This past year we made major breakthroughs in developing a unified understanding of the multifunctional molecular machinery of type IV pili involved in bacterial pathogenesis. Combined electron cryomicroscopy and crystallography allowed us to achieve the first view of these pili at high enough resolution to see surface grooves implicated in DNA and receptor binding and modifications that act in the escape from the host immune system (Fig. 2). Our cover article in the journal Molecular Cell provides a new understanding of how type IV pili can act in so many functions, including movement, attachment, and DNA binding. Because type IV pili are critical bacterial virulence factors, understanding their structure and function is critical to controlling cholera, pneumonia, gonorrhea, meningitis, and severe diarrhea. We also examined crystal and solution structures of the assembly ATPases and membrane anchor proteins involved in the assembly of fibers of pathogens. These discoveries provide new knowledge and new drug targets for bacterial infectious diseases.
In collaboration with E.D. Getzoff, we are defining the structures and mechanisms for the posttranslational modification responsible for functional modifications of green fluorescent protein. These studies are providing a unified understanding of green fluorescent protein with numerous implications for protein design and control of posttranslational modifications.
For DNA repair machines critical for repair of DNA damage, including double-strand breaks and chromosome aberrations, we are characterizing DNA damage responses and molecular machines relevant to cancer-related aspects of genome maintenance. These Skaggs-funded analyses of DNA repair machines are providing new concepts for the coordination of DNA repair and replication events relevant to understanding cancer initiation, aging, and neurodegenerative diseases. This research is relevant to both the cause and treatment of cancer, which caused the deaths of 553,768 persons in the United States in 2001, the most recent year for which such statistics are available.
Our research on the XPB helicase revealed how this enzyme can act in damage checking during transcription. We discovered that XPB has a novel damage-recognition domain that activates the helicase. Mutations of the DNA repair protein WRN can give rise to Werners syndrome, which is characterized by rapid aging, and mutations in XPB cause cancer and neuropathologic changes. Our work on the WRN nuclease structures provides an understanding of the roles of this enzyme in processing DNA ends for break repair. In other projects, we characterized a unique DNA base damage reversal enzyme involved in the resistance to chemotherapies and the protein subunit that holds the mitochondrial DNA polymerase on DNA to achieve efficient DNA replication in mitochondria. Inherited mitochondrial defects are associated with degenerative diseases and neuropathologic changes. Thus, our Skaggs-funded molecular characterizations of protein functional features and their disruption by disease-causing mutations are providing a molecular basis to connect inherited gene mutations to disease phenotypes.
Ayala, P., Wilbur, J.S., Wetzler, L.M., Tainer, J.A., Snyder, A., So, M. The pilus and porin of Neisseria gonorrhoeae cooperatively induce Ca2+ transients in infected epithelial cells. Cell. Microbiol. 7:1736, 2005.
Barondeau, D.P., Kassmann, C.J., Tainer, J.A., Getzoff, E.D. Understanding GFP posttranslational chemistry: structures of designed variants that achieve backbone fragmentation, hydrolysis, and decarboxylation. J. Am. Chem. Soc. 128:4685, 2006.
Barondeau, D.P., Tainer, J.A., Getzoff, E.D. Structural evidence for an enolate intermediate in GFP fluorophore biosynthesis. J. Am. Chem. Soc. 128:3166, 2006.
Craig, L., Volkmann, N., Arvai, A.S., Pique, M.E., Yeager, M., Egelman, E.H., Tainer, J.A. Type IV pilus structure by cryo-electron microscopy and crystallography: implications for pilus assembly and functions. Mol. Cell 23:651, 2006.
Doi, Y., Katafuchi, A., Fujiwara, Y., Hitomi, K., Tainer, J.A., Ide, H., Iwai, S. Synthesis and characterization of oligonucleotides containing 2′-fluorinated thymidine glycol as inhibitors of the endonuclease III reaction. Nucleic Acids Res. 34:1540, 2006.
Fan, L., Arvai, A., Cooper, P.K., Iwai, S., Hanaoka, F., Tainer, J.A. Conserved XPB core structure and motifs for DNA unwinding: implications for pathway selection of transcription or excision repair. Mol. Cell 22:27, 2006.
Fan, L., Kim, S., Farr, C.L., Schaefer, K.T., Randolph, K.M., Tainer, J.A., Kaguni, L.S. A novel processive mechanism for DNA synthesis revealed by structure, modeling and mutagenesis of the accessory subunit of human mitochondrial DNA polymerase. J. Mol. Biol. 358:1229, 2006.
Pascal, J.M., Tsodikov, O.V., Hura, G.L., Song, W., Cotner, E.A., Classen, S., Tomkinson, A., Tainer, J.A., Ellenberger, T. A flexible interface between DNA ligase and PCNA supports conformational switching and efficient ligation of DNA. Mol. Cell 24:279, 2006.
Perry, J.J.P., Yannone, S.M., Holden, L.G., Hitomi, C., Asaithamby, A., Han, S., Cooper, P.K., Chen, D.J., Tainer, J.A. WRN exonuclease structure and molecular mechanism imply an editing role in DNA end processing. Nat. Struct. Mol. Biol. 13:414, 2006.
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.
Sundheim, O., Vågbø, C.B., Bjørås, M., de Sousa, M.M.L., Talstad, V., Aas, P.A., Drabløs, F., Krokan, H.E., Tainer, J.A., Slupphaug, G. Human ABH3 structure and key residues for oxidative demethylation to reverse DNA/RNA damage. EMBO J. 25:3389, 2006.
Tsutakawa, S.E., Hura, G.L., Frankel, K.A., Cooper, P.K., Tainer, J.A. Structural analysis of flexible proteins in solution by small angle x-ray scattering combined with crystallography. J. Struct. Biol., in press.
Wood, T.I., Barondeau, D.P., Hitomi, C., Kassmann, C.J., Tainer, J.A., Getzoff, E.D. Defining the role of arginine 96 in green fluorescent protein fluorophore biosynthesis. Biochemistry 44:16211, 2005.