Antonija Tomić

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

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 kcat 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. Copyright

The first dipeptidyl peptidase III from a thermophile: Structural basis for thermal stability and reduced activity

Dipeptidyl peptidase III (DPP III) isolated from the thermophilic bacteria Caldithrix abyssi (Ca) is a two-domain zinc exopeptidase, a member of the M49 family. Like other DPPs III, it cleaves dipeptides from the N-terminus of its substrates but differently from human, yeast and Bacteroides thetaiotaomicron (mesophile) orthologs, it has the pentapeptide zinc binding motif (HEISH) in the active site instead of the hexapeptide (HEXXGH). The aim of our study was to investigate structure, dynamics and activity of CaDPP III, as well as to find possible differences with already characterized DPPs III from mesophiles, especially B. thetaiotaomicron. The enzyme structure was determined by X-ray diffraction, while stability and flexibility were investigated using MD simulations. Using molecular modeling approach we determined the way of ligands binding into the enzyme active site and identified the possible reasons for the decreased substrate specificity compared to other DPPs III. The obtained results gave us possible explanation for higher stability, as well as higher temperature optimum of CaDPP III. The structural features explaining its altered substrate specificity are also given. The possible structural and catalytic significance of the HEISH motive, unique to CaDPP III, was studied computationally, comparing the results of long MD simulations of the wild type enzyme with those obtained for the HEISGH mutant. This study presents the first structural and biochemical characterization of DPP III from a thermophile.