Scientific Report 2008
Structural Biology of Molecular Interactions
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
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).
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.
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
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.
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.
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.
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.