Human dipeptidyl peptidase III: insights into ligand binding from a combined experimental and computational approach

Abstract

Human dipeptidyl peptidase III (DPP III) is a zinc-exopeptidase with implied roles in protein catabolism, pain modulation, and defense against oxidative stress. To understand the mode of ligand binding into its active site, we performed molecular modeling, site-directed mutagenesis, and biochemical analyses. Using the recently determined crystal structure of the human DPP III we built complexes between both, the wild-type (WT) protein and its mutant H568N with the preferred substrate Arg-Arg-2-naphthylamide (RRNA) and a competitive inhibitor Tyr-Phe-hydroxamate (Tyr-Phe-NHOH). The mutation of the conserved His568, structurally equivalent to catalytically important His231 in thermolysin, to Asn, resulted in a 1300-fold decrease of k(cat) for RRNA hydrolysis and in significantly lowered affinity for the inhibitor. Molecular dynamics simulations revealed the key protein-ligand interactions as well as the ligand-induced reorganization of the binding site and its partial closure. Simultaneously, the non-catalytic domain was observed to stretch and the opening at the wide side of the inter-domain cleft became enhanced. The driving force for these changes was the formation of the hydrogen bond between Asp372 and the bound ligand. The structural and dynamical differences, found for the ligand binding to the WT enzyme and the H568N mutant, and the calculated binding free energies, agree well with the measured affinities. On the basis of the obtained results we suggest a possible reaction mechanism. In addition, this work provides a foundation for further site-directed mutagenesis experiments, as well as for modeling the reaction itself.

Synthesis, QSAR, and Molecular Dynamics Simulation of Amidino-substituted Benzimidazoles as Dipeptidyl Peptidase III Inhibitors

“…During the MD simulation, the zinc cation, Zn 2+ , was mostly six-coordinated by two histidines (His450 and His455), two glutamates (Glu451 and Glu508), and two water molecules, as also determined for the ligandfree DPPIII 27 . These results imply that the binding of inhibitor 1 has no significant effect on the zinc ion coordination.…”

“…Interestingly, the binding mode and the multiple interactions established during the MD simulations of this potent benzimidazole-based inhibitor in complex with DPP III, differ from those established for the reversible inhibitor Tyr-Phe-NHOH 27 and the opioide peptide tynorphin (Val-Val-Tyr-Pro-Trp) 3 (see Fig.7. and compare with the Supplemental Fig.…”

“…The same molecular dynamics (MD) simulation procedure was used as in our previous publication. 27 The structure of ligand-free DPP III obtained after 30 ns of MD simulations was selected as a starting point and a complex between the protein and the inhibitor was built. Prior to the MD simulations of the protein-inhibitor complex, protein geometry was optimized in four cycles with different constraints.…”

Novel dipeptidyl hydroxamic acids that inhibit human and bacterial dipeptidyl peptidase III

“…Although a significant knowledge on the molecular enzymology (structure-activity relationship) of the DPP III family accumulated in the last decade [19][20][21][22] , physiological roles of any of its members have not been elucidated in sufficient detail. This is partly due to the lack of specific inhibitors.…”

Yeast Ortholog of Peptidase Family M49: the Role of Invariant Glu461 and Tyr327

“…Most of the work has been performed in the human ortholog by using site-directed mutagenesis, X-ray crystallography and computational approaches, often in combination. [6][7][8] Much of the effort was directed towards defining the substrate binding site. Most recently, the first crystal structure of human M49 peptidase (dipeptidyl peptidase III, DPP III) in complex with the pentapeptide tynorphin was reported.…”