Structures of Δ79AAG bound to εC DNA. (A) Δ79AAG crystallized in the presence of ssεC DNA has two Δ79AAG molecules in the asymmetric unit: one that makes few contacts with DNA and represents a lower-affinity complex (green) and one that makes multiple contacts with DNA and represents a higher-affinity complex (orange). The two strands of ssεC DNA, which form a pseudoduplex, are shown as sticks with cyan carbons. Panel I displays Tyr162 (orange sticks) intercalating DNA while the εC lesion is flipped into the active site. Panel II depicts the lower-affinity interaction between Δ79AAG and DNA where Tyr162 (green sticks) stacks with nucleotide A2′. Atoms are colored as follows: red for oxygen, blue for nitrogen, and orange for phosphorus. A blue star denotes the location of the empty active site of lower-affinity AAG. (B) Schematic illustration of the interactions between the two strands of ssεC DNA and amino acid side chains (three-letter code) and main chains (mc) of the Δ79AAG molecules. Amino acid labels from the lower- and higher-affinity (pseudoduplex-bound) Δ79AAG molecules are colored green and orange, respectively. Hydrogen bonds are indicated by solid lines and van der Waals interactions by dashed lines. DNA bases are shown as rectangles containing one-letter codes and numbers that signify their respective positions in the oligonucleotide (5′ to 3′). All DNA bases contained in the nucleotide-flipped εC lesion strand are denoted with a prime. Disordered nucleotides are shown in dashed lines. (C) Nucleotide interactions near the lesion in Δ79AAG−εC:G dsDNA (PDB entry 3QI5) (yellow carbons). Relevant distances shown by dashed lines are given in angstroms. (D) Nucleotide interactions near the lesion in the pseudoduplex Δ79AAG structure (cyan carbons). (E) van der Waals interactions with G19 in the Δ79AAG−εC:G structure. (F) van der Waals interactions with A2 in the pseudoduplex Δ79AAG structure.

Comparison of the lower-affinity Δ79AAG structure with the high-affinity Δ79AAG−εA:T structure. (A) Δ79AAG (purple cartoon) bound to an εA lesion (stick form with cyan carbons) with active site residue His136 and intercalating Tyr162 represented in stick form with purple and yellow carbons, respectively (PDB entry 1F4R(8)). Oxygen atoms are colored red and nitrogen atoms blue. Regions that become disordered in the absence of a bound DNA adduct are circled and labeled 1–3. (B) Lower-affinity AAG (green) with Tyr162 (yellow). Loops 1–3 and other atoms are colored as in panel A. (C) Binding pocket for the εA lesion shown with van der Waals surfaces for protein residues (gray spheres) and εA lesion (cyan sphere). (D) Disrupted binding pocket in lower-affinity AAG with van der Waals surfaces colored as in panel B. (E) Electrostatic representation of Δ79AAG−εA:T calculated in the absence of DNA where blue surfaces are more positive, red surfaces more negative, and white surfaces near neutral. The position of Tyr162 is denoted with a yellow star. (F) Electrostatic representation of lower-affinity AAG with colors and symbols as in panel E. (G) Same depiction as panel E but with substrate DNA modeled (orange cartoon). (H) Same electrostatic depiction as panel F aligned and superimposed with a cartoon model (purple) of Δ79AAG−εA:T. Disordered regions that affect electrostatic potential are circled and represent the same loops as in panels A and B.

Tyr162 contacts in lower-affinity Δ79AAG. (A) Hydrogen bonding contacts (dashed lines, distances in angstroms) for lower-affinity Δ79AAG (green) with pseudoduplex DNA (cyan carbons), and non-carbon atoms colored as in Figure 1. (B) van der Waals radii for the protein and DNA are shown in gray spheres with all other representations and colors as in panel A. (C) Same depiction as panel B with the orientation changed slightly to show relevant distances (in angstroms) as depicted by dashed lines and to draw attention to the rotation of εC7 out of the pseudoduplex.

Proposal for how AAG can recognize DNA with two different affinities. The electrostatic representations from panels E and F of Figure 3 are displayed in panels A and B for Δ79AAG−εA:T and lower-affinity Δ79AAG, respectively, with the same coloring and symbols as in Figure 3. The orientations of the molecules start as in Figure 3 and are then rotated 120° counterclockwise (in two 60° steps) along the vertical axis such that the continuous positive surface can be visualized. (C) Cartoon depiction of the search on DNA by AAG, where blue and red represent positively and negatively charged surfaces, respectively. Relevant steps are labeled, and a lesion base is denoted as a green line. The AAG catalysis complex is a darker shade of blue to represent the more ordered positive surface visualized crystallographically (panel A vs panel B above).