Protonation Effects in Protein-Ligand Complexes - A Case Study of Endothiapepsin and Pepstatin A with Computational and Experimental Methods.

Protonation states serve as an essential molecular recognition motif for biological processes. Their correct consideration is key to successful drug design campaigns, since chemoinformatic tools usually deal with default protonation states of ligands and proteins and miss atypical protonation states. The protonation pattern for the Endothiapepsin/PepstatinA (EP/pepA) complex is investigated using different dry lab and wet lab techniques. ITC experiments revealed an uptake of more than one mole of protons upon pepA binding to EP. Since these experiments were performed at physiological conditions (and not at pH=4.6 at which a large variety of crystal structures is available), a novel crystal structure at pH=7.6 was determined. This crystal structure showed that only modest structural changes occur upon increasing the pH value. This lead to computational studies Poisson-Boltzmann calculations and constant pH MD simulation to reveal the exact location of the protonation event. Both computational studies could reveal a significant pKa shift resulting in non-default protonation state and that the catalytic dyad is responsible for the uptake of protons. This study shows that assessing protonation states for two separate systems (protein and ligand) might result in the incorrect assignment of protonation states and hence incorrect calculation of binding energy.