| Human O6-alkylguanine-DNA alkyltransferase (AGT) directly repairs alkylation damage at the O6-position of guanine in a unique, stoichiometric reaction. Since O6-alkylguanine lesions are the mechanism of cytotoxicity of numerous cancer chemotherapies, AGT confers resistance to these alkylation therapies and is therefore an appealing anticancer drug target. Crystal structures of active human AGT and its biologically and therapeutically relevant methylated and benzylated product complexes solved by multi-wavelength anomalous diffraction methods reveal an unexpected zinc-stabilized helical bridge joining a two-domain alpha/beta structure. The C-terminal domain bears a helix-turn-helix (HTH) motif that has been implicated in DNA-binding by structural and mutagenesis studies. Homology-based modeling of an AGT-DNA complex suggests that in the second helix of the HTH, the recognition helix, lies a conserved RAV(A/G) motif, whose "arginine finger" promotes flipping of the target nucleotide from the base stack. Thermodynamic, kinetic and fluorescence assays of AGT mutants support this hypothesis. Analysis of the active site and automated substrate docking demonstrate that recognition of the extrahelical guanine is predominantly through interactions with the protein backbone, while hydrophobic sidechains line the alkyl-binding pocket, as defined by product complexes of human AGT. Automated docking of ligands into this binding pocket using a modified genetic algorithm has allowed the design of inhibitors with, calculated binding energies significantly greater than those currently in use as potential adjuvant drugs. The irreversible dealkylation reaction is accomplished by an active-site cysteine that participates in a hydrogen bond network with invariant histidine and glutamic acid residues, reminiscent of the serine protease catalytic triad. Structural and biochemical results suggest that cysteine alkylation opens a domain-interfacing "Asn-hinge" that couples the active site to the recognition helix, providing both a mechanism for release of repaired DNA and a signal for the observed degradation of alkylated AGT. Crystal structures of AGT homologs from the three kingdoms of life reveal that despite their extremely low primary sequence homology, these structural motifs are invariant. |
