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Understanding substrate recognition and function of human dipeptidyl peptidase III

Dipeptidyl peptidase III (DPP III, EC 3.4.14.4) is a monozinc metalloexopeptidase that hydrolyzes dipeptides from the N-terminus of its substrates consisting of three or more amino acids. Widely distributed in mammalian tissue, DPP III has been considered to participate in intracellular protein catabolism but it is also indicated in pain modulation1 as well as in the stress response mechanism in mammals.2 DPP III contributes to the activation of transcription factor Nrf2, a constituent of the Nrf2-Keap1 signaling pathway, the main defense mechanism against many environmental toxic agents and carcinogens in cells.3 Until now several 3D structures of human DPP III (h.DPP III) have been determined, ligand free and in complexes with natural peptides (PDB_codes 3FVY, 5E33, and 3T6B, 3T6J, 5E3A, 5E2Q, 5EHH and 5E3C, respectively). H.DPP III structure consists of two domains, the catalytic one bearing the metal ion and the larger, “satellite” domain with a deep cleft in between. The presence of the interdomain cleft, together with promiscuous substrate specificity, 1 suggested that h.DPP III could experience significant internal motions. In order to rationalize the experimental data and to better understand its catalitical function we performed exhaustive in silico study of h.DPP III.4-9 Using molecular dynamics (MD) simulation techniques we investigated the conformational landscape of h.DPP III as well as the influence of ligand binding on the protein structure and dynamics.4-7 We found that h.DPP III can adopt a number of different forms in solution6, 7 wherein the most compact form was determined as the most stable. The substrate to be hydrolyzed binds preferably into a semi-closed conformation in a way to interact with both domains, influences the mutual orientation of domains and shifts the conformational equilibrium towards more compact protein form. In agreement with the experimental data we found that DPP III preferes ligands that adopt β-strand form and binds to the five-stranded β-core of the enzyme in an antiparallel fashion. Using QM/MM calculations we determined the Zn2+ coordination in different h.DPP III conformations, and showed that the most compact enzyme form is the catalytically active one.8 Based on this result we were able to determine the reaction mechanism of the peptide bond hydrolysis in the enzyme active site. Recently we have also studied h.DPP III binding to Keap1. In accord with the experimental data we showed that h.DPP III binds to the kelch domain via the flexible loop containg an “ETGE” motif. 9 1. Baršun M., Jajčanin N., Vukelić B., Špoljarić J. and Abramić M., Biol. Chem. 388 (2007) 343-348. 2. Liu Y., Kern J., Walker J.R., Johnson J.A., Schultz P.G., Luesch H., Proc. Natl .Acad. Sci. USA 104 (2007) 5205-5210. 3. Hast B.E., Goldfarb D., Mulvaney K.M., Hast M.A., Siesser P.F., Yan F., Hayes D.N. and Major M.B., Cancer Res. 73 (2013) 2199 -2210. 4. Tomić A., Abramić M., Špoljarić J., Agić D., Smith D.M., Tomić S., J. Mol. Recognit. 24 (2011) 804-814. 5. Špoljarić J., Tomić A., Vukelić B., Salopek-Sondi B., Agić D., Tomić S.and Abramić M., Croatica Chemica Acta 84 (2011) 259-268. 6. Tomić A, . Gonzalez M., Tomić S., J. Chem. Information Model. 52 (2012) ; 1583-1594. 7. Tomić A., Berynskyy M., Wade C. R., Tomić S., Mol. BioSyst. (2015) 11 3068-3080. 8. Tomić A., Tomić S., Dalton transactions. 43 (2014) 15503-155149. 9. Gundić M., Tomić A., Wade C.R., Tomić S., submitted for publication

Human dipeptidyl-peptidase III; enzyme reaction; computational chemistry; reaction mechanism; protein - protein interactions

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