Scientific Report 2005

Molecular Biology

Structural Molecular Biology of Interactions and Protein Design

J.A. Tainer, A.S. Arvai, D.P. Barondeau, M. Bjoras, B.R. Chapados, L. Craig, T.H. Cross, D.S. Daniels, G. DiVita, L. Fan, C. Hitomi, K. Hitomi, J.L. Huffman, C.J. Kassmann, I. Li, G. Moncalian, M.E. Pique, D.S. Shin, O. Sundheim, R.S. Williams, T.I. Wood, A. Yamagata

Our goals are to bridge the gap between the vastly improved tools and insights for structural cell biology at the molecular level and the applications of these advances for the molecular-based understanding of and eventual intervention in human diseases. Thus, our primary concern is the application of structural biology to fundamental questions of molecular and cellular biology relevant to human disease. Currently, we are investigating fundamental processes and principles of DNA repair, control of reactive oxygen species, control of the cell cycle, and pathogenesis. We think these processes have networked connections and common themes in terms of structural mechanisms and controls and medical implications. In general, our structural determination and design work involves hypothesis-driven studies; we focus on high-resolution structural analyses, functionally important conformational changes, and macromolecular interactions, including design of inhibitors and dynamic assemblies that act as macromolecular machines to control the fundamental processes of cell biology.

To accomplish our basic research, we use protein crystallography, solution x-ray scattering, fluorescence, biochemistry, mutagenesis, and protein expression. Our experimental work is complemented by efforts to develop new methods, particularly in structural analysis, protein and drug design, and the merging of crystal structures with x-ray solution structures and electron microscopy. These new experimental integrations involve the use of synchrotron radiation to bridge the size and resolution gap between high-resolution macromolecular structures and the multiprotein macromolecular machines and reversible interactions in the cell. For protein design, we have an active collaboration with E. Getzoff, Department of Molecular Biology, to understand and control the formation of self-synthesizing chromophores in green fluorescent protein and its homologs. We are increasingly interested in structure-based design of inhibitors that are relevant to the development of novel therapeutic agents and inhibitors that chemically knock out or block gene function to complement genes that are knocked out by removing the DNA. The synergy between basic research and advances in techniques is allowing us to contribute to the basic understanding and treatment of degenerative and infectious diseases and cancer.

Superoxide Dismutases

Superoxide dismutases (SODs) are master regulators for reactive oxygen species involved in injury, pathogenesis, aging, and degenerative diseases. In basic research on these enzymes, we are characterizing the activity of the mitochondrial SODs. We discovered a novel nickel ion SOD and characterized its hexameric assembly. For the human cytoplasmic copper, zinc SOD, we examined how single-site mutations cause the neurodegenerative Lou Gehrig disease or familial amyotrophic lateral sclerosis (FALS). We found that point mutations destabilize the copper, zinc SOD dimer and dramatically increase its propensity to aggregate and form filaments that resemble those seen in motor neurons of patients with FALS. These findings provide a molecular basis for the notion that a single FALS disease phenotype arises from diverse point mutations throughout the protein that reduce the structural integrity of copper, zinc SOD and lower the energy barrier for fibrous aggregation. Additionally, our new high-resolution structures of a related thermophilic copper, zinc SOD showed a trapped product complex. This novel finding helps define the enzyme’s mechanism of action and its susceptibility to inactivation by hydrogen peroxide.

DNA Repair

All life requires constant repair of DNA. Structural and mutational analyses of DNA repair enzymes provide a framework for understanding the molecular basis of genetic integrity and the loss of this integrity in cancer and degenerative diseases. We are interested in how specific types of damage are detected, how repair enzymes are coordinated within different pathways, and the nature and role of conformational change in proteins and DNA in repair pathways. We use electron microscopy, x-ray crystallography, small-angle x-ray scattering, and complementary in vitro and in vivo mutational analysis to go from enzyme structures to repair pathways and the coordination of repair with replication and transcription.

We focus on pathways for DNA base repair, DNA nick translation in repair and replication (Fig. 1), and repair of double-stranded breaks.

Fig. 1. Interactions between the complex consisting of flap endonuclease 1 (FEN-1), DNA, and proliferating cell nuclear antigen (PCNA) and the interface of DNA repair and replication. A, Nicked DNA is protected and repaired by the sequential activities of DNA polymerase δ(pol δ) and FEN-1 held to DNA by the "sliding clamp" PCNA. In the absence of FEN-1, a complex of pol δ and PCNA binds to and protects the nick (top). FEN-1 initiates nick translation by binding to PCNA (bottom), recognizing the 3´ DNA flap and cleaving the 5´ flap, generating a nick translated by 1 nucleotide. B. Structures of FEN-1 bound to DNA show that FEN-1 recognizes the 3´ flap in a sequence-independent manner. C, A composite model of the FEN-1–DNA–PCNA complex suggests how a kinked DNA intermediate might facilitate sequential activities of FEN-1 and pol δ.

Understanding the structural chemistry and cell biology of DNA repair is critical for designing specific inhibitors to increase the effectiveness of chemotherapy and also for assessing how DNA repair enzyme polymorphisms may affect diseases in humans. Currently, we are designing inhibitors of enzymes that repair alkylated and oxidized guanines. These enzymes are one of the body’s natural defenses against DNA damage, but they can also inadvertently protect cancer cells from chemotherapeutic agents. For example, the human repair protein O⁶-alkylguanine-DNA alkyltransferase, which acts in the repair of alkylated guanines, repairs damaged DNA inside human cells, and cancer cells can use it to repair DNA that has been damaged in the course of chemotherapy, thus making the chemotherapy ineffective.

Bacterial Pili

Type IV pili are essential virulence factors for many gram-negative bacteria, playing key roles in surface motility, adhesion, formation of microcolonies and biofilms, natural transformation, and signaling. We have determined structures for the type IV pilin subunits and for the assembled pilus fiber. Currently, we are investigating the type IV pilus assembly system, including the assembly ATPase, the membrane anchor protein interactions, and the assembled pilus fiber (Fig. 2).

Fig. 2. A schematic view of the assembly machinery of type IV pili: the electron cryomicroscopy structure of the pilus of Neisseria gonorrhoeae (GC); crystal structures of full-length Pseudomonas aeruginosa (P.a) pilin; BfpC, the binding partner protein to ATPase from enteropathogenic Escherichia coli; and GspE2, the hexameric assembly ATPase from Archaeoglobus fulgidus.

Our electron microscopy and x-ray structures of protein components and complexes are helping us understand the architecture and assembly mechanism as a basis for the design of antibacterial vaccines and therapeutic agents.

Publications

Ayala, I., Perry, J.P., Szczepanski, J., Tainer, J.A., Vala, M.T., Nick, H.S., Silverman, D.N. Hydrogen bonding in human manganese superoxide dismutase containing 3-fluorotyrosine. Biophys. J., in press.

Barondeau, D.P., Kassmann, C.J., Tainer, J.A., Getzoff, E.D. Understanding GFP chromophore biosynthesis: controlling backbone cyclization and modifying post-translational chemistry. Biochemistry 44:1960, 2005.

Crowther, L.J., Yamagata, A., Craig, L., Tainer, J.A., Donnenberg, M.S. The ATPase activity of BfpD is greatly enhanced by zinc and allosteric interactions with other Bfp proteins. J. Biol. Chem. 280:24839, 2005.

de Jager, M., Trujillo, K.M., Sung, P., Hopfner, K.P., Carney, J.P., Tainer, J.A., Connelly, J.C., Leach, D.R., Kanaar, R., Wyman, C. Differential arrangements of conserved building blocks among homologs of the Rad50/Mre11 DNA repair protein complex. J. Mol. Biol. 339:937, 2004.

Garcin, E.D., Bruns, C.M., Lloyd, S.J., Hosfield, D.J., Tiso, M., Gachhui, R., Stuehr, D.J., Tainer, J.A., Getzoff, E.D. Structural basis for isozyme-specific regulation of electron transfer in nitric-oxide synthase. J. Biol. Chem. 279:37918, 2004.

Hendrickson, E.A., Huffman, J.L., Tainer, J.A. Structural aspects of Ku and the DNA-dependent protein kinase complex. In: DNA Damage Recognition. Seide, W., Kow, Y.W., Doetsch, P.W. (Eds.). Taylor & Francis, New York, 2005, p. 629.

Huffman, J.L., Sundheim, O., Tainer, J.A. DNA base damage recognition and removal: new twists and grooves. Mutat. Res. 577:55, 2005.

Huffman, J.L., Sundheim, O., Tainer, J.A. Structural features of DNA glycosylases and AP endonucleases. In: DNA Damage Recognition. Seide, W., Kow, Y.W., Doetsch, P.W. (Eds.). Taylor & Francis, New York, 2005, p. 299.

Manuel, R.C., Hitomi, K., Arvai, A.S., House, P.G., Kurtz, A.J., Dodson, M.L., McCullough, A.K., Tainer, J.A., Lloyd, R.S. Reaction intermediates in the catalytic mechanism of Escherichia coli MutY DNA glycosylase. J. Biol. Chem. 279:46930, 2004.

Putnam, C.D.. Tainer, J.A. Protein mimicry of DNA and pathway regulation. DNA Repair (Amst.), in press.

Sarker, A.H., Tsutakawa, S.E., Kostek, S., Ng, C., Shin, D.S., Peris, M., Campeau, E., Tainer, J.A., Nogales, E., Cooper, P.K. Recognition of RNA polymerase II and transcription bubbles by XPG, CSB, and TFIIH: insights for transcription-coupled repair and Cockayne syndrome. Mol. Cell 20:187, 2005.

Simeoni, F., Arvai, A., Bello, P., Gondeau, C., Hopfner, K.P., Neyroz, P., Heitz, F., Tainer, J., Divita, G. Biochemical characterization and crystal structure of a Dim1 family associated protein: Dim2. Biochemistry 44:11997, 2005.

Tubbs, J.L., Tainer, J.A., Getzoff, E.D. Crystallographic structures of Discosoma red fluorescent protein with immature and mature chromophores: linking peptide bond trans-cis isomerization and acylimine formation in chromophore maturation. Biochemistry 44:9833, 2005.

Williams, R.S., Tainer, J.A. A nanomachine for making ends meet: MRN is a flexing scaffold for the repair of DNA double-strand breaks. Mol. Cell 19:724, 2005.