The Skaggs Institute
for Chemical Biology

Scientific Report 2006

Macromolecular Machines as Master Keys for Genome Integrity and Pathogenesis

J.A. Tainer, A.S. Arvai, D.P. Barondeau, M. Bjørås, R. Brudler, B.R. Chapados, C. Chahwan, L. Craig, D.S. Daniels, G. Divita, L. Fan, S. Han, C. Hitomi, K. Hitomi, J.L. Huffman, J.J. Perry, M.E. Pique, D.S. Shin, O. Sundheim, T.I. Wood, A. Yamagata

Skaggs funding especially affects our projects on the structure-function relationships for macromolecular machines, the design of proteins and inhibitors, and enhanced molecular-based understanding of human diseases relevant to informed interventions. Importantly, Skaggs funding is powerfully synergistic with our novel synchrotron beamline, Structurally Integrated Biology for Life Sciences, at the Advanced Light Source, University of California, Berkeley/Lawrence Berkeley National Laboratory. We designed the beamline to characterize protein complexes, conformations, and modifications that control pathway connections and coordination important to human health and disease.

In ongoing research, we are developing new technologies and facilities for detailed visualizations of protein complexes and modified proteins that change dynamically to respond to the internal and external environment of cells. During this past year, we developed and applied x-ray scattering to define protein structures in solution. This approach, termed small-angle x-ray scattering (SAXS), is sufficiently powerful to address the challenges of characterizing the dynamic microbial protein complexes and modified proteins responsible for many cellular functions. The combination of solution x-ray scattering with high-resolution structures defined by x-ray crystallography is central to our future Skaggs research because these technologies will allow us to investigate shape, conformational change, assembly states, and architectures for protein complexes and modified proteins.

Using SAXS to analyze the scatter of x-rays by proteins in solution provides critical characterizations of protein complexes and modified proteins by revealing mass, stoichiometry of subunits, radius of gyration, electron-pair distances, and maximum dimension. SAXS can be used to elucidate dynamic protein functional relationships and their alteration by assemblies, modifications, and metabolites because even small domain movements can be detected. Moreover, we are using SAXS to define protein shapes and to compare solution and crystal structures.

We discovered as part of this work how DNA ligase forms reversible complexes to achieve its functions. Ligase acts as a molecular watch band that can open into an extended complex and close into a ring-shaped conformation that catalyzes a DNA end-joining reaction stimulated by proliferating cell nuclear antigen (PCNA, Fig. 1). Thus, ligase architecture allows an extended ligase to bind to the PCNA platform for DNA replication and repair and then close into a ring around DNA for end joining.

Fig. 1. DNA ligase in its open, extended, and closed-ring conformations defined by combining SAXS and protein crystallography on our synchrotron beamline. In the absence of a nicked DNA molecule, the Sulfolobus solfataricus DNA ligase has an open, extended conformation by both crystallographic and SAXS studies. In the reversible complex amenable only to SAXS, the ligase retains an open, extended conformation and binds to a single PCNA3 subunit. Yet, a closed, ring-shaped conformation of ligase catalyzes a DNA end-joining reaction stimulated by PCNA.

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.

Fig. 2. The integrated electron cryomicroscopy fiber and type IV pilus subunit crystal structures provide the basis for understanding the multifunctionality and escape from the host immune system. We determined this 3-dimensional structure of the Neisseria gonorrhoeae type IV pilus at 12.5-Å resolution by using single-particle electron cryomicroscopy reconstruction and then generated a pseudoatomic model of the pilus filament by rigorous automated fitting of the reconstruction with a 2.3-Å-resolution x-ray crystallographic structure of the pilin subunit. The type IV pilus structure revealed a surprisingly corrugated surface with antigenically variable protrusions masking conserved functional features. Deep positively charged grooves appear as prime sites for DNA binding and potential targets for antibacterial therapeutic agents. The background is a scanning electron microscopy image of long thin type IV pili on N gonorrhoeae diplococci. Adapted from the cover photograph for 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.

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 Werner’s 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.

Publications

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.