Similarly to the G202R mutation, the N224D mutation is likely to have an electrostatic impact on TS stabilization, due to its location near the active site and the associated change in charge, as indicated also by calculations of Arrhenius parameters for the R1 and R4 variants. That said, our calculations show the mutation also impacts the conformational sampling of the active site. By analyzing the per feature scores obtained for the region around the N224D mutation site, we identified a network of hydrogen bonds present in the R1 variant that are correlated with the sampling of the W50 χ2 dihedral angle (Figure and Table ). Four of these interactions favor the "A" conformation and are all native interactions (i.e., they are present in the R1 X-ray structure, PDB ID: 5D2W 70 ), whilst 2/4 of the interactions that favor the "C" conformation are non-native interactions and require the H228-N224D interaction to break in order for them to be able to form (Figure and Table ). By comparing the per feature scores for these interactions in the R1 and R4 variants, we identified that the N224D mutation effectively abolishes this allosteric network, as the value of many of these interactions are close to 0 in the R4 variant (Table ). Given that native interactions in the R1 variant would appear to favor the "A" conformational state, the removal of this allosteric network would seem likely to help shift the population of W50 towards the "C" state.