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

Abstract

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.

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“…However, the long range conformational changes have not been traced during these additional 200 ns either (Supporting Information Figure S6). The structure of individual domains is conserved throughout the simulations (Supporting Information Figure S7), as previously reported for human and bacterial orthologues …”

“…Both carboxyl oxygen atoms of Glu444 and Glu501 entered the Zn 2+ coordination sphere at the very beginning of MD simulations. Such behavior is inconsistent with all currently known crystallographic structures of AFO, as well as with available crystallographic structures of the other DPP III family members, and suggests an overestimate of electrostatic interactions (Supporting Information Figure S2). On other hand, the tetrahedral model developed by Pang reproduced the active site architecture previously reported within the DPP III family to a certain degree .…”

“…Such behavior is inconsistent with all currently known crystallographic structures of AFO, as well as with available crystallographic structures of the other DPP III family members, and suggests an overestimate of electrostatic interactions (Supporting Information Figure S2). On other hand, the tetrahedral model developed by Pang reproduced the active site architecture previously reported within the DPP III family to a certain degree . However, within this model the active site was too rigid and the coordination with more than four ligands was not possible (Supporting Information Figure S2), which is inconsistent with the DPP III reaction mechanism requirements .…”

“…The crystal structure of the AFO (PDB code: 5YFB) was used as the starting point for MD simulations. Residues Met1–Val6, missing in the structure, have not been modeled since we assumed that they belong to the signaling peptide, in analogy with other DPP III orthologues …”

“…RMSD between the crystal structures of AFO and the closed structure of the human DPP III (PBD code: 3T6B) is about 1.0 Å. Both crystallographic and computational studies on DPPs III prove the existence of another conformation of the enzyme, the so‐called open form, with the two domains separated by a wide cleft . It is important to note that, in the case of the human orthologue, the open form is not catalytically active; both open and closed form exist in the solution, with the balance shifting toward the catalytically active closed form in the presence of a substrate .…”