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Scientific Report 2006
Molecular Biology
Structural Biology of Molecular Interactions and Design
J.A. Tainer, A.S. Arvai,
D.P. Barondeau, M. Bjoras, B.R. Chapados, L. Craig, T.H. Cross, L. Fan, C. Hitomi,
K. Hitomi, J.L. Huffman, C.J. Kassmann, I. Li, G. Moncalian, M.E. Pique, D.S.
Shin, O. Sundheim, R.S. Williams, T.I. Wood, A. Yamagata
Our
studies reveal overall themes and common relationships for fundamental principles
and processes of protein regulators and effectors of DNA damage responses, reactive
oxygen species, and pathogenesis. We combine x-ray crystallography and solution
small-angle x-ray scattering methods, often at our advanced synchrotron facility
SIBLYS, to gain a clear view of the structural chemistry that drives biology. Further
fusing these techniques with electron microscopy, we bridge the size gaps between
high-resolution macromolecular structures and lower resolution multiprotein machine
complexes. We then investigate the associated dynamic reversible interactions within
cells, potential for structure-based design of inhibitors relevant to the development
of novel therapeutic agents and chemical tools, and structural implications by biochemistry
and mutagenesis. For DNA repair, we collaborate with P. Russell and N. Boddy, Department
of Molecular Biology, to couple our structures with genetics and phenotypes. For
protein design, we collaborate with E. Getzoff, Department of Molecular Biology,
to understand and control the formation of self-synthesizing chromophores in green
fluorescent protein and its homologs.
Protein Modifications and Function
The finding that the number of protein-coding
genes in the human genome is more than 10-fold lower than the number of proteins
found in human cells by the Human Genome Project is surprising. This huge increase
in protein diversity must primarily be due to alternative splicing and posttranslational
modification of proteins. A particularly important and intriguing posttranslational
modification is the spontaneous peptide backbone cyclization and oxidation chemistry
required to convert 3 amino acids into a fluorophore for the family of green fluorescent
proteins.
Reactive Oxygen and Xenobiotic Control Enzymes
Superoxide dismutases and nitric
oxide synthases are master regulators for reactive oxygen species involved in injury,
pathogenesis, aging, and degenerative diseases. We are characterizing the hydrogen-bonding
networks that underlie the activity of mitochondrial manganese superoxide dismutases.
For human copper, zinc superoxide dismutase, we are probing how single-site mutations
cause the neurodegeneration in Lou Gehrig disease or familial amyotrophic lateral
sclerosis. For nitric oxide synthases, we are examining the structure and chemistry
that control levels of nitric oxide, which acts as an important signal and cytotoxin
with implications for inflammatory and neurodegenerative diseases.
DNA Repair and Genetic Evolution
All the information for heredity
is encoded in DNA molecules that are constantly under attack from sunlight, ionizing
radiation, and other environmental carcinogens. Surprisingly, however, most DNA
damage is due to chemical reactions and free radicals that arise from normal cellular
metabolism that is necessary for life. Thus, paradoxically, life is impossible even
in the absence of environmental toxins unless coupled to DNA repair. Mutations that
cause defects in DNA repair systems may cause cancer and degenerative diseases associated
with aging, but fortunately the mutations can also be exploited for cancer therapy.
Aging and the WRN Structure
Mutation of the DNA repair protein
WRN can give rise to Werner syndrome, which is characterized by rapid aging and
cancer disorders. We have characterized the structure of the WRN nuclease component
(Fig. 1). This component is an editing nuclease resembling those found in DNA polymerases.
Furthermore, the editing of DNA ends by the WRN exonuclease is stimulated for broken
DNA end joining by the Ku DNA end-binding complex. Our findings suggest how the
editing of DNA ends during DNA damage responses can critically affect aging and
carcinogenesis.
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| Fig. 1. Hexameric ring model for the WRN nuclease (WRN exo) component. A, WRN x-ray crystal structures
aligned as a ring by homology comparisons. B, DNA processing is altered in the WRN
Trp145A mutant. C, Electron density map (3σ, 5σ) of dGMP bound
to WRN exo. D, The similar internal and external dimensions of Ku70/80 (left) and
the WRN exo hexamer model (right). |
Nucleotide Excision Repair
Nucleotide excision repair, a critical
defense mechanism that removes DNA lesions caused by the mutational effects of sunlight
(ultraviolet radiation) and toxic chemicals, is also central to the success of anticancer
drugs such as cisplatin. We have focused on understanding the mechanisms of nuclear
excision repair for potential improvements in cancer treatment. We determined the
crystal structure of an enzyme called xeroderma pigmentosum group B (XPB) helicase
(Fig. 2). We found several unexpected functions of XPB helicase in nuclear excision
repair. These findings helped us address important questions about the enzyme’s
role in DNA transcription and repair. XPB helicase recognizes DNA damage that causes
blockages in reading the DNA code and aids initiation of efficient repair.
Bacterial Pili and Infectious Diseases
Type IV pili are essential virulence
factors for many gram-negative bacteria, such as Neisseria gonorrhoeae and
Neisseria meningitidis. Pili play key roles in surface motility, adhesion,
formation of microcolonies and biofilms, natural transformation, and signaling.
We are characterizing structures of type IV pilin subunits: the assembled pilus
fiber, the pilus membrane protein partners, and the assembly ATPase. Pili induce
a calcium influx in host cells that plays a role in pathogenesis by altering endocytic
trafficking and lysosome homeostasis in infected cells. Because calcium is a central
second messenger that regulates several signal cascades, pilus-induced calcium bursts
most likely influence bacterial infectivity in key ways. For infections caused by
N meningitidis, these calcium bursts are expected to activate neuronal nitric
oxide synthases, resulting in toxic levels of nitric oxide that may in part explain
the fatal effects of N meningitidis infections of the brain.
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| Fig. 2.Fig. 2. Conserved XPB helicase core and DNA-induced open-to-closed
conformational changes. XPB contains 4 conserved functional domains: the damage
recognition domain (DRD), 2 helicase domains (HD1 and HD2), and a thumb insert (ThM).
The interaction of the helicase with DNA may induce a rotation of about 170°
of domain HD2 and ThM to form the closed conformation as observed in the crystal
structure of hepatitis C virus (HCV) NS3 helicase bound to a single-stranded DNA. |
Publications
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.
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.
Fan, L., Perry, J.J.P., Tainer,
J.A. Reactive oxygen control and DNA repair structural
biology: implications for aging and neuropathology. Neuroscience, in press.
Hitomi, K., Iwaia, S., Tainer,
J.A. The intricate structural chemistry of base
excision repair machinery: implications for DNA damage recognition, removal, and
repair. DNA Repair (Amst.), in press.
Ivanov, I., Chapados, B.R.,
McCammon, J.A., Tainer, J.A. Proliferating cell
nuclear antigen loaded onto double-stranded DNA: dynamics, minor groove interactions
and functional implications. Nucleic Acids Res. in press.
Pascal, J.M., Tsodikov, O.V.,
Hura, G.L., Song, W., Cotner, E.A., Classen, S., Tomkinson, A.E., Tainer, J.A.,
Ellenberger, T. A flexible interface between DNA
ligase and PCNA supports conformational switching and efficient ligation of DNA.
Mol. Cell. 24:279-91, 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.
Putnam, C.D., Hura, G.L., Tainer,
J.A. Combining x-ray solution and crystal diffraction
and scanning force microscopies to characterize reversible macromolecular interactions
and conformational states. Q. Rev. Biophys., in press.
Putnam, C.D., Tainer, J.A.
Protein mimicry of DNA and pathway regulation. DNA Repair (Amst.) 4:1410, 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., Tainer, J.A.
Combined methods of SAXS and crystallography to characterize dynamic protein conformations
at atomic resolution. 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.
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