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Scientific Report 2008


Molecular Biology




Structural Biology of Molecular Interactions and Design


J.A. Tainer, A.S. Arvai, B.R. Chapados, L. Fan, E. Garcin, G. Guenther, C. Hitomi, K. Hitomi, M.D. Kroeger, J.J. Perry, M.E. Pique, D.S. Shin, J.L. Tubbs, R.S. Williams

We are developing new technologies and systems to close the gaps from proteins to pathways and from interaction networks to biological outcomes in cells. We focus on molecular mechanisms and relationships for proteins that control DNA damage responses, reactive oxygen species, protein modifications, and pathogenesis. Our results have relevance for improved understanding and therapeutic approaches for cancer, aging, and degenerative diseases and for bacterial pathogens.

Small-Angle X-Ray Scattering in Solution Combined with Crystallography and Computation

Aided by our synchrotron facility SIBLYS, we are developing and applying technologies to develop accurate structures of protein conformation, assembly, and interactions in solution by combining x-ray scattering with x-ray crystallography and computation. We recently developed methods for high-throughput analyses via small-angle x-ray scattering. Small-angle x-ray scattering, crystallography, and computation together allow multiscale modeling and fundamental insights to allosteric mechanisms, self-assemblies, and dynamic molecular machines acting as master keys to cell biology.

Protein Modifications and Function

The ubiquitin-like protein family of posttranslational modifications consists primarily of ubiquitin and the small ubiquitin modifier SUMO. In collaboration with M.N. Boddy, Department of Molecular Biology, we have helped discover an intriguing family of proteins, SUMO-targeted ubiquitin ligases (STUbLs), that directly connect the ubiquitination and sumoylation pathways. Uniquely, STUbLs use SUMO interaction motifs to recognize their sumoylated targets. STUbLs act as global regulators of protein sumoylation levels, and cells lacking STUbLs have associated genomic instability and hypersensitivity to genotoxic stress; the human STUbL RNF4 is implicated in cancer.

Reactive Oxygen Control Enzymes

Superoxide dismutases (SODs) and nitric oxide synthases are master regulators for reactive oxygen species involved in injury, pathogenesis, aging, and degenerative diseases. We tested if Alvinella pompejana, a deep-sea hydrothermal vent worm, could be a eukaryotic source for thermostable and humanlike proteins and whether information on SOD mechanism and stability could be obtained by examining A pompejana SOD. We discovered that the worm SOD has a remarkably high sequence identity with other mammalian SOD enzymes but is substantially more stable than human SOD. Moreover, crystals from initial conditions diffracted just beyond 1-Å resolution. These results extend knowledge of SOD stability and catalysis and also suggest that A pompejana may be a unique resource of macromolecules of enhanced stability for science and technology.

For human copper, zinc SOD, we are examining single-site mutations that cause the neurodegeneration in Lou Gehrig disease or familial amyotrophic lateral sclerosis. Our structures show a key role for the zinc ion in the defects associated with the disease.

For the nitric oxide synthases, our combined solution scattering and crystallographic methods are revealing regulatory mechanisms responsible for controlling nitric oxide levels, which act as an important signal and as a cytotoxin, with implications for inflammatory and neurodegenerative diseases. Our structures of the synthases, determined in collaboration with E.D. Getzoff, Department of Molecular Biology, are enabling us to design new inhibitors to directly control nitric oxide levels for the treatment of human diseases.

DNA Repair and Genetic Evolution

Structural knowledge allows possible selective inhibition of certain DNA repair pathways for new cancer therapies. Endonuclease IV is an archetype for an endonuclease superfamily critical for DNA-base excision repair. Our structures of endonuclease IV revealed a mechanism for binding to and incising areas of DNA damage that involves 3 metal ions and explained how the chemistry avoids the release of toxic and mutagenic repair intermediates (Fig. 1).


Fig. 1. Endonuclease IV E261Q DNA substrate—bound and DNA-free x-ray structures. A, Apurinic-apyrimidinic (AP)-DNA complex stereo shows the 3-metal-ion active site (green spheres); residues R37, Y72, and Q261 (pink); and bound DNA substrate with both the AP site sugar and phosphate moieties and the cognate nucleotide (orange) flipped out from normal duplex DNA. The 2Fo-Fc electron density map is contoured at 1 σ (blue mesh). B, DNA-free structure with active-site phosphate and 3 zinc ions coordination. Omit map is contoured at 2 (light blue) and 4 (dark blue) σ for the bound phosphate group. C, DNA substrate complex binding to active-site metal ions. High-quality omit map (contoured at 2 σ, pink mesh) shows the intact phosphodiester bond (black arrow) that constrains the Zn3 to Cyt6 O3′ distance to 2.7 Å. Based on Garcin, E.D., Hosfield, D.J., Desai, S.A., et al., DNA apurinic-apyrimidinic site binding and excision by endonuclease IV. Nat. Struct. Mol. Biol. 15:515, 2008.

Mutations in XPD helicase cause 3 distinct phenotypes: cancer-prone xeroderma pigmentosum and the aging disorders Cockayne syndrome and trichothiodystrophy. To clarify molecular differences that underlie these diseases, we determined crystal structures of the XPD helicase catalytic core from Sulfolobus acidocaldarius and measured mutant enzyme activities. Mutations associated with xeroderma pigmentosum map along the ATP-binding edge and DNA-binding channel and impair helicase activity essential for nucleotide excision repair. Mutations associated with xeroderma pigmentosum and Cockayne syndrome both impair helicase activity and likely affect functional movement. Mutations association with trichothiodystrophy lose or retain helicase activity but map to sites in all 4 domains expected to cause framework defects affecting the integrity of transcription factor IIH.

These new results broaden our understanding of how structural changes in the XPD helicase might affect cancer risks or result in developmental or aging phenotypes (Fig. 2). In general, the structural biology of proteins such as XPD, which control reactive oxygen species and DNA repair, may provide master keys to brain abnormalities, cancer, and aging.
Fig. 2. Structural placement of disease-causing mutations in XPD helicase. Mapping the 3 classes of mutations onto the SaXPD structure reveals patterns associated with each disease defect. A, Stereo pair mapping the distribution of disease-causing mutations on a XPD Cα< trace. Disease-causing mutation sites (Cα colored sphere): red (XP), greenish yellow (XP/CS), and purple (TTD). Residue F136 is also shown (cyan). B, XPDcc fold and domain architecture (ribbons) with labeled disease-causing mutation sites as spheres colored as in A. C, XP mutations affect DNA- and ATP-binding regions. D, XP/CS mutations affect HD1-HD2 conformational changes. E, TTD mutations affect the overall framework stability. Reprinted from Fan, L., Fuss, J.O., Cheng, Q.J., Arvai, A.S., Hammel M., Roberts, V.A., Cooper, P.K., Tainer, J.A. XPD helicase structures and activities: insights into the cancer and aging phenotypes from XPD mutations. Cell 133:789, 2008. Copyright 2008, with permission from Elsevier.

Bacterial Pili and Infectious Diseases

Type IV pili are essential virulence factors for many bacterial pathogens and therefore act in many important infectious diseases. Functions of type IV pili include motility, formation of microcolonies and biofilms, adhesion to host cells, and natural transformation. Because they are prominently exposed on bacterial surfaces, pili are attractive targets for the host immune response and for vaccines and therapeutic reagents.

Our studies are providing an integrated understanding of the assembly and disassembly of type IV pili. This understanding suggests new approaches to drug and vaccine design for bacterial pathogens, including Francisella tularensis, a highly virulent microorganism that causes tularemia. Because of its high infectivity and potential airborne transmission, F tularensis is designated a category A bioterrorism agent.

Publications

Chrencik, J.E., Brooun, A., Zhang, H., Mathews, I.I., Hura, G.L., Foster, S., Perry, J.J.P., Streiff, M., Ramage, P., Widmer, H., Bokoch, G.M., Tainer, J.A., Weckbecker, G., Kuhn, P. Structural basis of guanine nucleotide exchange mediated by the T-cell essential Vav1. J. Mol. Biol. 380:828, 2008.

Fan, L., Fuss, J.O., Cheng, Q.J., Arvai, A.S., Hammel M., Roberts, V.A., Cooper, P.K., Tainer, J.A. XPD helicase structures and activities: insights into the cancer and aging phenotypes from XPD mutations. Cell 133:789, 2008.

Garcin, E.D., Hosfield, D.J., Desai, S.A., Haas, B.J., Björas, M., Cunningham, R.P., Tainer, J.A. DNA apurinic-apyrimidinic site binding and excision by endonuclease IV. Nat. Struct. Mol. Biol. 15:515, 2008.

Perry, J.J., Tainer, J.A., Boddy, M.N . A SIM-ultaneous role for SUMO and ubiquitin. Trends Biochem. Sci. 33:201, 2008.

Perry, J.J.P., Tainer, J.A. Structural biology of Cockayne syndrome proteins, their interactions and insights into DNA repair mechanisms. In: Molecular Mechanisms of Cockayne Syndrome. Ahmad, S.I. (Ed.). Landes Bioscences, Austin, TX, in press.

Putnam, C.D., Hammel, M., Hura, G.L., Tainer, J.A. X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution. Q. Rev. Biophys. 40:191, 2007.

Prudden, J., Pebernard, S., Raffa, G., Slavin, D.A., Perry, J.J., Tainer, J.A., McGowan, C.H., Boddy, M.N. SUMO-targeted ubiquitin ligases in genome stability. EMBO J. 26:4089, 2007.

Roberts, B.R., Tainer, J.A., Getzoff, E.D., Malencik, D.A., anderson, S.R., Bomben, V.C., Meyers, K.R., Karplus, P.A., Beckman, J.S. Structural characterization of zinc-deficient human superoxide dismutase and implications for ALS. J. Mol. Biol. 373:877, 2007

Tubbs, J.L., Pegg, A.E., Tainer, J.A. DNA binding, nucleotide flipping, and the helix-turn-helix motif in base repair by O6-alkylguanine-DNA alkyltransferase and its implications for cancer chemotherapy. DNA Repair (Amst.) 6:1100, 2007.

 

John A. Tainer, Ph.D.
Professor



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