Dynamics, Hydration, and Motional Averaging of a Loop-Gated Artificial Protein Cavity:  The W191G Mutant of Cytochrome c Peroxidase in Water as Revealed by Molecular Dynamics Simulations^†

Five molecular dynamics simulations of the W191G cavity mutant of cytochrome c peroxidase in explicit water reveal distinct dynamic and hydration behavior depending on the closed or open state of the flexible loop gating the cavity, the binding of (K⁺ or small molecule) cations, and the system temperature. The conformational spaces sampled by the loop region and by the cavity significantly reduce upon binding. The largest ordering factor on water dynamics is the presence of the K⁺ ion occupying the gated cavity. Considerable water exchange occurs for the open-gate cavity when no ligand or cation is bound. In all cases, good correspondence is found between the calculated (ensemble-averaged) location of water molecules and the water sites determined by X-ray crystallography experiments. However, our simulations suggest that these sites do not necessarily correspond to the presence of bound water molecules. In fact, individual water molecules may repeatedly exchange within the cavity volume yet occupy on average these water sites. Four major conclusions emerge. First, it seems misleading to interpret the conformation of protein loop regions in terms of single dominant structures. Second, our simulations support the general picture of Pro 190 cis−trans isomerization as a determinant of the loop-opening mechanism. Third, receptor flexibility is fundamental for ligand binding and molecular recognition, and our results suggest its importance for the docking of small compounds to the artificial cavity. Fourth, after validation against the available experimental data, molecular dynamics simulations can be used to characterize the dynamics and exchange of water molecules and ions, providing atomic level and time-dependent information otherwise inaccessible to experiments.