The Skaggs Institute
for Chemical Biology

Macromolecular Machines as Master Keys for Genome Integrity, the Cell Cycle, Control of Reactive Oxygen Species, and Pathogenesis

J.A. Tainer, A.S. Arvai, D.P. Barondeau, R. Brudler, B.R. Chapados, L. Craig, G. Divita, L. Fan, C. Hitomi, K. Hitomi, J.L. Huffman, J.J.P. Perry, D.S. Shin, O. Sundheim, J.L. Tubbs, T.I. Wood, R.S. Williams, A. Yamagata

Through funding from the Skaggs Institute, we are doing research on human health and disease by concentrating on gaps in the knowledge between structural and cellular biology. We focus on the determination and functional understanding of structures of proteins that are key in cellular processes, particularly macromolecular cellular machines, and in the design of both novel proteins and novel inhibitors.

Our studies on the control of reactive oxygen species by the enzymes superoxide dismutase and nitric oxide synthase are continuing. These proteins are relevant to the oxidative damage associated with inflammation, as well as with cancer and aging. More generally the results with superoxide dismutase address the central paradox of how mutations can cause the fatal neurodegenerative disease familial amyotrophic lateral sclerosis (Lou Gehrig disease). Recent successes include studies on the human mitochondrial superoxide dismutase, which may help protect neurons and mitochondria from oxidative damage and cell death (Fig. 1).

Fig. 1. To understand in detail the activity of superoxide dismutases in defending cells against oxidative damage, we combined high-resolution crystal structures with 3-fluorotyrosine to examine the hydrogen-bonding interactions important in the activity of human mitochondrial superoxide dismutase. Shown here is an overlay of the key active-site residues and hydrogen-bonding scheme (depicted by gray spheres) of the structures of wild-type and fluorinated manganese superoxide dismutase.

In our studies of pathogenesis, we extended our analyses of type IV pilins from bacterial pathogens to characterize the type IV pilin system, including the assembly and functional interactions. Because type IV pilins are critical bacterial virulence factors, understanding the structure and function of the pilins is critical to controlling cholera, pneumonia, gonorrhea, meningitis, and severe diarrhea. We are now completing structures of the ATPases and membrane anchor proteins that act in the assembly of the fibers of pathogens. Thus, we are now able to characterize type IV pilins and related systems while investigating several different key drug targets.

Our studies on 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. In particular, studies on DNA repair are directly relevant to the cause and treatment of cancer, which causes the death of more than half a million persons each year in the United States alone. Major advances include our characterizations of helicases that couple DNA repair to DNA replication and transcription. Mutations in these helicases are associated with rapid aging and with predisposition to cancer.

In addition, we recently characterized key enzymes that deal with oxidized DNA base damage and DNA strand breaks. These results are providing a prototypical set of key structures, including enzyme complexes with DNA and with protein partners, to define the critical interactions for DNA repair. These molecular characterizations of functional features and the disruption of the features by disease-causing mutations are providing a molecular basis to connect inherited mutations with disease phenotypes.

The anticipated outcome of these cross-disciplinary experiments is a molecular picture of the protein-DNA complexes, protein-protein interactions, and functional states that orchestrate sensing of double-strand DNA breaks for repair and signaling events. This picture will provide the molecular foundation for a detailed understanding of human diseases and cancer predisposition linked to the proteins involved in repairing double-strand breaks. Collaborative interactions with the cancer centers at the University of California, San Francisco, and other locations will provide mechanisms to facilitate the transfer of structural information, including the nature of key interactions that direct progression along the repair pathway and DNA damage signaling, to achieve informed therapeutic interventions.

Funding from the Skaggs Institute has also allowed us to greatly use the Computational Center for Macromolecular Structure at Scripps Research and our new synchrotron beamline, Structurally Integrated Biology for Life Sciences, at the Advanced Light Source, University of California, Berkeley/Lawrence Berkeley National Laboratory. We designed and built the beamline to specifically characterize protein-protein and protein-DNA complexes, protein conformational changes, and macromolecular machines, all critical to cellular processes.

We are developing and applying small-angle x-ray scattering (SAXS), in addition to protein crystallography, to aid in our studies with the beamline. SAXS can provide critical characterizations by revealing overall mass, stoichiometry of subunits, radius of gyration, electron-pair distances, and maximum dimension. Furthermore, SAXS can be used to elucidate dynamic protein functional relationships and alterations in the relationships by assemblies, modifications, and metabolites, because conditionally induced domain movements as small as 2 Å can be detected. SAXS will generate low-resolution forms that provide structural information on flexible regions. Therefore, we are also defining disorder-to-order transitions that occur upon assembly with partner proteins and showing that this conformational switching enables differential assembly with multiple protein partners. Thus, our SAXS technology provides a tremendous opportunity for comprehensive characterizations of protein complexes and key conformational switching states.

We are also developing new technologies to aid our visualization of this structural information, including a database we call ISIS (integrated structures imaged in solution; Fig. 2). Uses of the database include rapid searches for structural homologs indicated by experimentally obtained SAXS curves.

Fig. 2. Examples of structures in the ISIS database of solution structures integrated with detailed x-ray crystallographic structures.

Overall, we have especially made substantial progress in understanding the molecular machinery that acts in cellular processes related to pathogenesis, cancer susceptibility, and aging. Published and pending articles on research funded by the Skaggs Institute include articles on the characterization of DNA damage responses; protein modifications that occur after translation; and enzymes and molecular machines relevant to control of reactive oxygen species, pathogenesis, and cancer-related aspects of genome maintenance.

Publications

Arthur, L.M., Gustausson, K., Hopfner, K.P., Carson, C.T., Stracker, T.H., Karcher, A., Felton, D., Weitzman, M.D., Tainer, J., Carney, J.P. Structural and functional analysis of Mre11-3. Nucleic Acids Res. 32:1886, 2004.

Ayala, I., Perry, J.P., Szczepanski, J., Tainer, J.A., Vala, M.T., Nick, H.S., Silverman, D.N. Hydrogen bonding in human manganese superoxide dismutase containing 3-fluorotyrosine. Biophys. J. 89:4171, 2005.

Barondeau, D.P., Kassmann, C.J., Tainer, J.A., Getzoff, E.D. Understanding GFP chromophore biosynthesis: controlling backbone cyclization and modifying post-translational chemistry. Biochemistry 44:1960, 2005.

Connolly, S., Aberg, A., Arvai, A., Beaton, H.G., Cheshire, D.R., Cook, A.R., Cooper, S., Cox, D., Hamley, P., Mallinder, P., Millichip, I., Nicholls, D.J., Rosenfeld, R.J., St-Gallay, S.A., Tainer, J., Tinker, A.C., Wallace, A.V . 2-Aminopyridines as highly selective inducible nitric oxide synthase inhibitors: differential binding modes dependent on nitrogen substitution. J. Med. Chem. 47:3320, 2004.

Crowther, L.J., Yamagata, A., Craig, L., Tainer, J.A., Donnenberg, M.S. The ATPase activity of BfpD is greatly enhanced by zinc and allosteric interactions with other Bfp proteins. J. Biol. Chem. 280:24839, 2005.

Hendrickson, E.A., Huffman, J.L., Tainer, J.A. Structural aspects of Ku and the DNA-dependent protein kinase complex. In: DNA Damage Recognition. Siede, W., Doetsch, P., Kow, Y.W. (Eds.). CRC Press, Boca Raton, FL, 2005, p. 626.

Huffman, J.L., Sundheim, O., Tainer, J.A. DNA base damage recognition and removal: new twists and grooves. Mutat. Res. 577:55, 2005.

Huffman, J.L., Sundheim, O., Tainer, J.A. Structural features of DNA glycosylases and AP endonucleases. In: DNA Damage Recognition. Siede, W., Doetsch, P., Kow, Y.W. (Eds.). CRC Press, Boca Raton, FL, 2005, p. 299.

Putnam, C.D., Tainer, J.A. Protein mimicry of DNA and pathway regulation. DNA Repair (Amst.) 4:1410, 2005.

Simeoni, F., Arvai, A., Bello, P., Gondeau, C., Hopfner, K.P., Neyroz, P., Heitz, F., Tainer, J., Divita, G. Biochemical characterization and crystal structure of a Dim1 family associated protein: Dim2. Biochemistry 44:11997, 2005.

Tubbs, J.L., Tainer, J.A., Getzoff, E.D. Crystallographic structures of Discosoma red fluorescent protein with immature and mature chromophores: linking peptide bond trans-cis isomerization and acylimine formation in chromophore maturation. Biochemistry 44:9833, 2005.

Williams, R.S., Tainer J.A. A nanomachine for making ends meet: MRN is a flexing scaffold for the repair of DNA double-strand breaks. Mol. Cell 19:724, 2005.