Abstract
Alkyltransferase-like proteins (ATLs) are a novel class of DNA repair proteins related to O(6)-alkylguanine-DNA alkyltransferases (AGTs) that tightly bind alkylated DNA and shunt the damaged DNA into the nucleotide excision repair pathway. Here, we present the first structure of a bacterial ATL, from Vibrio parahaemolyticus (vpAtl). We demonstrate that vpAtl adopts an AGT-like fold and that the protein is capable of tightly binding to O(6)-methylguanine-containing DNA and disrupting its repair by human AGT, a hallmark of ATLs. Mutation of highly conserved residues Tyr(23) and Arg(37) demonstrate their critical roles in a conserved mechanism of ATL binding to alkylated DNA. NMR relaxation data reveal a role for conformational plasticity in the guanine-lesion recognition cavity. Our results provide further evidence for the conserved role of ATLs in this primordial mechanism of DNA repair.
Highlights
Structural statistics were computed for the ensemble of 20 deposited structures (PDB entry, 2KIF). r.m.s., root mean square; r.m.s.d., r.m.s. deviation
Swapping Trp[54] for an alanine (Trp formational dynamics within the recognition cavity of apoand Ala are present in Ϸ89 and Ϸ9%, respectively, of ATLs; ATL, revealed for the first time by this structural NMR study, Pfam 23.0) results in an ATL that exhibits some affinity for confer functional plasticity that may be essential for providing methylated DNA, less than that for wild type vpAtl its wider range of guanine lesion specificity
To our knowledge, this is the first mutagenesis study examining these critical residues in ATLs and their roles in binding to O6-mG DNA and blocking human AGT activity
Summary
Complete details of the methods used in this work are provided in the supplemental material. Sample Preparation—The cloning, expression, and purification of isotopically enriched protein samples of a 100-residue construct from the A79_1377 gene of V. parahaemolyticus AQ3810 (Northeast Structural Genomics code, VpR247; vpAtl) plus C-terminal affinity tag (LEHHHHHH) was performed function out of 100 in the final cycle calculated were further refined by restrained molecular dynamics in explicit water with CNS 1.2 (19, 20). The global goodness-of-fit of the final structure ensembles with the nuclear Overhauser effect spectroscopy (NOESY) peak list data were determined using the RPF analysis program (23). Single backbone 15N relaxation and 1H-15N heteronuclear NOE data residue mutations of vpAtl (Y23A, Y23F, R37A, and W54A) using the Modelfree 4.20 program (25, 26) assuming an isotrowere cloned using the QuikChange site-directed mutagenesis pic model for molecular motion. Ten microliters of purified hAGT (0.5 pmol) were added to the above reaction mixture, and incubation was continued for 60 min at 37 °C and assayed for alkyltransferase activity
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