title: 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.
authors.affiliation: Division of Physical Chemistry, Rudjer Bošković Institute Bijenička 54,10002 Zagreb, Croatia.
authors.forename: Antonija
authors.initials: A
authors.lastname: Tomić
authors.forename: Marija
authors.initials: M
authors.lastname: Abramić
authors.forename: Jasminka
authors.initials: J
authors.lastname: Spoljarić
authors.forename: Dejan
authors.initials: D
authors.lastname: Agić
authors.forename: David M
authors.initials: DM
authors.lastname: Smith
authors.forename: Sanja
authors.initials: S
authors.lastname: Tomić
mesh_terms: D020134:Catalytic Domain / Q000235:genetics / Q000502:physiology; D004151:Dipeptides / Q000737:chemistry / Q000378:metabolism; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D017433:Protein Structure, Secondary
chemical_list: D004151:Dipeptides; C040852:arginylarginine 2-naphthylamide; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C100152:dipeptidyl peptidase III