News and Publications
The Skaggs Institute for Chemical Biology
Scientific Report 1999-2000
Master Keys for Chemical Molecular Biology From Macromolecular Structures
J.A. Tainer, A.S. Arvai, Y. Bourne, C. Bruns, B. Crane, D. Daniels, K.P. Hopfner,
D. Hosfield, T.P. Lo, C. Mol, S. Parikh, C. Putnam, D. Shin
Proteins That Regulate Genomic Stability and Variation
Life is a delicate balance between maintaining genomic stability and allowing
the genetic variation that drives evolutionary selection. DNA repair proteins
maintain genomic fidelity by detecting, removing, and repairing specific lesions
in DNA that otherwise cause mutations that are critical early events in tumorigenesis.
Our crystal structures of enzymes and enzyme-DNA complexes for enzymes that directly
reverse DNA damage or act in the base-excision repair pathway are providing key
insights into the structural chemistry and cell biology of DNA repair. These
findings typify our efforts to develop new cancer treatments predicated on the
structure-based design of inhibitors that specifically disrupt DNA repair.
During the past year, we established the mechanism for recognition and removal
of damaged bases by human O6-alkylguanine alkyltransferase (AGT), which directly
removes promutagenic alkyl lesions from the O6 position of guanine in an irreversible
and stoichiometric reaction. Because many cancer chemotherapeutic agents achieve
their cytotoxic effects through alkylated bases, such as O6-methylguanine, numerous
tumor cell lines develop resistance to alkylation therapies by increasing expression
of AGT. Correspondingly, inhibitors of AGT can sensitize tumors to alkylation
therapies and thereby potentiate current chemotherapies.
We determined the structure of native human AGT to 2.0-Å resolution
(Fig. 1), as well as the structures of AGT-product complexes with O6-methylguanine
and the suicide inhibitor O6-benzylguanine. AGT is a 2-domain α/ß protein
with a hydrophobic cleft that recognizes alkylated guanine bases by a system
of complementary hydrogen bonds. The location of conserved residues and kinetic
data on point mutations suggest that AGT uses an "arginine finger" to flip alkylated
guanines into the AGT pocket. A structure-based reaction mechanism indicates
that after alkyltransfer to a conserved AGT cysteine residue, steric collisions
most likely destabilize the intradomain interface, leading to release of the
repaired DNA and clearance of alkylated AGT from the cell.
These studies on direct DNA repair complement our work on base-excision repair
enzymes such as uracil-DNA glycosylase (UDG), which specifically recognizes and
cleaves uracil from DNA to create the central base-excision repair intermediate:
an apurinic/apyrimidinic, or abasic, site. We extended our structure-function
analyses of human UDG by determining 1.8- and 2.0-Å resolution cocrystallized
structures of human UDG bound to cleaved product and uncleaved substrate analogs.
The results suggest that the energy produced by binding of enzyme to the DNA
substrate at the macromolecular interface is funneled into catalytic power at
the active site (Fig. 2). A transformation from open to closed in the conformation
of UDG enforces distortions of the target uracil and deoxyribose in the flipped-out
nucleotide substrate that are relieved by cleavage of glycosylic bonds.
This experimentally defined substrate stereochemistry implies an enzyme-induced
alteration of 3 orthogonal electron orbitals into conformations that favor electron
transpositions for cleavage of glycosylic bonds. This coupling of the anomeric
effect to a delocalization of the glycosylic bond electrons into the uracil aromatic
system resolves apparent paradoxes concerning the electron transpositions among
orbitals and the retention of catalytic efficiency despite mutational removal
of active-site functional groups. These new UDG-DNA structures imply dissociative
excision chemistry that may apply to other multistep DNA biological reaction
pathways that require coordination of complex chemical transformations.
The abasic sites generated either spontaneously or by UDG and other DNA repair
glycosylases are recognized by apurinic/apyrimidinic endonucleases, which cleave
the DNA backbone to create a free 3´-OH end to prime DNA repair synthesis.
We determined the structures of abasic DNA cocrystallized with APE1, the primary
human apurinic/apyrimidinic endonuclease. We found that enzyme loops insert into
both the DNA major and minor grooves, severely kinking the DNA to bind a flipped-out
apurinic/apyrimidinic site in a pocket that excludes DNA bases. The geometry
of the active site of APE1 and of a ternary complex consisting of the enzyme,
cleaved apurinic/apyrimidinic DNA, and metal ion support a distinct structure-based
Both the larger APE1-DNA interface and the induced DNA kink suggest how APE1
may enhance glycosylase activity by completely displacing DNA glycosylases from
their tightly bound apurinic/apyrimidinic DNA products. Furthermore, mutagenesis
results unexpectedly revealed that human APE1 is structurally optimized to retain
the cleaved DNA product, suggesting that APE1 acts in vivo to coordinate the
orderly transfer of unstable DNA-damage intermediates between the excision and
synthesis steps of DNA repair.
Endonuclease IV is a zinc ion-dependent apurinic/ apyrimidinic endonuclease
distinct from the APE1 enzyme family. The ultra-high-resolution, 1.0-Å structure
of endonuclease IV and a 1.5-Å resolution structure of a complex consisting
of endonuclease IV and abasic DNA revealed that the endonuclease smoothly bends
apurinic/apyrimidinic DNA about 90° and flips both the target abasic and
opposite nucleotides out of the DNA helix. These results provide the structural
basis for recognition of apurinic/apyrimidinic sites and suggest a testable 3-metal-ion
mechanism for cleavage of phosphodiester bonds.
Coupled with the findings of biochemical studies, mutagenesis, and DNA-binding
and enzyme kinetics, the results of these structural examinations of wild-type
and mutant DNA repair enzymes and of complexes consisting of the enzymes and
damaged DNA suggest new aspects of the structural chemistry that regulate recognition
of DNA damage, progression along the repair pathway, and avoidance of destructive
interference between different steps and pathways of DNA repair.
Reactive Oxygen Signals and Defensive Cytotoxins
The human superoxide dismutase enzymes protect DNA and other cellular components
from oxidative damage associated with degenerative diseases such as amyotrophic
lateral sclerosis and with cancer and aging. As an important enhancement of our
work on these enzymes, we solved structures of human catalase, an enzyme that
protects cells against oxidative damage and cell death mediated by hydrogen peroxide.
These structures helped define the catalase mechanism, suggested a role for the
cofactor NADPH, and established the basis for activity of an important heterocycle
A second major focus of our research on the regulation of reactive oxygen
is structural studies of nitric oxide synthases (NOSs), done in collaboration
with E. Getzoff, the Skaggs Institute. These enzymes oxidize arginine to make
nitric oxide, a key intercellular signal and defensive cytotoxin in the nervous,
cardiovascular, and immune systems. Overproduction of nitric oxide by inducible
NOS in macrophages can lead to many abnormalities, including juvenile diabetes,
arthritis, aneurysms, neurodegenerative disorders, and septic shock. Consequently,
specific inhibitors of inducible NOS have great therapeutic potential. The structures
of a series of ligands bound to NOS suggest new aspects of the enzyme mechanism
and aid in the design of specific inhibitors.
Cabelli, D.E., Guan, Y., Leveque, V., Hearn, A.S., Tainer, J.A., Nick,
H.S., Silverman, D.N. Role of tryptophan 161 in catalysis by human manganese
superoxide dismutase. Biochemistry 38:11686, 1999.
Crane, B.R., Arvai, A.S., Ghosh, D.K., Getzoff, E.D., Stuehr, D.J., Tainer,
J.A. Structures of the Nw-hydroxy-L-arginine complex of
inducible nitric oxide synthase dimer with active and inactive pterins. Biochemistry
Crane, B.R., Rosenfield, R.A., Arvai, A.S., Ghosh, D.K., Tainer, J.A.,
Stuehr, D.J., Getzoff, E.D. N-terminal domain swapping and metal ion binding
in nitric oxide synthase dimerization. EMBO J. 18:6271, 1999.
Daniels, D.S., Mol, C.D., Arvai, A.S., Kanugula, S., Pegg, A.E., Tainer
J.A. Active and alkylated human AGT structures: A novel zinc site, inhibitor,
and extrahelical base binding. EMBO J. 19:1719, 2000.
Daniels, D.S., Tainer, J.A. Conserved structural motifs governing
the stoichiometric repair of alkylated DNA by O(6)-alkylguanine-DNA alkyltransferase.
Mutat. Res. 460:151, 2000.
Ghosh, D., Crane, B.R., Ghosh, S., Wolan, D., Gachhui, R., Crooks, C.,
Presta, A., Tainer, J.A., Getzoff, E.D., Stuehr, D.J. Inducible nitric oxide
synthase: Role of the N-terminal ß-hairpin hook and pterin-binding segment
in dimerization and tetrahydrobiopterin interaction. EMBO J. 18:6260, 1999.
Hosfield, D.J., Daniels, D.S., Mol, C.D., Putnam, C.D., Parikh, S.S.,
Tainer, J.A. DNA damage recognition and repair pathway coordination revealed
by the structural biochemistry of DNA repair enzymes. Prog. Nucleic Acids Res.
Mol. Biol., in press.
Hosfield, D.J., Guan, Y., Haas, B.J., Cunningham, R.P., Tainer, J.A. Structure
of the DNA repair enzyme endonuclease IV and its DNA complex: Double-nucleotide
flipping at abasic sites and three-metal-ion catalysis. Cell 98:397, 1999.
Mol, C.D., Hosfield, D.J., Tainer, J.A. Abasic site recognition by
two apurinic/apyrimidinic endonuclease families in DNA base excision repair:
The 3´ ends justify the means. Mutat. Res. 460:211, 2000.
Mol, C.D., Izumi, T., Mitra, S., Tainer, J.A. DNA-bound structures
and mutants reveal abasic DNA binding by APE1 and DNA repair coordination. Nature
Parikh, S.S., Putnam, C.D., Tainer, J.A. Lessons learned from structural
results on uracil-DNA glycosylase. Mutat. Res. 460:183, 2000.
Parikh, S.S., Walcher, G., Jones, G.D., Slupphaug, G., Krokan H.E., Blackburn,
G.M., Tainer, J.A. Uracil-DNA glycosylase-DNA substrate and product structures:
Conformational strain promotes catalytic efficiency by coupled stereoelectronic
effects. Proc. Natl. Acad. Sci. U. S. A. 97:5083, 2000.
Pellequer, J.-L., Chen, S.W., Roberts, V.A., Tainer, J.A., Getzoff, E.D. Unraveling
the effect of changes in conformation and compactness at the antibody VL-VH interface
upon antigen binding J. Mol. Recognit. 12:267, 1999.
Putnam, C.D., Arvai, A.S., Bourne, Y., Tainer, J.A. Active and inhibited
human catalase structures: Ligand and NADPH binding and catalytic mechanism.
J. Mol. Biol. 296:295, 2000.
Putnam, C.D., Tainer, J.A. The food of sweet and bitter fancy. Nat.
Struct. Biol. 7:17, 1999.
Ramilo, C.A., Leveque, V., Guan, Y., Lepock, J.R., Tainer, J.A., Nick,
H.S., Silverman, D.N. Interrupting the hydrogen bond network at the active
site of human manganese superoxide dismutase. J. Biol. Chem. 274:27711, 1999.