Multi scale computational approaches to understand a structure, dynamics and activity of thehuman dipeptidyl-peptidase III

Abstract

Sažetak Dipeptidil-peptidaza III (DPP III) je o cinku ovisna egzopeptidaza koja katalizira reakciju hidrolize peptidne veze odcjepljujući dipeptid s N-kraja svojih supstrata. U okviru ove doktorske disertacije detaljno je istražena struktura i dinamika ljudske DPP III i njenih kompleksa primjenom računalnih pristupa različitih stupnjeva složenosti. Modeliranje je bilo temeljeno na trodimenzionalnim strukturama enzima određenim rentgenskom strukturnom analizom. Molekulsko-dinamičke (MD) simulacije slobodnog (bez vezanog liganda) enzima otkrile su veliku fleksibilnost proteina, a simulacije kompleksa DPP III sa sintetskim i peptidnim supstratima pridonijele su razumijevanju široke supstratne specifičnosti ovoga enzima, te u kombinaciji s računom slobodne energije omogućile određivanje najvjerojatnijeg i kemijski aktivnog načina vezanja supstrata. Aktivna konformacija enzima dodatno je potvrđena primjenom različitih MD metoda i hibridnim kvantno-mehaničkim molekulskomehaničkim (QM/MM) računima različitih načina koordinacije cinka. Mehanizam reakcije hidrolize određen je na modelnom sustavu sastavljenom od supstrata, aminokiselinskih ostataka koji neposredno sudjeluju u procesu katalize i ostataka koji koordiniraju ion cinka.The dipeptidyl-peptidase III (DPP III) is a zinc-exopeptidase that hydrolyzes dipeptide from the N-terminus of its substrates. In this thesis a structure and dynamics of ligand-free enzyme and its complexes is investigated in detail by multi scale computational approaches. Modeling study was based on the three-dimensional enzymes structures obtained by X-ray diffraction analysis. Molecular dynamics (MD) simulations of the ligand-free enzyme revealed large flexibility of the protein, while the simulations of DPP III in the complexes with synthetic and peptide substrates enabled understanding of its broad substrate specificity, and, in combination with the free energy calculations, helped in determination of the most probable and chemically active ligand binding mode. The active enzyme conformation was confirmed by different MD approaches as well as by the hybrid quantum mechanicsmolecular mechanics (QM/MM) calculation of the different zinc ion coordinations. The reaction mechanism was determined using the model system consisting of substrate, amino acids participating in catalysis and the residues that coordinate the zinc ion