Changes in Protonation States of In-Pathway Residues can Alter Ligand Binding Pathways Obtained From Spontaneous Binding Molecular Dynamics Simulations.

Protein-ligand binding processes often involve changes in protonation states that can be key to recognize and orient the ligand in the binding site. The pathways through which (bio)molecules interplay to attain productively bound complexes are intricate and involve a series of interconnected intermediate and transition states. Molecular dynamics (MD) simulations and enhanced sampling techniques are commonly used to characterize the spontaneous binding of a ligand to its receptor. However, the effect of protonation state changes of in-pathway residues in spontaneous binding MD simulations remained mostly unexplored. Here, we used molecular dynamics simulations to reconstruct the trypsin-benzamidine binding pathway considering different protonation states of His57. This residue is part of the trypsin catalytic triad and is located more than 10 Å away from Asp189, which is responsible for benzamidine binding in the trypsin S1 pocket. Our MD simulations showed that the binding pathways that benzamidine follow to target the S1 binding site are critically dependent on the His57 protonation state. Binding of benzamidine frequently occurs when His57 is protonated in the delta nitrogen while the binding process is significantly less frequent when His57 is positively charged. Constant-pH MD simulations retrieved the equilibrium populations of His57 protonation states at trypsin active pH offering a clearer picture of benzamidine recognition and binding. These results indicate that properly accounting for protonation states of distal residues can be important in spontaneous binding MD simulations.