News and Publications

Master Keys for Chemical Molecular Biology From Macromolecular Structures

We use experimental and computational structural analysis to address central questions at the interface of cellular and molecular biology with 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 enhanced our structural studies by increasing our ability to pursue in-depth biological understanding and long-term goals for the development of new treatments for inflammatory, degenerative, and infectious diseases; high blood pressure; stroke; and cancer.

Regulation of Genome Stability and Variation

Proteins involved in DNA repair balance genome fidelity with genome variability. Specific mutations leading to decreased genome stability are critical early events in tumorigenesis, and DNA-repair enzymes are key components in maintaining genome stability. Yet, genetic variation provides the basis for evolutionary selection. An unusual but important type of DNA repair involves the direct reversal of damage to DNA bases, such as is accomplished by the alkyltransferases. These enzymes help humans resist alkylating agents, including agents used in chemotherapy. We are doing high-resolution diffraction studies of the human alkylguanine-DNA alkyltransferase and have begun designing inhibitors; the latter work includes crystallization of the enzyme with bound synthetic inhibitors.

A principle type of DNA repair is removal of damaged bases from DNA by excision. We are solving the structures of enzymes that initiate and thereby control base-excision repair in humans (Fig. 1).

Our findings have suggested a new model for enzyme-DNA recognition that involves nucleotide flipping to expose a damaged DNA base for recognition of specific damage by repair enzymes. Examination of high-resolution cocrystallized structures of DNA in complex with wild-type or mutant human uracil-DNA glycosylase, plus the structure of the unbound glycosylase, has resolved some of the remaining fundamental issues in the initiation of DNA base-excision repair. These issues include detection of damage, nucleotide flipping vs extrahelical capture of nucleotides, avoidance of abasic site toxicity, and coupling of damage-specific and damage-general steps in base-excision repair.

Nucleotide-excision repair, which is essential for the 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. We are continuing diffraction studies on RuvB, a key helicase involved in recombination repair.

We have also begun studies on another protein key to eukaryotic genome integrity: the multifunctional enzyme flap endonuclease, or FEN-1. Mutations in FEN-1 result in defects in DNA duplication 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 has revealed new motifs for DNA binding and has defined the motif for formation of complexes of FEN-1 with the processivity factor for DNA polymerase, termed proliferating cell nuclear antigen.

DNA repair and genome fidelity are tightly controlled by checkpoints in the cell cycle that link DNA repair and replication. Our structures of the regulatory human Cks proteins, which are essential to progression of the cell cycle, suggest novel mechanisms for regulation of the cycle that involve a unique conformational switch that controls 2 distinct Cks folds and assemblies. Working with S. Reed, Department of Molecular Biology, The Scripps Research Institute, we have now tied Cks to DNA-repair checkpoints and are pursuing structural studies on these complexes.

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. These hairlike fibers not only allow pathogenic bacteria to crawl across host cells but also participate in the entry of foreign DNA containing new pathogenicity genes into the bacterial cell. With support from the Skaggs Institute, we have broadened our studies on Neisseria gonorrhoeae pili to include studies on Neisseria meningitidis. During the past year, we characterized structural and functional roles for phosphorylation and glycosylation of pili.

Reactive Oxygen Signals and Defensive Cytotoxins

Mutations of the enzyme superoxide dismutase cause an inherited form of the neurodegenerative disease amyotrophic lateral sclerosis or Lou Gehrig disease. Our crystal structures of these and other mutant superoxide dismutases reveal much about the properties of the enzymes and the defects underlying Lou Gehrig disease. Recently, we detected an unexpected link between superoxide dismutase and the sporadic form of Lou Gehrig disease.

A major new focus for our research on the regulation of reactive oxygen is structural studies of the enzyme nitric oxide synthase (NOS, Fig. 2) in collaboration with E. Getzoff, the Skaggs Institute. NOSs 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.

Our structures of monomeric murine iNOS reveal a novel topology and heme environment. We recently solved 3 structures of fully functional, pterin-loaded, oxygenase domain dimers of iNOS that reveal how dimerization, the tetrahydrobiopterin cofactor, and arginine binding complete the catalytic center for synthesis of nitric oxide. The ligand-bound NOS structures suggest that NOS catalysis has evolved to select between 2 different reductive activations of dioxygen, depending on the protonation state of the reactant.

Publications

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.

Crane, B.R., Arvai, A.S., Gachhui, R., Wu, C., Ghosh, D.K., Getzoff, E.D., Stuehr, D.J., Tainer, J.A. The structure of NO synthase oxygenase domain and inhibitor complexes. Science 278:425, 1997.

Crane, B.R., Arvai, A.S., Ghosh, D.K., Wu, C.P., Getzoff, E.D., Stuehr, D.J., Tainer, J.A. Structure of nitric oxide synthase oxygenase dimer with pterin and substrate. Science 279:2121, 1998.

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 29:103, 1997.

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. 16:6548, 1997.

Guan, Y., Hickey, M.J., Borgstahl, G.E.O., Hallewell, R.A., Lepock, J.R., O'Connor, D., Hsieh, Y., Nick, H.S., Silverman, D.N., Tainer, J.A. The crystal structure of Y34F mutant human mitochondrial manganese superoxide dismutase and the functional role of tyrosine 34. Biochemistry 37:4722, 1998.

Hsieh, Y., Guan, Y., Tu, C., Bratt, P.J., Angerhofer, A., Lepock, J.R., Hickey, M.J., Tainer, J.A., Nick, H.S., Silverman, D.N. Probing the active site of human manganese superoxide dismutase: The role of glutamine 143. Biochemistry 37:4731, 1998.

Marccau, M., Forest, K., Bertti, J.-L., Tainer, J.A., Nassif, X. Consequences of the loss of O-linked glycosylation of meningococcal type IV pilin on piliation and pilus-mediated adhesion. Mol. Microbiol. 27:705, 1998.

Mol, C.D., Parikh, S.S., Lo, T.P., Tainer, J.A. Structural phylogenetics of DNA base-excision repair. Nucleic Acids Mol. Biol., in press.

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 DNA-binding kinetics of human uracil-DNA glycosylase bound to DNA. EMBO J., in press.

Parikh, S.S., Mol, C.D., Tainer, J.A. Base excision repair enzyme family portrait: Integrating the structure and chemistry of an entire DNA repair pathway. Structure 5:1543, 1997.

Shen, B., Qiu, J., Hosfield, D., Tainer, J.A. Flap endonuclease homologues in Archaebacteria exist as independent proteins. Trends Biochem. Sci., in press.

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 encoded domain is not required for the toxic function of mutant SOD1 but is essential for dismutase activity: Identification and characterization of two new SOD1 mutations associated with familial amyotrophic lateral sclerosis. Neurogenetics 1:65, 1997.