STRUCTURAL MOLECULAR BIOLOGY AND PROTEIN DESIGN

John A. Tainer, Ph.D.

Professor, Department of Molecular Biology
and The Skaggs Institute for Chemical Biology
J. A. Tainer, M. Aoyagi, A. S. Arvai, D. P. Barondeau, G. E. O. Borgstahl , S. L. Bernstein, Y. Bourne, C. M. Bruns, J. M. Castagnetto, B. R. Crane, T. H. Cross, D. S. Daniels, G. DiVita, C. L. Fisher, K. T. Forest, Y. Guan, R. A. Hallewell, S. Han, F. R. Henderson, M. J. Hickey, K.-P. Hopfner, D. J. Hosfield, A. Karcher, C. K. Koike, T. P. Lo, T. J. Macke, C. D. Mol, S. S. Parikh, J. L. Pellequer, M. E. Pique, C . D. Putnam, S. M. Redford, D. S. Shin, M. M. Thayer.

Other Useful Information: Grant Deadlines, Synchroton Deadlines, Dawn's Page

Key words: DNA-repair, metalloenzyme, oxidative damage, degenerative disease, infectious disease, Lou Gehrig's disease, superoxide dismutase, cell cycle, cancer, protein design

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Visit some of our collaborators at TSRI: [E. D. Getzoff] [V. A. Roberts] [Metalloprotein Structure and Design Group]

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Movie of reaction AP Endonuclease DNA repair enzyme

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DNA Damage Excision Enzyme Structures

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Structure of Nitric Oxide Synthase Oxygenase Dimer with Pterin and Substrate.

B.R. Crane, A.S. Arvai, D.K. Ghosh, C. Wu, E.D. Getzoff, D.J. Stuehr and J.A. Tainer.

Science 279, 2121-21216, 1998. Visit Science Magazine Online

Check out our NOS Steroeviews and Figures.

We focus on four classes of proteins that interface structural biology with cellular chemistry:

  1. Enzymes regulating reactive oxygen and xenotoxin control (superoxide dismutases, catalase, and glutathione transferase);
  2. Enzymes controlling DNA repair and evolution enzymes;
  3. Pilus fiber motility and adherence proteins for bacterial pathogens;
  4. Cell cycle control proteins.
Current structural and design results are contributing to an understanding of these systems that should contribute to new treatments for infectious disease, degenerative diseases, and cancer.

Bacterial pilus structure, assembly, and antigenicity.

We seek to improve understanding and to develop new treatments for the many bacterial pathogens that use long fibers termed Type IV pili for attachment and mobility. We solved the crystallographic structure of the pilus fiber- forming pilin protein essential to the virulence of these pathogens, which includes Neisseria meningitidis, Neisseria gonorrhoeae, Pseudomonas aeroginosa, Dichlobacter nodosus, Moraxella bovis, Vibrio cholerae, and enterotoxogenic Escherichia coli. The pilin subunit is ladle- shaped with an extended a-helix spine wrapped at one end by b-sheet. A few key residue interactions allow the remaining part of a hypervariable region to undergo extreme antigenic variation to escape t he host immune response. The assembled fiber shows extreme sequence variation plus glycosylation and phosphorylation at the survace. 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 Cu,Zn superoxide dismutases, the mitochondrial Mn superoxide dismutases, and schistosomal glutathione transferases are improving our understanding of reactive oxygen and xenobiotic control within cells. Superoxide dismutases (SODs) are master regulators for reactive oxygen species involved in injury, pathogenesis, aging and degenerative diseases. For Cu,Zn SOD, we have defined the active site structural chemistry responsible for the rapid reaction. We are now exam ining how single site mutations cause degenerative disease such as Lou Gehrig's disease or familial amyotrophic lateral sclerosis (FALS). For Mn SOD, single site mutations can destabilize the tetramer and also reduce stability and activity in ways that ma y cause degenerative diseases. Structures of glutathione-S-transferase (GST), which is an essential detoxification enzyme in all organisms, show how the leading anti-schistosomal drug praziquantel binds to GST. This information may allow the design of new drugs to overcome the growing resistance to current schistosomal drugs. Protein design on GST enzymes includes using random libraries for the loop regions surrounding the active site, thereby developing glubodies as a new class of binding proteins in bio technology.

DNA-repair and genetic evolution.

Cells must balance DNA repair to preserve fidelity with DNA variation allowing evolutionary changes. As over 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 and chemotherapies for cancer by specifically killing cancer cells which unlike normal cells will often undergo DNA synthesis and cell division with unrepaired DNA resulting in their death. Our DNA repair enzyme structures show how damaged DNA bases are recognized and removed in atomic detail. These enzymes repair DNA by flipping the DNA nucleotides out from the double helix and into specific binding pockets (Figure), which are ideal for the design of inhibitors for anti-cancer therapies. We confirmed our understanding of these binding pockets by deliberately altering the specificity of the DNA repair enzyme uracil-DNA glycosylase to 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 APendonuclease, which cuts DNA at sites where bases are missing, defines its active site and identifies 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 thymic-less cell death from cycles of uracil misincorporation and removal that would generate multiple DNA strand breaks and eventual cell death. Atomic structures of dUTPase with bound nucleotides reveal 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 anti-cancer drugs.

    • Cell cycle control.

    Together with Steve Reed's group, we are working to define structural controls on cell cycle progression. Structures of the Cks or suc1 proteins which are essential to cell cycle progression, provide clues for new mechanisms for cell cycle regulation via a conformational switch that controls two distinct Cks folds and assemblies. A straight b-hinge conformation of Cks, which forms a dimer of swapped b-strands blocks binding to the cell cycle kinase Cdk2. Formation of a closed, bent b- hinge conformation creates a single domain fold that promotes Cdk2 binding (Figure). Preliminary experiments by Steve Reed's group show that blocking Cks expression results in cell death for several types of cancer cells suggesting this is a useful cancer drug target.

    • Publications

    Boissinot, M., Karnas, S., Lepock, J. R., Cabelli, D. E., Tainer, J. A., Getzoff, E. D., Hallewell, R. A. (1997). "Function of the Greek key connection analysed using circular permutants of superoxide dismutase", EMBO J., 16, 2171-2178.

     Crane, B. R., Arvai, A. S., Gachhui, R., Wu, C., Ghosh, D. K. P., Getzoff, E. D., Stuehr, D. J. and Tainer, J. A. (1997). "The structure of NO synthase oxygenase domain and inhibitor complexes", Science, 278, 425-431.

     Crane, B. R., Arvai, A. S., Ghosh, D. K. P., Wu, C. P., Getzoff, E. D., Stuehr, D. J. and Tainer, J. A. (1998). "Structure of nitric oxide synthase oxygenase dimer with pterin and substrate", Science, 279, 2121-2126.

     Fisher, C. L., Cabelli, D. E., Tainer, J. A., Hallewell, R. A., and Getzoff, E. D. (1997). "Computational, pulse-radiolytic and structural investigations of lysine 136 and its role in the electrostatic triad of human of Cu,Zn superoxide dismutase", Proteins: Structure, Function and Genetics, 29, 103-112.

     Forest, K. T. and Tainer, J. A. (1997). "Type IV pilus structure: outside to inside and top to bottom", Gene, 192, 165-169.

     Gorman, M. A., Morera, S., Rothwell, D. G., La Fortelle, E. D., Mol, C. D. and Tainer, J. A. (1997). "The crystal structure of the human DNA-repair enzyme endoclease HAP1 suggests the recognition of extra-helical deoxyribose at DNA abasic sites", EMBO J. 16, 6548-5448.

     Guan, Y., Hickey, M. J., Borgstahl, G. E. O., Hallewell, R. A., Lepock, J. R., O'Oconner, D., Hsieh, Y., Nick, H. S., Silverman, D. N. and Tainer, J. A. (1998). "The crystal structure of Y34F mutant human mitochondrial manganese superoxide dismutase and the functional role of Tyrosine 34", Biochemistry, 37, 4722-4730.

     Hsieh, Y., Guan, Y., Tu, C., Bratt, P. J., Angerhofer, A., Lepock, J. R., Hickey, M. J., Tainer, J. A., Nick, H. S. and Silverman, D. N. (1998). "Probing the active site of human manganese superoxide dismutase: The role of Glutamine 143", Biochemistry, 37, 4731-4739.

     Marccau, M., Forest, K., Bertti, J. -L., Tainer, J. A. and Nassif, X. (1998). "Role of O-linked glycosylation of meningococcal type IV pilin for piliation and pilus-mediated adhesion", Mol. Microbiol., 27, 705-715.

     Parikh, S. S., Mol, C. D. and Tainer, J. A. (1997). "Base excision repair enzyme family portrait: integrating the structure and chemistry of an entire DNA repair pathway", Structure, 5, 1543-1550.

    Roberts, V. A., Nachman, R. J., Coast, G. M., Hariharan, M., Chung, J. S., Holman, G. M., Williams, H., and Tainer, J. A. (1997). "Consensus chemistry and b-turn conformation of the active core of the myotropic/diuretic insect neuropeptide family", Chem. and Biol., 4, 105-117.

    Shen, B., Qiu, J., Hosfield, D. and Tainer, J. A. (1998). "Flap endonuclease homologues in Archebacteria exist as independent proteins", Trends in Biol. Sci., in press.

     Tong, W., Burdi, D., Riggs-Gelasco, P., Chen, S., Edmondson, D., Huynh, B. H., Stubbe, J., Han, S., Arvai, A. S. and Tainer, J. A. (1998). "Characterization of Y122F R2 of Escherichia coli ribonucleotide reductase by time-resolved physical biochemical methods and X-ray crystallography", Biochemistry, 37, 5840-5848.

     Zu, J. S., Deng, H-X, Lo, T. P., Mitsumoto, H., Ahmed, M. S., Hung, W-Y., Cai, Z-J., Tainer, J. A., and Siddique, T. (1997). "Exon5 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 amyotropic lateral sclerosis", Neurogenetics, 1, 65-71.