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

Abstract

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 target. Crystal structures of active human AGT and its biologically and therapeutically relevant methylated and benzylated product complexes reveal an unexpected zinc-stabilized helical bridge joining a two-domain alpha/beta structure. An asparagine hinge couples the active site motif to a helix-turn-helix (HTH) motif implicated in DNA binding. The reactive cysteine environment, its position within a groove adjacent to the alkyl-binding cavity and mutational analyses characterize DNA-damage recognition and inhibitor specificity, support a structure-based dealkylation mechanism and suggest a molecular basis for destabilization of the alkylated protein. These results support damaged nucleotide flipping facilitated by an arginine finger within the HTH motif to stabilize the extrahelical O(6)-alkylguanine without the protein conformational change originally proposed from the empty Ada structure. Cysteine 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.

Fig. 1. Human AGT secondary structure, two-domain fold, zinc site, and location of structurally and catalytically critical residues. (A) Overall fold, displaying the active site cysteine (yellow) and its surrounding hydrogen-bond network, zinc (purple) ligands and Arg128 ‘arginine finger’. A central interdomain cleft separates the N-terminal α/β roll (α–helices in royal blue, β-strands in orange) from the C-terminal domain, which contains the active site and HTH DNA-binding motif (sky blue). For continuity, the location of the internal disordered loop (purple) has been interpolated. (B) Primary sequence, secondary structure and residue function of human AGT aligned with the E.coli Ada-C protein. Identity between the two sequences is shown with an asterisk, while corresponding residues that lie within 4.5 Å in the overlaid structures are given in upper case letters. The conserved active site motif (yellow boxes) is flanked by residues defining the O⁶–alkylguanine-binding channel (green boxes), anticipated DNA-binding residues (orange boxes), and HTH motif, with its associated Arg128 (sky blue). Colors distinguish residues participating in the active site hydrogen-bond network (red), zinc ligands (purple), α–helices (royal blue) and 3₁₀–helices (navy blue).

Fig. 2. Identification of a zinc-binding site in human AGT. X-ray fluorescence of non-zinc-supplemented crystals of AGT was measured as a function of incident radiation. The position of the inflection point in the intensity (I) measurement and the maximum in the first derivative curve (dI/dE) correspond well to that expected for the K absorption edge for zinc (vertical line).

Fig. 3. The methylated and benzylated AGT adducts identify the alkyl-binding pocket and suggest the bases for species-dependent substrate specificity. (A) Methylated AGT active site and electron density from a σ_A-weighted 2F_o – F_c map (2σ, purple; 4σ, red) and omit electron density excluding the methyl adduct (2σ, green; 3σ, gold). Four ordered water molecules adjacent to the active site cysteine have replaced the repaired guanine. (B) Omit electron density for benzylated AGT (2σ, white; 6σ, red) is shown for maps calculated excluding the benzyl adduct. The benzyl group stacks between the ring of Pro140 and the Cβ of Ser159, and is flanked by the side chains of Tyr158 and Asn137 (oxygens, red; nitrogens, blue; carbons, green). (C) The structural basis for differential affinity of O⁶–BG between human AGT (royal blue) and E.coli Ada-C (gray). The AGT alkyl-binding pocket, shown by the benzylated cysteine (yellow and sky blue), is partially filled by Trp161 in Ada-C. Additionally, alteration of Pro138 and Pro140 (sky blue) of AGT to Lys and Ala (gray), respectively, results in a narrowing of the alkyl-binding pocket.

Fig. 4. Structurally inferred guanine- and DNA-binding mode of AGT. (A) Guanine inserts into the active site channel along the recognition helix, stacking the aromatic base against Met134 and Gly131. Guanine-specific hydrogen bonds occur between the Watson–Crick base-pairing atoms of guanine and protein main chain atoms. The benzyl lesion is positioned adjacent to the active site cysteine, and oriented for optimal S_N2 displace– ment. (B) Overlay of AGT and the HTH-containing DNA-binding region of the catabolite gene activator protein bound to DNA (both red), and proposed analogous binding of DNA (purple) by AGT. Arg128, extending from the HTH motif (sky blue), penetrates the base stack and is ideally positioned to facilitate flipping of target O⁶–alkylguanine nucleotides. (C) Potential DNA-contacting residues of AGT (same color scheme as for Figure 1A), rotated by 90° about the vertical axis relative to (B). The HTH motif (sky blue), lying within the major groove, provides several potential hydrophilic and electrostatic DNA contacts. The adjacent ‘wing’, corresponding to the turn between B6 and B7, contacts the minor groove through Ser151 and Ser152. (D) The electrostatic surface of AGT, oriented as (C) and colored by Coulombic electrostatic potential (kT/e^–), displays a positively charged region centered around Arg128 complementary to the DNA phosphate backbone (oxygens, red; phosphates, yellow). Arg128, the ‘arginine finger’, facilitates damage repair by entering the base stack and displacing the extrahelical O⁶–alkylguanine nucleotide.

Fig. 5. AGT active site structure, dealkylation mechanism and stability of mutants mimicking alkylated AGT. (A) Active site hydrogen bond network, required for both stability and activity. Electron density from a σ_A-weighted 2F_o – F_c map contoured at 2σ is shown. (B) A proposed reaction mechanism for AGT. His146, acting as a water-mediated general base, deprotonates Cys145 to facilitate attack at the O⁶–alkyl carbon, with concomitant protonation of N3 by Tyr114. (C) Distance difference matrix plot of benzylated (upper right) and methylated (lower left) versus native AGT showing 0.5–1.5 Å shifts of helix H6 (residues 125–136) and the guanine-binding loop (residues 153–160) away from the N-terminal domain. This opening of the tertiary structure, accommodating the alkyl adducts, distorts the DNA-binding surface. (D) Instability of Cys145 mutants. Wild-type and C145F and C145L mutants were expressed in E.coli lacking endogenous AGT. Following arrest of protein synthesis, the presence of AGT was measured as a function of time by immunoblotting of cell lysates with anti-AGT antibodies. C145F and C145L mutants, which mimic alkylated C145, demonstrate in vivo instability.