O(6)-alkylguanine-DNA alkyltransferase (AGT), or O(6)-methylguanine-DNA methyltransferase (MGMT), prevents mutations and apoptosis resulting from alkylation damage to guanines. AGT irreversibly transfers the alkyl lesion to an active site cysteine in a stoichiometric, direct damage reversal pathway. AGT expression therefore elicits tumor resistance to alkylating chemotherapies, and AGT inhibitors are in clinical trials. We report here structures of human AGT in complex with double-stranded DNA containing the biological substrate O(6)-methylguanine or crosslinked to the mechanistic inhibitor N(1),O(6)-ethanoxanthosine. The prototypical DNA major groove-binding helix-turn-helix (HTH) motif mediates unprecedented minor groove DNA binding. This binding architecture has advantages for DNA repair and nucleotide flipping, and provides a paradigm for HTH interactions in sequence-independent DNA-binding proteins like RecQ and BRCA2. Structural and biochemical results further support an unpredicted role for Tyr114 in nucleotide flipping through phosphate rotation and an efficient kinetic mechanism for locating alkylated bases.

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.

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Table of Contents I. Peptide Synthesis and Purification SI-2 i. Table SI-1. Peptide Sequences SI-2 ii. Table SI-2. Peptide calculated and observed masses SI-3 II. Peptide Characterization SI-4 A. Circular Dichroism SI-4 i. Figure SI-1. Thermostability of aPP and variants SI-5 ii. Figure SI-2. Thermostability of YY2 and variants SI-6 iii. Table SI-3. Peptide Melting Temperatures SI-7 B. Flow Cytometry SI-7 C. Confocal microscopy SI-8 D. Cell Viability SI-8 i. Figure SI-3. Cell Viability SI-9 III. References Cited SI-9 Supplemental Information SI-2 I. Peptide Synthesis and PurificationTable SI-1. Sequences of peptides and miniature proteins described in this work. aPP GPSQPTYPGDDAPVEDLIRFYNDLQQYLNVVTRHRY 1 aPP 4R1 GPSQPTYPGDDAPVRDLIRFYRDLQRYLNVVTRHRY aPP 5R1 GPSQPTYPGDDAPVRDLIRFYRDLRRYLNVVTRHRY aPP 6R1 GPSQPTYPGDDAPVRDLRRFYRDLRRYLNVVTRHRY YY2 APPLPPRNRGEDASPEELSRYYASLRHYLNLVTRQRY 2 YY2 3R1 APPLPPRNRGEDASPEELSRYYRSLRHYLNLVTRQRY YY2 4R1 APPLPPRNRGEDASPRELSRYYRSLRHYLNLVTRQRY YY2 5R1 APPLPPRNRGEDASPRELRRYYRSLRHYLNLVTRQRY R 8 Y RRRRRRRRY Tat (48-60) GRKKRRQRRRPPQY 3 Penetratin RQIKIWFQNRRMKWKK 4 Transportan GWTLNSAGYLLKINLKALAALAKKIL 5 KLA KLALKLALKALKAALKLA 6Synthesis. All peptides were synthesized using standard solid-phase Fmoc chemistry on a 25 µmol scale with a Symphony ® multi-channel solid phase synthesizer (Protein Technologies, Inc., Tuscon, AZ). All α-amino acids and resins were purchased from Novabiochem (San Diego, CA) and solvents were purchased from American Bioanalytical (Natick, MA). All peptides were synthesized to carry free amines at their N-termini and carboxamides at their C-termini. Peptides were labeled on their N-termini on resin for at least one hour with fluorescein-5-EX, succinimidyl ester (Invitrogen, Carlsbad, CA, Cat. # F-6130), which was dissolved in 1 mL DMF and added to the reaction vessel with 20 µL N,N-diisopropylethylamine (EDIPA).Purification. Peptides were purified by reverse-phase HPLC using Grace Vydac C8 preparative or semi-preparative scale columns (300 silica, 10 µm particle size, 22 mm x 250 mm) and water/acetonitrile gradients containing 0.1% TFA. Peptide identity was confirmed by mass spectrometry on an Applied Biosystems Voyager-DE Pro MALDI-TOF mass spectrometer

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