Role of Water Molecules in Protein–Ligand Dissociation and Selectivity Discrimination: Analysis of the Mechanisms and Kinetics of Biomolecular Solvation Using Molecular Dynamics

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The mechanism by which water molecules modulate biomolecular interactions and the time scale of microscopic solvation processes are usually not known. This is particularly problematic as it prevents the incorporation of effects of water molecules into the design of drug molecules with optimal binding kinetics and selectivity. We investigated this crucial problem of drug discovery using trypsin and thrombin in complex with benzamidine and N-amidinopiperidine. For these systems, we studied the mechanism and time scale of solvation using molecular dynamics and umbrella sampling calculations. In thrombin, water molecules are seemingly stable in the apo binding pocket and have an exchange rate on the nanosecond time scale. On the contrary, water molecules in apo trypsin exchange approximately one order of magnitude faster than in thrombin. This difference in the exchange rate is due to internal water channels that are only found in thrombin linking the interior of the binding pocket with bulk solvent. These cause the exchange rate of water molecules to be independent of the ligand molecule. However, in the case of trypsin, the solvent exchange rate greatly varies between the two complexes, indicating a strong dependence on the ligand molecule. Furthermore, the binding mechanism of the ligand molecules critically depends on water molecules that intercalate between key amino acids and the ligand, leading to enhanced water residence times in intermediate dissociation steps. Our findings strongly indicate a selectivity discriminating role of water molecules for these two proteins and underline the functional interplay between water channels and binding affinity of ligand molecules.