Unveiling prolyl oligopeptidase ligand migration by comprehensive computational techniques.

Prolyl oligopeptidase (POP) is a large 80 kDa protease, which cleaves oligopeptides at the C-terminal side of proline residues and constitutes an important pharmaceutical target. Despite the existence of several crystallographic structures, there is an open debate about migration (entrance and exit) pathways for ligands, and their coupling with protein dynamics. Recent studies have shown the capabilities of molecular dynamics and classical force fields in describing spontaneous binding events and nonbiased ligand migration pathways. Due to POP's size and to the buried nature of its active site, an exhaustive sampling by means of conventional long enough molecular dynamics trajectories is still a nearly impossible task. Such a level of sampling, however, is possible with the breakthrough protein energy landscape exploration technique. Here, we present an exhaustive sampling of POP with a known inhibitor, Z-pro-prolinal. In >3000 trajectories Z-pro-prolinal explores all the accessible surface area, showing multiple entrance events into the large internal cavity through the pore in the β-propeller domain. Moreover, we modeled a natural substrate binding and product release by predicting the entrance of an undecapeptide substrate, followed by manual active site cleavage and nonbiased exit of one of the products (a dipeptide). The product exit shows preference from a flexible 18-amino acid residues loop, pointing to an overall mechanism where entrance and exit occur in different sites.