Active and alkylated human AGT structures: a novel zinc site, inhibitor and extrahelical base binding

Human O-6-alkylguanine-DNA alkyltransferase (AGT), which directly reverses endogenous alkylation at the O-6-position of guanine, confers resistance to alkylation chemotherapies and is therefore an active anticancer drug targe t. Crystal structures of active human AGT and its biologically and therapeu tically relevant methylated and benzylated product complexes reveal an unex pected zinc-stabilized helical bridge joining a two-domain alpha/beta struc ture. An asparagine hinge couples the active site motif to a helix-turn-hel ix (HTH) motif implicated in DNA binding. The reactive cysteine environment , its position within a groove adjacent to the alkyl-binding cavity and mut ational analyses characterize DNA-damage recognition and inhibitor specific ity, support a structure-based dealkylation mechanism and suggest a molecul ar basis for destabilization of the alkylated protein. These results suppor t damaged nucleotide flipping facilitated by an arginine finger within the HTH moth to stabilize the extrahelical O-6-alkylguanine without the protein conformational change originally proposed from the empty Ada structure. Cy steine alkylation sterically shifts the HTH recognition helix to evidently mechanistically couple release of repaired DNA to an opening of the protein fold to promote the biological turnover of the alkylated protein.