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Ligand binding to human DPP III and its mutant H568N - computational analyses

Human dipeptidyl-peptidase III (DPP III) hydrolyses distinctive synthetic substrate Arg-Arg-2-naphthylamide and a number of biologically active peptides (e.g. endomorphins) by cleaving dipeptides from their N-termini. Like the other members of recently recognized metallopeptidase family M49[1], it has HEXXXH structural motif important for zinc ligation and catalytic activity. Besides its contribution in normal intracellular protein catabolism, there are evidences for its regulatory and pathophysiological role as well[2]. Human DPP III was recognized by the group in Zagreb as a biochemical indicator of endometrial and ovarian malignancies[3]. Others indicated the role for DPP III in the endogenous pain-modulatory system, defense against oxidative stress and in cataractogenesis[4][5][6]. We used the crystal structure of ligand-free human DPP III (PDB ID:3FVY) and by combining docking with molecular dynamics (MD) simulations we determined binding modes for two synthetic ligands, inhibitor Tyr-Phe-hydroxamate (YFNHOH) and substrate Arg-Arg-2-naphthylamide (RRNA). In order to study changes in the protein active site and protein conformation induced by ligands binding, we performed series of 10 ns long MD simulations for the wild type (WT) enzyme, its H568N mutant and their complexes. In the crystal structure of DPP III the central zinc ion is coordinated by two histidines (His-450, His-455), one glutamate (Glu-508) and one water molecule. MD simulations revealed the octahedral coordination of Zn2+, for example in the complexes with ligands (RRNA and YFNHOH) it is additionally coordinated by Glu-451 and by carbonyl oxygen belonging to the second peptide bond from the substrate N terminus. RRNA binds similarly to H565N as well. However, inhibitor in the complex with H568N moves away from Zn2+ and after about 8 ns of MD simulations it stabilizes in the new position closer to the 'lower' domain. According to Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) calculations, and in agreement with the experimental data, the binding affinity of YFNHOH is significantly lower for the mutant than for the WT DPP III. Based on the results of the simulations we proposed two possible reaction mechanisms for RRNA hydrolysis. In one Glu-508 behaves as general base and His-450 as the proton carrier, and in the other Glu-451 plays dual role of both the water activator and the proton carrier.

metaloenzyme; Human dipeptidyl-peptidase III; peptidaze; molecular modeling; molecular dynamics simulations; docking

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