News and Publications

Master Keys for Chemical Molecular Biology From Macromolecular Structures

We address central structural questions at the interface of cellular and molecular biology and chemistry. We wish to characterize and understand protein structures in order to develop chemical regulators as master keys for controlling fundamental biological processes associated with reactive oxygen signals and cytotoxins, pathogenesis, DNA repair, genetic variation, and the cell cycle. Interactions with staff at the Skaggs Institute have transformed our research by allowing in-depth characterization of the structure and chemical biology of human enzymes and initiation of long-term therapeutic goals for new treatments of inflammatory, degenerative, and infectious diseases; high blood pressure; stroke; and cancer.

Proteins Regulating Genome Stability and Variation

DNA repair proteins balance genome fidelity with the genome variability that underlies evolutionary selection. Specific mutations leading to decreased genome stability are critical early events in tumorigenesis, and DNA repair enzymes are key components in maintaining genome stability. Our new structures of complexes formed by base-excision repair enzymes and DNA and of complexes consisting of proteins that directly reverse DNA damage and the removed alkylation damage are thus providing key insights to the structural chemistry of tumorigenesis relevant to genotoxicology.

Our structures and kinetics of wild-type and mutant complexes formed by DNA repair enzymes and damaged DNA that initiate different pathways of DNA repair suggest new aspects of the structural chemistry that regulates recognition of DNA damage, progression along the repair pathway, and avoidance of destructive interference between different DNA repair steps and pathways. During this past year, for example, we established the mechanism for recognition and removal of damaged bases by the HhH superfamily of DNA glycosylases typified by enodnuclease III, which removes oxidized pyrimidines, and MutY, which removes adenine-guanine mismatches due to oxidized guanine (Fig. 1). Recognition of damaged bases by these DNA glycosylases appears to provide a structural basis for specific recruitment of subsequent enzymes that are not recruited by proteins that act in single-step repair.

Single-step repair is an important type of DNA repair that involves the direct reversal of damage to DNA bases, such as the reversal accomplished by the alkyltransferases. Our studies of alkyltransferases typify our structure-based efforts to develop new cancer treatments that use inhibitors designed to specifically disrupt DNA repair. O6-alkylguanine alkyltransferase (AGT) is the human DNA repair protein that removes the promutagenic lesions caused by alkylation of guanine at the O6 position. Many cancer chemotherapies involve DNA alkylation, and the cytotoxic effects of the agents used are due to generation of alkylated bases, such as O6-methylguanine. Because AGT can repair such lesions through a direct and irreversible alkyl transfer reaction, numerous tumor cell lines develop resistance to alkylation therapies by increasing expression of AGT. Correspondingly, inhibitors of AGT can sensitize tumors to alkylation therapies and thereby potentiate current chemotherapies.

To begin an iterative cycle of the structure-based design of inhibitors, we determined the structure of native human AGT to 2.0-Å resolution. The structure reveals a 2-domain α/ß protein and identifies the active site. Our structure of a product complex of AGT with the suicide inhibitor O6-benzylguanine provides the first look at recognition of damage by the active site of AGT and the specificity of the active site for damaged areas. Using this complex, the location of evolutionarily conserved residues, and available kinetic data on point mutations, we inferred the basis for substrate recognition. AGT presents a hydrophobic cleft that recognizes alkylated guanine bases with a system of complementary hydrogen bonds. Comparison of the AGT structure with the structures of DNA base excision enzymes and DNA methyltransferases suggests how AGT may combine different methods of protein-DNA interactions observed in DNA repair enzymes and DNA methyltransferases.

DNA repair of bulky lesions requires enzymes such as hexameric DNA helicases. These helicases are amazing molecular motors that unwind DNA for replication, recombination, and repair. Our diffraction studies on the helicase RuvB will characterize one of the key helicases involved in recombination repair.

We are also studying another protein key to eukaryotic genome integrity, the multifunctional enzyme flap endonuclease (FEN-1). Mutations in FEN-1 result in DNA duplication defects that occur in human tumors and inherited human diseases. FEN-1 is a structure-specific nuclease necessary for DNA repair and for processing the 5´ ends of Okazaki fragments during the synthesis of lagging strands of DNA. Structural analysis of FEN-1 revealed new motifs for DNA binding and defined the motif for formation of complexes of FEN-1 with the processivity factor for DNA polymerase, termed proliferating cell nuclear antigen (Fig. 2).

DNA repair and genome fidelity are tightly controlled by checkpoints in the cell cycle that link DNA repair and replication. Our structures of proteins in humans that regulate the cell cycle, proteins essential to progression of the cycle, suggest mechanisms for regulation that involve a unique conformational switch that controls kinase recognition. Working with S. Reed, The Scripps Research Institute, we are examining the interactions between radiation-induced DNA damage and cell-cycle checkpoints.

Genomic variation of human pathogens creates new threats to health in the form of emerging infectious diseases. Our research in this area focuses on pili, fibers that not only allow pathogenic bacteria to crawl across host cells but also act in taking into the cell large regions of foreign DNA containing new pathogenicity genes. With funding from the Skaggs Institute, we have broadened our studies to include pili of Vibrio cholerae and Neisseria meningitidis. In this research, in collaboration with M. Yeager and R. Milligan, The Scripps Research Institute, we are bridging the resolution gap between protein structures and supermolecular assemblies by developing new techniques for combining the results of electron microscopy and x-ray diffraction.

Reactive Oxygen Signals and Defensive Cytotoxins

Human superoxide dismutases protect DNA and other cellular components from the oxidative damage caused by superoxide that occurs in degenerative diseases such as Lou Gehrig disease, cancer, and aging. As part of our work on these enzymes, we solved structures of human catalase, an enzyme that protects cells against oxidative damage and cell death caused by hydrogen peroxide. These structures help define the catalase mechanism, suggest a role for the cofactor NADPH, and establish the basis for activity of an important heterocycle inhibitor.

A second major focus for our research on the regulation of reactive oxygen is structural studies of nitric oxide synthases (NOSs), done in collaboration with E. Getzoff, the Skaggs Institute. These enzymes oxidize arginine to make nitric oxide, a key intercellular signal and defensive cytotoxin in the nervous, cardiovascular, and immune systems. Overproduction of nitric oxide by inducible NOS (iNOS) in macrophages can lead to many abnormalities, including juvenile diabetes, arthritis, aneurysms, neurodegenerative disorders, and septic shock. Consequently, specific inhibitors of iNOS are of great therapeutic potential. The structures of ligands bound to NOS suggest new aspects of the enzyme mechanism and aid in the design of specific inhibitors.

Publications

Guan, Y., Manuel, R.C., Arvai, A.S., Parikh, S.S., Mol, C.D., Miller, J.H., Lloyd, S., Tainer, J.A. MutY catalytic core, mutant and bound adenine structures define specificity for DNA repair enzyme superfamily. Nat. Struct. Biol. 5:1058, 1998.

Hosfield, D.J., Frank, G., Weng, Y., Tainer, J.A., Shen, B. Newly discovered archaebacterial flap endonucleases show a structure-specific mechanism for DNA substrate binding and catalysis resembling human flap endonuclease-1. J. Biol. Chem. 273:27154, 1998.

Hosfield, D.J., Mol, C.D., Shen, B., Tainer, J.A. Structure of the DNA repair and replication endonuclease and exonuclease FEN-1: Coupling DNA and PCNA binding to FEN-1 activity. Cell 95:135, 1998.

Lo, T.P., Thayer, M.M., Koike, C.K., Hallewell, R.A., Getzoff, E.D., Tainer, J.A. Variability among multiple subunits in the high-resolution structure of human Cu,Zn superoxide dismutase. In: Superoxide Dismutases: Recent Advances and Clinical Applications. Edeas, M.A. (Ed.). Editions Mel Paris, Paris, France, 1999, p. 22.

Mol, C.D., Parikh, S.S., Lo, T.P., Tainer, J.A. Structural phylogenetics of DNA base excision repair. Nucleic Acids Mol. Biol. 12:29, 1998.

Parikh, S.S., Mol, C.D., Hosfield, D.J., Tainer, J.A. Envisioning the molecular choreography of DNA base excision repair. Curr. Opin. Struct. Biol. 9:37, 1999.

Parikh, S.S., Mol, C.D., Slupphaug, G., Bharati, B., Krokan H.E., Tainer, J.A. Base excision repair initiation revealed by crystal structures and binding kinetics of human uracil-DNA glycosylase with DNA. EMBO J. 17:5214, 1998.

Putnam, C.D., Shroyer, M.J.N., Lundquist, A.J., Mol, C.D., Arvai, A.S., Mosbaugh, D.W., Tainer, J.A. Protein mimicry of DNA from crystal structures of the uracil-DNA glycosylase inhibitor protein and its complex with Escherichia coli uracil-DNA glycosylase. J. Mol. Biol. 287:331, 1999.

Shen, B., Qiu, J., Hosfield, D., Tainer, J.A. Flap endonuclease homologs in archaebacteria exist as independent proteins. Trends Biochem. Sci. 25:171, 1998.