Figure 6
D206N HtxB crystallises in a partially open complex with sulphate. (A) The central cleft of D206N HtxB (blue), showing the location of a sulphate molecule, which binds at the N-terminal end of helix 3, forming hydrogen bonds with the mainchain amides of P71, W72 and G73, the sidechain of T129 and four water molecules. An omit map surrounding the sulphate moiety (black mesh, contoured at 1.5 σ) is also shown. (B) A comparison with WT HtxB in complex with hypophosphite (beige) shows the spatial relationship of the two ligand binding sites (ligands shows as spheres) and the difference in the position of the binding pocket residues in the mutant versus the WT protein. Hydrogen bonds are represented as orange dashed lines. (C,D) A comparison between the D206N HtxB (blue), D206A HtxB (green) and WT HtxB structures (beige) (superimposed on the large domain: N-F96 and P204-C) shows how the D206N mutation changes the hydrogen bonding across the beta-sheet of this domain. In the WT and D206A HtxB structures the carbonyl of residue 206 hydrogen bonds with the mainchain amide of V89 (orange dashes, 3.0 Å). In the D206N HtxB structure, the sidechain oxygen of N206 sits within hydrogen bonding distance (2.9 Å) of the V89 amide and the N206 carbonyl is rotated by 50° (black arrow). The N205 carbonyl flips 180° (black asterisk), altering the rotamer of N205 in the D206N HtxB structure and resulting in a new hydrogen bond being formed with R178. The acetate molecule from the D206A HtxB structure (cyan) is shown for reference. (E) Two orthogonal views of superimposed HtxB-like proteins in closed (HtxB WT with hypophosphite; beige), partially-open (HtxB D206N mutant with sulphate; blue) and fully open (apo-PtxB from Pseudomonas stutzeri; purple) conformations. The superposition is carried out on the smaller of the two domains (residues 99–200 in HtxB), containing the important loop that recognises the oxygen atoms of the cargo (HtxB; N128-S130, PtxB; S124-S126).
