FIGURE 2.

Crystal structures of CVB3 3D^pol motif D mutants. A, mutations that do not alter the wild type conformation of the motif D loop include A341G (blue), A345V (orange), F364Y (pink), and F364W (magenta). The F364Y mutation may stabilize this conformation via a new hydrogen bond to the backbone carbonyl of Met³⁵⁵ (black dashed line), and F364W may reinforce the interaction with Pro³⁵⁷ by providing a larger aromatic surface area. YGDD marks the active site β-turn within motif C. B, mutation of Phe³⁶⁴ to small hydrophobic and β-branched residues precludes the stacking interaction with Pro³⁵⁷, inducing a downward movement of Pro³⁵⁷. F364A is shown in yellow, F364V is in cyan, F364L is in red, and F364I is in teal. C–E, mutations to Phe³⁶⁴ result in a closing of the active site in the absence of RNA and NTPs as shown in these top views of the active site motifs. C, the open conformation seen with A341G, A345V, and the aromatic F364Y and F364W mutations matches the structure of the wild type 3D^pol (gray). The open conformation is characterized by partial hydrogen bonding (yellow dashes) between motifs A and C and an ≈6.5-Å distance between Asp³²⁹ C^α in Motif C and Tyr²³⁴ C^α in motif A. D, The F364A, F364L, and F364V mutant structures show closed active sites characterized by an ≈0.5-Å movement of motif A toward motif C that is stabilized by the formation of three new hydrogen bonds (black dashes). The hydrogen bonds between motifs A and D are maintained (gray dashes), demonstrating the concerted movement of motifs A and D to close the active site. The open wild type CVB3 (gray) and closed poliovirus (PV) (green) conformation structures are shown for comparison. E, the F364I mutant (teal with nitrogen atoms in blue and oxygen atoms in red) is in a partially closed state where the hydrogen bonding network of the closed active site has not fully formed. The open wild type (gray), closed F364A (yellow), and F232L (green) structures are shown for comparison.