News and Publications

Master Keys for Chemical Molecular Biology From Macromolecular Structures

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 catalytic mechanism.

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 inhibitor.

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.

Publications

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 N^w-hydroxy-L-arginine complex of inducible nitric oxide synthase dimer with active and inactive pterins. Biochemistry 39:4608, 2000.

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 403:451, 2000.

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 V_L-V_H 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.