Structural basis of O6-alkylguanine recognition by a bacterial alkyltransferase-like DNA repair protein

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.

FIGURE 1.

Solution NMR structure of vpAtl. A, structure-based sequence alignment of vpAtl, spAtl1, and hAGT (residues 91–176). The sequence numbering for vpAtl and the secondary structural elements found in its solution NMR structure are shown above the alignment. Identical residues are shown in red. Residues in the helix-turn-helix and active site sequence motifs are boxed in blue and yellow, respectively. Key highly conserved, functionally important residues (Tyr²³, Arg³⁷, and Trp⁵⁴ in vpAtl) are denoted by triangles below the alignment. B, stereoview into the putative alkyl-binding site in the lowest energy (CNS) conformer from the final solution NMR structure ensemble of vpAtl. The α-helices and β-strands are shown in cyan and magenta, respectively. Side chains of Tyr²³, Arg³⁷, and Trp⁵⁴ are shown in red, blue, and yellow, respectively. C, ConSurf (28) image showing the conserved residues in the alkyl-binding site of vpAtl (same view as in B). Residue coloring ranges from magenta (highly conserved) to cyan (variable) and reflects the degree of residue conservation across ATL sequences extracted from the entire O⁶-alkylguanine-DNA methyltransferase protein domain family (PF01035, Pfam 23.0). D, DelPhi (29) electrostatic surface potential of vpAtl showing negative (red), neutral (white), and positive (blue) charges.

FIGURE 2.

Backbone dynamics of vpAtl. A, plots of backbone amide ¹⁵N R₁ and R₂ relaxation rates, ¹H-¹⁵N heteronuclear NOEs, and generalized order parameters, S², versus residue number obtained on [U(5%)-¹³C, (100%)-¹⁵N]-vpAtl at a ¹⁵N Larmor frequency of 60.8 MHz. Order parameters were computed using the Modelfree 4.20 program (25, 26) assuming an isotropic model, yielding an overall rotational correlation time, τ_c, of 7.9 ns. B, backbone dynamics of vpAtl mapped onto its structure. Residues with S² ≤0.7, indicative of enhanced backbone flexibility, are in red, and residues with weak or missing ¹H-¹⁵N heteronuclear single quantum coherence resonances are colored yellow. Prolines are shown in gray, and the substrate binding pocket and binding pocket cap are indicated.

FIGURE 3.

Conserved structure and DNA binding properties of vpAtl. A, overlay of the vpAtl structure (cyan) and the crystal structure of spAtl1 bound to O⁶-mG-DNA (Protein Data Bank code 3GX4; magenta) (10). The side chains of Tyr²³, Arg³⁷, and Trp⁵⁴ are shown in red, blue, and yellow, respectively, and the O⁶-mG in the bound spAtl1 structure is shown in gray. B, percent activity of hAGT as a function of ATL concentration for vpAtl (triangles) and spAtl1 (circles). C, effect of mutating Tyr²³ in vpAtl on hAGT activity; wild type vpAtl (black), [Y23A]-vpAtl (red triangles), and [Y23F]-vpAtl (red circles). D, effect of mutating Arg³⁷ and Trp⁵⁴ in vpAtl on hAGT activity; wild type vpAtl (black), [R37A]-vpAtl (blue), and [W54A]-vpAtl (gold).