Figure 3
Several mutants highlight dynamics within the active site.
(a) Superposition of R307Q with the H309S substrate complex and the V321A product complex demonstrate that in R307Q, the product 2′-phosphate can adopt different conformations, linked to the β5-α7 loop conformation. (b) Comparison of the surface electrostatics between wild-type CNPcat and R307Q reveals changes in the immediate proximity of the active site (arrow), caused by the absence of the positively charged Arg307. The catalytic site has been indicated with an orange dot. (c) The conformations of Arg307 in the sulphate complex and Gln307 in R307Q suggest that Arg307 has a dynamic role, which is possibly related to the adjacent anion-binding pocket. In these two structures, this pocket is occupied by sulphate (cyan) and chloride (red), respectively. Conformational variability of Arg307 is evident also in the P296G structure. For clarity, only the substrate from the H309S structure is shown in the active site. (d) The presence of the 5′-phosphate causes the ligand to retract slightly from the active site in V321A, but the β5-α7 loop remains open and the 2′-phosphate is bound under it (arrow). (e) In P296G, the β5-α7 loop adopts the open conformation, similarly to the V321A structures, showing that loop opening is not caused by the absence of Val321. (f) Mutations from Pro to Gly at positions 225 and 296 affect loops α3-β2 and α6-β5, respectively. Loop α6-β5 is partially disordered in many structures.
