: Outside to inside and top to bottom. Gene 192:165, 1997.
Gorman, M.A., Morera, S., Rothwell, D.G., La Fortelle, E.D., Mol, C.D., Tainer, J.A., Hickson, I.D., Freemont, P.S. The crystal structure of the human DNA-repair enzyme endonuclease HAP1 suggests the recognition of extra-helical deoxyribose at DNA abasic sites. EMBO J., in press.
Mol, C.D., Harris, J.M., McIntosh, E.M., Tainer, J.A. Crystal structures of free and nucleotide-bound complexes of human dUTP pyrophosphatase: Uracil recognition by a ß-hairpin and active sites formed by three separate subunits. Structure 4:1077, 1996.
Roberts, V.A., Nachman, R.J., Coast, G.M., Hariharan, M., Chung, J.S., Holman, G.M., Williams, H., Tainer, J.A. Consensus chemistry and ß-turn conformation of the active core of the myotropic/diuretic insect neuropeptide family. Chem. Biol. 4:105, 1997.
Slupphaug, G., Mol, C.D., Kavli, B., Arvai, A.S., Krokan H.E., Tainer, J.A. A nucleotide flipping mechanism from the structure of human uracil-DNA glycosylase bound to DNA. Nature 384:87, 1996.
Watson, M.H., Bourne, Y., Arvai, A.S., Hickey, M.J., Santiago, A., Bernstein, S.L., Tainer, J.A., Reed, S.I. A mutation in the human cyclin-dependent kinase interacting protein, CksHs2, interferes with cyclin-dependent kinase binding and biological function, but preserves protein structure and assembly. J. Mol. Biol. 261:646, 1996.
Zu, J.S., Deng, H.-X., Lo, T.P., Mitsumoto, H., Ahmed, M.S., Hung, W.-Y., Cai, Z.-J., Tainer, J.A., Siddique, T. Exon 5 is not required for the toxic function of mutant SOD1 but essential for dismutase activity: Identification and characterization of two new SOD1 mutations associated with familial amyotrophic lateral sclerosis. Neurogenetics, in press.
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D.E. McRee, C. Bruns, Y. Cao, M. Israel, N. Jourdan, V. Shridhar, P. Williams
Our laboratory studies the structure, function, and catalysis of metalloproteins, with particular emphasis on iron metalloproteins. One question being investigated is how proteins give iron diverse biological roles ranging from electron transfer to oxygen activation to iron transport. We are using high-resolution protein crystallography, protein engineering, and biochemistry to answer this question.
Iron is an essential growth requirement of all organisms, and using lactoferrin and transferrin to sequester iron from invading pathogens is one means of antibacterial defense in humans. However, some bacterial pathogens, notably Neisseria and Haemophilus, have evolved to turn adversity into advantage; they have receptors for transferrins that enable the bacteria to steal the host iron. This iron is transported into the pathogens by a bacterial protein, iron-binding protein. We have solved the structure of the iron-binding protein to high resolution (Fig. 1)
and have started protein engineering studies. Our eventual goal is to find a way to knock out this protein in bacteria and thus produce a bacteriostatic agent.
Cytochrome c552 is the physiologic electron transfer partner of cytochrome c oxidase in Thermus thermophilus. We are solving the structure of cytochrome c552 in collaboration with E. Stura, Department of Molecular Biology, and J. Fee, University of California, San Diego. We also have crystals of the CuA fragment of T. thermophilus cytochrome c oxidase, and the combination of this fragment and cytochrome c552 provides a unique opportunity to study an electron transfer system. We will do protein engineering studies in collaboration with J. Fee.
VERY HIGH-RESOLUTION METAL-SITE STRUCTURES
With the advent of freezing devices and synchrotron radiation sources, very high-resolution structures (<1.4 Å) are now routinely accessible. At these resolutions, it becomes possible to refine structures with the rigorous methods used in small-molecule crystallography. This refinement includes adding hydrogens and anisotropic thermal factors and using full-matrix least-squares analyses to determine the standard uncertainties of the atom positions. Because metal centers scatter more than the lighter carbon atoms do, the metal centers are even better determined.
In a high-resolution 1.35-Å refinement of Azotobacter seven-iron ferredoxin, we have determined the positions of the iron and sulfur atoms with a standard uncertainty of 0.01 Å (Fig. 2).
This uncertainty is within the limit of molecular orbital calculations and will lead to a better coupling of theoretical calculations on metal centers with structure. We have also refined cytchrome c peroxidase to 1.38-Å resolution. These more accurate coordinates will allow improved calculations of the enzyme's mechanism. In the coming years, we plan to build a database of high-resolution metalloprotein structures in conjunction with the Scripps Metalloprotein Structure and Design Group (http://www.scripps.edu/pub/dem-web/metallo/), of which our group is a member.
As part of a project funded by the National Science Foundation to improve visualization and computational tools for protein crystallography, we have developed a software package called XtalView (http://www.scripps.edu/pub/dem-web/toc.html). XtalView uses the power of modern workstations to solve protein crystallographic problems by using a visual, graphical user interface. XtalView is available from the Computational Center for Macromolecular Structure (http://www.sdsc.edu/CCMS/) and has been downloaded by more than 1000 groups.
Cao, Y., Musah, R.A., Wilcox, S.A., Goodin, D.B., McRee, D.E. Protein conformer selection by ligand binding observed with protein crystallography. Protein Sci., in press.
Genick, U.K., Borgstahl, G.E.O., Ng, K., Ren, Z., Pradervand, C., Burke, P.M., Srajer, V., Teng, T., Shildkamp, W., McRee, D.E., Moffat, K., Getzoff, E. D. Millisecond time-resolved Laue crystallography: Structure of a protein photocycle intermediate. Science 275:1471, 1997.
Israel, M., McRee, D.E. XtalView. In: Crystallographic Computing 7. Bourne, P., Watenpaugh, K. (Eds.). Oxford Press, New York, in press. Available at http://www.sdsc.edu/projects/Xtal/IUCr/CC/School96/IUCr.html
Prasad, G.S., McRee, D.E., Stura, E.A., Levitt, D.G., Lee, H.C., Stout, C.D. Crystal structure of Aplysia ADP ribosyl cyclase, a homologue of the bifunctional ectozyme CD38. Nature Struct. Biol. 3:957, 1996.
Prasad, G.S., Stura, E.A., McRee, D.E., Laco, G.S., Hasselkus-Light, C., Elder, J.H., Stout, C.D. Crystal structure of dUTP pyrophosphatase from feline immunodeficiency virus. Protein Sci. 5:2429, 1996.
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C.D. Stout, G.S. Prasad, S.J. Lloyd, P.J. Shim, N. Kresge, A. Muhlberg, V. Sridhar
This laboratory focuses on experimental x-ray crystallography of macromolecules. Several fundamental questions are being addressed through structure determination of key proteins involved in biological processes. This research often involves collaboration with scientists at TSRI and other institutions. The experimental work has several stages, including biochemical preparation, crystallization, and collection and analysis of x-ray diffraction data. Once a structure is solved, experiments are designed to study relationships between structure and function. These experiments entail preparation of site-directed mutants and protein-ligand complexes, structure analysis, and assays of biological function. Several projects focused primarily on iron-sulfur proteins and enzymes, fertilization proteins, and enzymes that use nucleotides are in progress.
In collaboration with J. Elder and E. Stura, Department of Molecular Biology, we have determined at high resolution the structure of dUTPase of feline immunodeficiency virus and of dUTPase in complex with dUMP. The enzyme dUTPase is an important target for drug design, and a series of mutant and inhibitor complexes are being analyzed to understand the affinity and specificity of small-molecule ligands in the active site and the catalytic mechanism. Experiments also are in progress to study the binding of substrates to ADP ribosyl cyclase, an enzyme that synthesizes the secondary messenger cyclic ADP ribose. ADP ribosyl cyclase appears to use a novel dual-site mechanism. Understanding this reaction is relevant to the function of the cyclase homolog, CD38, a widely distributed cell-surface ectozyme in the immune system. A third nucleotide-processing enzyme being studied is dynamin, a critical component in cellular endocytosis. Through collaboration with S. Schmid, Department of Molecular Biology, we are determining the structure of the domains of this complex multifunctional GTPase.
Continuing our focus on proteins involved in fertilization, in collaboration with V. Vacquier, Scripps Institution of Oceanography, we are determining the structure of the 16,000 and 18,000 molecular weight lysins from green abalone. These structures will provide important insights into the molecular details of the interaction between sperm and egg. The 16,000 molecular weight protein is a species-specific homolog of red abalone lysin, for which we have two crystal structures; the 18,000 molecular weight lysin is a novel protein involved in fusion of the gamete plasma membranes. In collaboration with J. Bleil, Department of Molecular Biology, experiments are in progress to determine the structure of the octameric mouse sperm cell-surface protein, sp56, which functions in recognition and binding of egg oligosaccharides.
Detailed study of the mechanism of the iron-sulfur enzyme aconitase, a dehydratase that uses an iron-sulfur cluster in catalysis, is continuing with the determination of the structures of five site-directed mutants of active-site residues in eight complexes with substrates and inhibitors. A large number of crystallization trials are in progress with cytosolic aconitase and the stem-loop RNA molecule, the iron regulatory element, to which aconitase binds.
In collaboration with B. Burgess, University of California, Irvine, an ongoing study of the structure and function of the seven-iron ferredoxin from Azotobacter vinelandii has led to additional structures of mutants. One of the mutants has a cysteine persulfide, a possible intermediate in the biosynthesis of iron-sulfur clusters. A series of six structures of the seven-iron ferredoxin oxidized with ferricyanide have been refined to study the chemical decomposition of iron-sulfur clusters. In collaboration with D. McRee, Department of Molecular Biology, the seven-iron ferredoxin has been refined at 1.35-Å resolution, providing an unprecedented level of accuracy in the structural details of the [3Fe-4S] and [4Fe-4S] clusters (see Fig. 2 in D. McRee's report). The redox partner of ferredoxin is NADPH:ferredoxin oxidoreductase; determination of the crystal structure of this enzyme, nearly complete, will allow docking of the proteins to model the electron transfer reaction.
Beinert, H., Kennedy, M.C., Stout, C.D. Aconitase as iron-sulfur protein, enzyme, and iron-regulatory protein. Chem. Rev. 96:2335, 1996.
Kemper, M.A., Stout, C.D., Lloyd, S.J., Prasad, G.S., Fawcett, S., Armstrong, F.A., Shen, B., Burgess, B.K. Y13C Azotobacter vinelandii ferredoxin I: A designed [Fe-S] ligand motif contains a cysteine persulfide. J. Biol. Chem. 272:15620, 1997.
Lauble H., Kennedy M.C., Emptage M.H., Beinert H., Stout, C.D. The reaction of fluorocitrate with aconitase and the crystal structure of the enzyme-inhibitor complex. Proc. Natl. Acad. Sci. U.S.A. 93:13699, 1996.
Prasad, G.S., McRee, D.E., Stura, E.A., Levitt, D.G., Lee, H.C., Stout, C.D. Crystal structure of Aplysia ADP ribosyl cyclase, a homologue of the bifunctional ectozyme CD38. Nature Struct. Biol. 3:957, 1996.
Prasad, G.S., Stura, E.A., McRee, D.E., Laco, G.S., Hasselkus-Light, C., Elder, J.H., Stout, C.D. Crystal structure of dUTP pyrophosphatase from feline immunodeficiency virus. Protein Sci. 5:2429, 1996.
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Nuclear Magnetic Resonance Investigations of the Three-Dimensional Structure and Dynamics of Proteins in Solution
P.E. Wright, H.J. Dyson, B. Duggan, M. Foster, S. Holmbeck, R. Kriwacki, J. Love, J. Laity, B. Lee, G. Legge, G. Perez-Alvarado, J. Pikkemaat, I. Radhakrishnan, J. Xu, L. Zhu, L.L. Tennant, M. Martinez-Yamout, J. Chung, M. Gearhart, D.A. Case, H.J. Dyson, J. Gottesfeld, U. Hommel,* T. Huang**
* Novartis Pharmaceuticals, Basel, Switzerland
** Institute of Biomedical Sciences, Taipei, Taiwan, Republic of China
We use multidimensional nuclear magnetic resonance (NMR) spectroscopy to investigate the structures and dynamics of proteins in solution. Such studies are essential for understanding the mechanisms of action of these proteins and for elucidating structure-function relationships.
PROTEIN STRUCTURE DETERMINATION IN SOLUTION
Solution three-dimensional structures have been determined for a number of proteins and protein complexes of molecular weight up to more than 20 kD. These include plastocyanin, thioredoxin, the human anaphylatoxin C3a, enzyme IIAglc, myoglobin, rusticyanin, and various DNA-binding domains and protein-DNA complexes. We are beginning heteronuclear three- and four-dimensional NMR experiments with proteins labeled with 2H, 13C, or 15N to extend use of the NMR structure determination method to even larger proteins and protein complexes. In addition, new computational methods are being developed to facilitate structure determination and refinement.
TRANSCRIPTION FACTOR--DNA COMPLEXES
NMR methods are being used to determine the three-dimensional structures and intramolecular dynamics of various DNA-binding motifs from eukaryotic transcriptional regulatory proteins, both free and complexed with the target DNA. Structures determined include that of the HMG domain of the lymphoid enhancer-binding factor 1 (LEF-1) bound to its cognate DNA sequence and the DNA complex formed by the three amino-terminal zinc fingers of transcription factor IIIA (TFIIIA). Ongoing work on LEF-1 has focused on the structural characteristics of the free protein and on the role of the C-terminal basic tail in DNA binding and bending. In addition, studies of the complex of LEF-1 with a Holliday junction are in progress.
Three-dimensional structures have been determined for the complex of zf1-3, a protein containing the three N-terminal zinc fingers of TFIIIA, with the cognate 15-bp oligonucleotide duplex. The three zinc fingers bind in the DNA major groove, thus validating parts of our earlier model of the TFIIIA-DNA complex deduced from biochemical experiments. The structures contain several novel features and show 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.
Each zinc finger contacts 4--5 bp, and the repertoire of base contact residues is expanded in the zf1-3--DNA complex. Lysine and histidine side chains involved in base recognition are dynamically disordered in the solution structure, thereby minimizing the entropic cost of DNA binding. On DNA binding, the protein forms an ordered globular structure with substantial protein-protein interactions between adjacent fingers. Dynamics measurements show that the linker regions that connect the zinc-finger domains lose their intrinsic flexibility on binding to DNA. The linkers appear to play an active structural role in stabilization of the protein-DNA complex. Contributions to high-affinity binding come from both direct protein-DNA contacts and from indirect protein-protein interactions associated with structural organization of the linkers and formation of well-packed interfaces between adjacent zinc fingers in the DNA complex.
In addition to its role in binding to and regulating the 5S RNA gene, TFIIIA also forms a complex with the 5S RNA transcript. The minimal set of zinc fingers required for 5S RNA recognition has been determined and consists of fingers 4--6. The minimal region of the 5S RNA needed to bind these fingers has also been mapped, and NMR studies of the protein-RNA complex have been started to gain insights into the structural basis for 5S RNA recognition. Work is also in progress to determine the structure of finger 6 and examine its interactions with RNA. In a further new direction, NMR studies of the Wilms' tumor zinc-finger protein and the zinc finger--DNA complex have commenced.
In collaboration with R. Evans, Salk Institute, the solution structure of the DNA-binding domain of the nuclear hormone receptor RXR, which is activated by 9-cis retinoic acid, has been refined by using 13C,15N-labeled protein. The refined structures confirm the existence of the novel third helix, which appears to play a role in homodimer formation and DNA binding. NMR structural studies of the DNA-binding domain of ERR, a member of a class of nuclear hormone receptors that bind DNA as monomers, are also in progress in collaboration with Dr. Evans. The protein has been uniformly labeled with 13C and 15N, and resonance assignments have been made for free ERR and for the complex with its cognate DNA.
PROTEIN-PROTEIN INTERACTIONS IN TRANSCRIPTIONAL REGULATION
Activated transcription in eukaryotes relies on protein-protein interactions between DNA-bound factors and coactivators that, in turn, interact with the basal transcription machinery. The nuclear factor cyclic AMP response element binding protein (CREB) activates transcription of target genes, in part through direct interactions with the KIX domain of the coactivator CREB-binding protein (CBP) in a phosphorylation-dependent manner. In collaboration with M. Montminy, Harvard Medical School, we recently determined the structure of the complex formed by the phosphorylated kinase-inducible domain (pKID) of CREB and the KIX domain of CBP (Fig. 1).
The structure reveals that pKID undergoes a coil-to-helix folding transition on binding to KIX, forming two -helices in the process. The amphipathic helix B of pKID interacts with a large hydrophobic patch defined by helices 1 and 3 of KIX. The other pKID helix, A, interacts with a different face of the 3 helix of KIX. The critical phosphoserine residue of pKID is in a position to interact favorably with Y658 of KIX. The structure provides a model for interactions between other transactivation domains and their targets.
A novel method has been developed, in collaboration with G. Siuzdak, Department of Molecular Biology, for probing protein-protein interactions by using mass spectrometry and isotopic labeling. Application of this method led to detection of the kinase inhibitory domain of the cyclin-dependent kinase inhibitor p21Waf1/Cip1/Sdi1. NMR studies of this important cell-cycle regulatory protein established that it is disordered in the free state but adopts a stable folded structure when bound to cyclin-dependent kinase 2. These observations challenge the generally accepted view that stable tertiary structure is a prerequisite for biological activity and suggest that a broader view of protein "structure" should be considered in the context of structure-function relationships.
INTERACTIONS BETWEEN DOMAINS OF CELL ADHESION MOLECULES
We recently completed resonance assignments for the I (inserted) domain of lymphocyte function--associated antigen-1, and structure calculations are in progress. Our interest in this system is in the understanding of the interactions between lymphocyte function--associated antigen-1 and its physiologic partner intracellular adhesion molecule-1. This research is being done in parallel with a collaborative effort with B. Cunningham, Department of Neuropharmacology, to probe the interactions between the immunoglobulin-like domains of the neural cell adhesion molecule. These projects illustrate the excellent sensitivity of the NMR method as a tool to probe protein-protein interactions. The NMR spectrum can be used directly to map the sites of interaction in a straightforward way, once assignments are available.
Chen, Y., Case, D.A., Reizer, J., Saier, M.H., Jr., Wright, P.E. High-resolution solution structure of Bacillus subtilis IIAglc. Proteins, in press.
Foster, M.P., Wuttke, D.S., Radhakrishnan, I., Case, D.A., Gottesfeld, J.M., Wright, P.E. Domain packing and dynamics in the DNA complex of the amino-terminal zinc fingers of transcription factor IIIA. Nature Struct. Biol., in press.
Gippert, G.P., Wright, P.E., Case, D.A. Distributed torsion angle grid search in high dimensions: A systematic approach to NMR structure determination. J. Biomol. NMR, in press.
Kriwacki, R.W., Hengst, L., Tennant, L., Reed S.I., Wright, P.E. Structural studies of p21 Waf1/Cip1/Sdi1 in the free and Cdk2-bound state: The p21 NH2-terminus folds on binding to Cdk2. Proc. Natl. Acad. Sci. U.S.A. 93:11504, 1996.
Kriwacki, R.W., Wu, J., Tennant, L., Wright P.E., Siuzdak, G. Probing protein structure using biochemical and biophysical methods: Proteolysis, MALDI mass analysis, HPLC, and gel-filtration chromatography of p21 Waf1/Cip1/Sdi11. J. Chromatogr., in press.
Markley, J.L., Bax, A., Arata, Y., Hilbers, C.W., Kaptein, R., Sykes, B.D., Wright, P.E., Wüthrich, K. Recommendations for the presentation of NMR structures of proteins and nucleic acids. Pure Appl. Chem., in press.
Sem, D.S., Casimiro, D.R., Kliewer, S.A., Provencal, J., Evans, R.M., Wright, P.E. NMR spectroscopic studies of the DNA-binding domain of the monomer-binding nuclear orphan receptor, human ERR2: The carboxy-terminal extension to the zinc-finger region is unstructured in the free form of the protein. J. Biol. Chem. 272:18038, 1997.
Wright, P.E. Smith, J.L. Biophysical methods: Faster and bigger. Curr. Opin. Struct. Biol. 6:583, 1996.
Wuttke, D.S., Foster, M.P., Case, D.A., Gottesfeld, J.M., Wright, P.E. Solution structure of the first three zinc fingers of TFIIIA bound to the cognate DNA sequence: Determinants of affinity and sequence specificity. J. Mol. Biol., in press.
Zhu, L., Dyson, H.J., Wright, P.E. A NOESY-HSQC simulation program, SPIRIT. J. Biomol. NMR, in press.
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Folding of Proteins and Protein Fragments
P.E. Wright, H.J. Dyson, Y. Bai, S. Cavagnero, D. Donne, D. Eliezer, C. Garcia-Gonzalez, S. Prytulla, J. Viles, J. Yao, O. Zhang, J. Chung, L.L. Tennant, S. Lahrichi, V. Tsui
The molecular mechanism by which proteins fold into their three-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. We have used NMR methods to show that many peptide fragments of proteins tend to adopt folded conformations in water solution. The observation 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.
APOMYOGLOBIN FOLDING PATHWAY
A major program in our laboratory is to establish a structural and mechanistic description of the folding pathway of apomyoglobin. We used quenched-flow pulse-labeling methods in conjunction with two-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 adopt hydrogen-bonded secondary structure is formed within 6 msec of the initiation of refolding. Folding proceeds by stabilization of structure in the B-helix and then the C- and E-helices. Using time-resolved small-angle x-ray scattering, in collaboration with S. Doniach and K.O. Hodgson, Stanford University and Stanford Synchrotron Radiation Laboratory, we showed that apomyoglobin folds into a highly compact state within 20 msec after initiation of refolding. We are now using carefully selected myoglobin mutants and both optical stopped-flow spectroscopy and hydrogen-exchange pulse-labeling methods to further probe the kinetic folding pathway. We have detected mutations that increase the rate of folding and apparently influence the folding pathway.
Apomyoglobin provides a unique opportunity for detailed characterization of the incremental development of structure and of the changes in dynamics that accompany compaction of a protein during folding. By careful manipulation of the pH, a number of partially folded states that are directly implicated in folding can be stabilized under conditions suitable for direct study by multidimensional NMR. Backbone resonance assignments have now been completed for an apomyoglobin molten globule intermediate, formed at pH 4.1. Analysis of 13C and other chemical shifts has provided the first "high resolution" insights into the structure of this state at the level of individual amino acid residues. In addition, 15N relaxation measurements have shown that the terminal regions of the polypeptide chain form a tightly packed hydrophobic core, whereas the central parts retain considerable intrinsic flexibility.
In earlier work, we used peptides to model the initiation events in the folding of apomyoglobin. Peptides covering the entire sequence of myoglobin were synthesized, and their propensities for spontaneous formation of secondary structure were examined by using NMR and circular dichroism spectroscopy. We have now fully assigned the polypeptide backbone resonances for the acid-denatured state of apomyoglobin. The NMR data show formation of helical secondary structure in regions that form the A- and H-helices in the folded protein. The acid-denatured state 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.
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.
Structural characterization of an unfolded state of the ß-sheet protein apoplastocyanin is also in progress. Apoplastocyanin offers a unique opportunity to study an unfolded protein under nondenaturing conditions, because it forms an unfolded state at neutral pH under low-salt conditions. Extensive resonance assignments have been completed, revealing that the unfolded polypeptide has a marked propensity to populate the ß-region of dihedral angle space. NMR relaxation measurements indicate that the polypeptide backbone is highly fluctional on a timescale shorter than 1 nsec.
FRAGMENTS OF PRION PROTEINS
A number of proteins are unfolded or only partly folded before they bind to their biological receptors. Although scientists have known for many years that this fact applies to peptide hormones, it is now evident that on binding to their receptors, the activation domains of some transcription factors, the p21 cyclin-dependent kinase inhibitors, and certain other proteins undergo transitions to folded states. Furthermore, folding has been implicated in a number of abnormalities, such as Alzheimer's disease and the prion diseases such as bovine spongiform encephalopathy (mad cow disease).
We recently used NMR methods to characterize the structure and dynamics of the full-length prion protein. The N-terminal region, residues 29--124, including the octapeptide repeats, is highly disordered and undergoes rapid backbone fluctuations on a subnanosecond timescale. This highly flexible region probably provides the plasticity required for the conformational transition of the cellular form into the infectious scrapie state.
Bai, Y., Karimi, A., Dyson, H.J., Wright, P.E. Absence of a stable intermediate on the folding pathway of protein A. Protein Sci. 6:1449, 1997.
Donne, D.G., Viles, J.H., Groth, D., Mehlhorn, I., James, T.L., Cohen, F.E., Prusiner, S.B., Wright, P.E., Dyson, H.J. Structure of the recombinant full-length hamster prion protein PrP(29--231): The N-terminus is highly flexible. Proc. Natl. Acad. Sci. U.S.A., in press.
Eliezer D., Wright, P.E. Is apomyoglobin a molten globule? Structural characterization by NMR. J. Mol. Biol. 263:531, 1996.
Prytulla, S., Dyson, H.J., Wright, P.E. Gene synthesis, high level expression and assignment of backbone 15N and 13C resonances of soybean leghemoglobin. FEBS Lett. 399:283, 1996.
Reymond, M.T., Huo, S., Duggan, B., Wright, P.E., Dyson, H.J. Contribution of increased length and intact capping sequences to the conformational preference for helix in a 31-residue peptide from the C-terminus of myohemerythrin. Biochemistry 36:5234, 1997.
Reymond, M.T., Merutka, G., Dyson, H.J., Wright, P.E. Folding propensities of peptide fragments of myoglobin. Protein Sci. 6:706, 1997.
Yao, J., Dyson, H.J., Wright, P.E. Chemical shift dispersion and secondary structure prediction in unfolded and partly-folded proteins. FEBS Lett., in press.
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Nuclear Magnetic Resonance Studies of the Structure and Mechanism of Enzymes
H.J. Dyson, P.E. Wright, J. Chung, S. Huo, G. Kroon, M. Martinez-Yamout, M.J. Osborne, M. Reymond, S. Scrofani, B. Xia, L.L. Tennant, S.J. Benkovic,* A. Holmgren**
* Pennsylvania State University, University Park, PA
** Karolinska Institute, Stockholm, Sweden
We use site-specific information from nuclear magnetic resonance (NMR) spectroscopy to further the understanding of enzyme function through study of structure and dynamics.
Thioredoxin, a small (108-residue) thiol-disulfide oxidoreductase, has a multitude of functions in the cell, including the vital reduction of ribonucleotides to form deoxyribonucleotides for DNA synthesis. Thioredoxins occur in all living organisms, including viruses. Mammalian thioredoxins have a vital role in cellular control mechanisms and have been implicated in human diseases; serum levels of the enzyme are elevated in AIDS patients. One of the primary functions of thioredoxin in the cell is as a protein disulfide reductase, a function vital for the prevention of misfolded proteins in vivo.
The Escherichia coli thioredoxin system has been fully characterized by NMR, including the calculation of high-resolution structures for both the oxidized (disulfide) and the reduced (dithiol) forms of the protein. Backbone dynamics and amide proton hydrogen exchange enabled us to determine that functional differences in phage systems between oxidized and reduced thioredoxin were due to differences in the flexibility of the molecules, rather than to structural differences.
We have also delineated the mechanism of action of E. coli thioredoxin. The reduction reaction of thioredoxin depends critically on the movement of protons during the two-electron--two-proton transfer reaction as a substrate disulfide is reduced. An extensive series of experiments on the pH-dependence of the NMR spectrum has given us important insights into the complex relationship between the mechanism of action of this enzyme and local structure at the active site, including the presence of conserved, buried charged residues. This project has recently been extended to include a structural study by NMR of a related protein, glutaredoxin 2, whose structure and function are unknown.
DYNAMICS IN ENZYME ACTION
The relationship between dynamics of the polypeptide chain and enzyme catalysis is being explored. We hypothesize that efficient enzyme catalysis requires flexibility at the active site and that enzymes have therefore evolved to incorporate this flexibility. To test these hypotheses, we are using mutagenesis coupled with detailed characterization of changes in enzymatic function, structure, and dynamics in two very different enzyme systems: dihydrofolate reductase, for which we already have clear evidence that dynamics play a role in catalysis, and a metallo-ß-lactamase, for which evidence from the crystal structure indicates flexibility in the region of the active site.
NMR relaxation measurements on substrate and cofactor complexes of dihydrofolate reductase, in collaboration with S. Benkovic, Pennsylvania State University, are providing novel insights into the relationship between dynamics and enzyme activity. Motions on a wide range of time scales (picoseconds to milliseconds) have been detected and can be correlated with enzyme function. Both of these enzymes are clinically important drug targets, for anticancer drugs in the case of dihydrofolate reductase and for prevention of antibiotic resistance in the case of the lactamase. Our approach is leading to new insights into the role of dynamics in catalysis.
DESIGN AND SYNTHESIS OF PROTEINS
We have developed an efficient method of overproducing proteins for NMR that involves designing and synthesizing genes specifically for expression in E. coli. This method is exceptionally useful in the production of several proteins, including Thiobacillus ferrooxidans rusticyanin and soybean leghemoglobin. The method is also easily adaptable for the production of site-specific mutants, an important aspect of the investigation of the relationship between structure and function.
DESIGN OF A CATALYTIC ANTIBODY
Related to the issue of design for catalysis is a long-standing project on the characterization and redesign of the Fv fragment of a catalytic antibody. This work is extremely promising for understanding the influence of structure on function for enzymes. Because they have evolved over millions of years, most enzymes are exquisitely tuned to the reactions they catalyze and may also be tolerant of mutations. By contrast, catalytic antibodies have much lower efficiency and specificity. A knowledge of the local structure and dynamics of the catalytic site will allow novel insights into the mechanisms of antibody catalysis and will guide future work aimed at enhancing the catalytic efficiency. These studies will also provide valuable insights into the structural and functional evolution of enzymes.
Bender, C.J., Casimiro, D., Dyson, H.J. Electron spin envelope modulation spectra of Thiobacillus ferrooxidans rusticyanin and a mutant lacking one of the copper ligands. J. Chem. Soc. [Faraday], in press.
Casimiro, D., Wright, P.E., Dyson, H.J. PCR-based gene synthesis for protein over-production. Structure, in press.
Dyson, H.J., Jeng, M.-F., Slaby, I., Lindell, M., Cui, D.-S., Holmgren, A. Effects of buried charged groups on cysteine thiol ionization and reactivity in Escherichia coli thioredoxin: Structural and functional characterization of mutants of Asp 26 and Lys 57. Biochemistry 36:2622,1997.
Ghiara, J.B., Ferguson, D., Satterthwait, A.C., Dyson, H.J., Wilson, I.A. Structure-based design of a constrained-peptide mimic of the HIV-1 V3 loop neutralization site. J. Mol. Biol. 266:31,1996.
Zhu, L., Dyson, H.J., Wright, P.E. A NOESY-HSQC simulation program SPIRIT. J. Biomol. NMR, in press.
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Structural Biology of Protein and Oligonucleotide Recognition, Binding, and Signal Transduction
W.J. Chazin, M. Akke,* G. Bifulco, D. Boger,** O. Crescenzi, P.A. Fagan, S. Forsén,* L. Gomez-Paloma,*** H. Hidaka,**** B. Huang,***** M.J. Hunter, R.R. Ketchem, S. Linse,* M. Lubienski, J.C. Madsen, L. Mäler, M. Nelson, K.C. Nicolaou,** S. Parikh, B.C. Potts, J. Rydzewski, M. Sastry, J.A. Smith, E. Thulin,* T. Tsudo,**** C. Weber, B.T. Wimberly
* University of Lund, Lund, Sweden
** Department of Chemistry, TSRI
*** University of Naples, Naples, Italy
**** Nagoya University, Nagoya, Japan
***** Department of Cell Biology, TSRI
Our laboratory uses nuclear magnetic resonance spectroscopy to examine the solution structure and dynamics of proteins and oligonucleotides, free in solution and in complexes with cellular targets or drugs. Highlights in the past year include solving the structure of the calcium-activated state of calcyclin, an important calcium sensor from the S100 subfamily of calcium-binding proteins (CaBPs), and characterizing the related heterodimeric S100 protein complex, MRP8/MRP14, which has been implicated in inflammatory rheumatic disease and cystic fibrosis.
CALCIUM SIGNAL TRANSDUCTION
Calcium and CaBPs play a central role in intracellular signal transduction and are associated with a wide range of effects on health and disease. Our goal is to understand the molecular basis for the activation of CaBPs and the concomitant transduction of calcium signals. Ultimately, we hope to control binding properties and interactions with target proteins, thereby enabling the design of specific biological activities and therapeutic strategies relevant to calcium-mediated disease. We are using nuclear magnetic resonance spectroscopy and computational methods to determine the three-dimensional structures and internal dynamics of specific CaBPs in the presence and absence of calcium of cellular targets. Comparative structural analysis and protein engineering experiments are being used to elucidate the fundamental principles that govern the calcium-induced activation of these proteins.
We are determining the structure of several novel CaBPs. One of these is caltractin, an essential component of the microtubule organizing center in the centrosome, which is required for accurate chromosomal segregation during the M stage (mitosis) of the cell cycle. Among CaBPs, this protein has the unique property of being phosphorylated in vivo, apparently in a cell cycle--dependent manner. Interest in caltractin is high because of its potential role in coupling calcium and phosphorylation signaling pathways. In collaboration with B. Huang, Department of Cell Biology, we have developed high-level expression systems for the intact protein and for the two separate domains and have extensively characterized the biophysical properties of all three. Determination of the structure of caltractin in the presence and absence of calcium is in progress.
We have placed a particular emphasis on the S100 subfamily of CaBPs. A growing body of evidence indicates that these proteins provide cell type--specific transduction of calcium signals. Members of the S100 subfamily have been implicated in a variety of disease states and have apparent cellular activities ranging from growth and proliferation to apoptosis. The S100 protein calcyclin is preferentially expressed in the G1 phase of the cell cycle, shows deregulated expression in association with cell transformation, and is found in high abundance in certain cancer cell lines. We determined the structure of native calcyclin in the apo state and found a novel organization of the calcium-binding domains (Fig. 1).
We propose that all other S100 proteins will have a similar structure and that their mode of signal transduction is clearly distinct from classical calmodulin-like calcium sensors. The structure of the calcium-activated state of calcyclin has recently been solved and is being refined. We are also characterizing interactions with one of calcyclin's cellular targets, annexin XI.
A new effort involves the MRP8 and MRP14 proteins, which constitute a unique two-chain S100 system that is expressed by myeloid cells during inflammatory reactions. Inflammatory disorders such as chronic bronchitis, cystic fibrosis, and rheumatoid arthritis are associated with elevated levels of MRP8/MRP14. Substantial progress has been made in the characterization of the pairing of the subunits and in the production of isotope-enriched protein samples for structure determination.
The comparison of different CaBPs is highly informative but is not sufficient to provide a fundamental understanding of the driving forces and the molecular basis for calcium activation. For this purpose, we are analyzing all known CaBP structures, determining the structure of calbindin D9k at ultrahigh resolution, and doing protein engineering experiments. In an effort to increase our understanding of calcium activation, we are attempting to rationally remodel calbindin D9k so that it undergoes a calmodulin-like opening when it binds calcium, thereby creating the novel protein calbindomodulin.
STRUCTURE-BASED DESIGN OF ANTITUMOR DRUGS
We are using three-dimensional structures and molecular design to determine the structural basis of the antitumor activity of ligands that bind in the minor groove of duplex DNA. From this foundation, we are establishing a rational basis for the design of new antitumor drugs. Projects involve determining, at the molecular level, the factors that govern binding affinity, specificity, and chemical reactivity of specific antitumor agents. Current efforts are focused on duocarmycin DNA alkylating agents and calicheamicin DNA scission agents, compounds that are distinguished by chemical reactivity with the DNA substrate and by exceptionally high potency.
We recently completed new structures of DNA complexes from each of these families. A high-resolution structure of (+)-duocarmycin-SA bound to a high-affinity binding site has been obtained (Fig. 2).
This and related duocarmycin structures reveal a DNA binding--induced twist in the ligand that catalyzes the alkylation reaction, slows the rate of the back reaction, and stabilizes the resulting adduct. The structures of the head-to-head and head-to-tail dimers of the calicheamicin oligosaccharide domain in complex with double-site DNA duplexes have shown the crucial role of the oligosaccharide in recognition and binding. We are probing the molecular basis for the high binding affinity and substantially increased inhibitory activity of these chemically linked oligosaccharide oligomers.
Eis, P.S., Smith, J., Rydzewski, J., Case, D.A., Boger, D.L., Chazin, W.J. High resolution solution structure of a DNA duplex alkylated by the antitumor agent duocarmycin SA. J. Mol. Biol. 272:237, 1997.
Kördel, J., Pearlman, D.A., Chazin, W.J. Protein solution structure calculations in solution: Solvated molecular dynamics refinement of calbindin D9k. J. Biomol. NMR, in press.
Lee, L.K., Rance, M., Chazin, W.J., Palmer, A.G. III. Rotational diffusion anisotropy of proteins from simultaneous analysis of 15N and 13C nuclear spin relaxation. J. Biomol. NMR 9:287, 1997.
Nelson, M., Chazin, W.J. Calmodulin as a calcium sensor. In: Calmodulin and Signal Transduction. van Eldik, L.J., Watterson, D.M. (Eds.). Academic Press, San Diego, in press.
Nelson, M., Chazin, W.J. An interaction-based analysis of calcium-induced conformational changes in Ca2+ sensor proteins. Protein Sci., in press.
Nelson, M., Weber, C., Chazin, W.J. Calcium-binding proteins. In: Encyclopedia of Molecular Biology. Creighton, T. (Ed.). Wiley, New York, in press.
Potts, B.C.M., Chazin, W.J. Chemical shift homology in proteins. J. Biomol. NMR, in press.
Skelton, N.J., Chazin, W.J. Solution structure determination of proteins using NMR spectroscopy. In: Peptide and Protein Analysis. Reid, R. (Ed.). Marcel Dekker, New York, in press.
Smith, J., Paloma, L.G., Case D.A., Chazin, W.J. Molecular dynamics docking driven by NMR derived restraints to determine the structure of the calicheamicin 1I oligosaccharide domain complexed to duplex DNA. Magn. Reson. Chem. 34:S147, 1996.
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Structure and Dynamics of Oligonucleotide Intermediates in Recombination and Repair
W.J. Chazin, D.P. Millar, R. Fee, D. Gonzales, S. Mangahas, G. Mer, S. Miick, M. Otto, A.G. Palmer III*
* Columbia University, New York, NY
The purpose of our multidisciplinary research program is to understand the structural biology of unusual oligonucleotide structures formed in the course of genetic recombination and repair. Our efforts to date have focused on a specific four-arm DNA crossover structure, the Holliday junction, which is formed as a transient intermediate during genetic recombination and repair. The structure and flexibility of these DNA crossovers appear to be critical to their recognition and processing into parental or recombinant products (Fig. 1).
The principal goals of our research are to determine the three-dimensional structure of Holliday junctions, to establish the dependence on sequence, and, ultimately, to understand how the cellular recombination machinery distinguishes Holliday junctions of different sequence.
We use nuclear magnetic resonance (NMR) and time-resolved fluorescence resonance energy transfer (tr-FRET) spectroscopies to study 32-bp model Holliday junctions. Computation and molecular modeling are used in data interpretation and analysis. Our studies have shown that the key sequence-dependent structural property of a Holliday junction is the equilibrium distribution between the two crossover structural isomers (Fig. 2).
In the past year, this research was highlighted by the discovery of a previously unrecognized dependence of the structure and dynamics of Holliday junctions on the sequence adjacent to the branching point. In addition, we developed new methods for the production of DNA oligomers and adapted a protocol that uses a DNA polymerase strategy to produce duplexes and Holliday junctions enriched in NMR active isotopes (13C, 15N). These labeled DNA molecules are being used to facilitate NMR analysis of complexes formed with recombination and repair enzymes.
NMR studies have provided complete 1H assignments for three synthetic 32-bp Holliday junctions and have established base pairing, local conformation in the region of the junction, and the presence and relative ratio of crossover isomers. Using tr-FRET, we have determined the global folding arrangement of the four duplex arms and characterized the unique conformational flexibility of these structures. An NMR selective isotope-based strategy and a new implementation of tr-FRET data analysis have been developed to quantitate the ratio of crossover isomers. Molecular dynamics calculations with restraints for NMR- and tr-FRET--derived distance ranges are being used to generate representations of the three-dimensional structures in solution. As a first step toward a biophysical analysis of Holliday junctions that can undergo branch migration, tr-FRET is being used to examine monomobile Holliday junctions designed to permit a single step of branch migration.
Together, these results are providing evidence to distinguish between various structural models proposed for Holliday junctions and to determine how the sequence at the branching point modulates the global folding, local molecular conformation, and internal dynamics of these four-arm DNA crossover structures.