Hunting the human DPP III active conformation: combined thermodynamic and QM/MM calculations

Abstract

Multiple choices of the protein active conformations in flexible metalloenzymes complicate study of their catalytic mechanism. We used three different conformations of human dipeptidyl-peptidase III (DPP III) to investigate the influence of the protein environment on ligand binding and the Zn(2+) coordination. MD simulations followed by calculations of binding free energy components accomplished for a series of DPP III substrates, both synthetic and natural, revealed that binding of the β-strand shaped substrate to the five-stranded β-core of the compact DPP III form (in antiparallel fashion) is the preferred binding mode, in agreement with the experimentally determined structure of the DPP III inactive mutant-tynorphin complex (Bezerra et al., Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 6525). Previously it was proposed that the catalytic mechanism of DPP III is similar to that of thermolysin, which assumes exchange of five and four coordinated Zn(2+), and activation of Zn-bound water by a nearby Glu. Our QM/MM calculations, performed for a total of 18 protein structures with different zinc ion environments, revealed that the 5-coordinated metal ion is more favourable than the 6-coordinated one in only the most compact DPP III form. Besides, in this structure E451 is H-bonded to the metal ion coordinating water. Also, our study revealed two constraints for the broad substrate specificity of DPP III. One is the possibility of the substrate adopting the β-strand shape and the other is its charged N-terminus. Altogether, we assume that the human DPP III active conformation would be the most compact form, similar to the "closed X-ray" DPP III structure.

Oxidase or peptidase? A computational insight into a putative aflatoxin oxidase from Armillariella tabescens

“…It is important to stress that the observed transition from mono‐ to bidentate coordination is reversible. Such a high active site flexibility has previously been reported in human and bacterial DPP III homologues as well. Perhaps the most interesting behavior was noticed in the third run, where Glu444 left the Zn 2+ coordination sphere.…”

“…The inability to observe the protein opening in classical and accelerated MD simulations might be explained by inadequate sampling of the conformational space but also with the rigidity of the inter‐domain linker (hinge) as well as with the stabilizing inter‐domain interactions. Similar behavior was also determined for the human DPP III, despite quite exhausting conformational search. The crystal packing environment might be an explanation why AFO appears exclusively in the closed form in crystal structures (Supporting Information Figure S25).…”

“…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 . Crystallization experiments performed on AFO did not yield an open structure, which was attributed to structural differences in the hinge region …”

Validation of flavonoids as potential dipeptidyl peptidase III inhibitors: Experimental and computational approach

“…Substrates preferably bind to a semiclosed hDPP III and boost its closure. In such a compact enzyme structure, the catalytic zinc ion, bound to the upper domain at the “roof” of the cleft, interacts with the carbonyl oxygen from the scissile amide bond of the substrate molecule that bounds to the lower protein domain . Specific inhibitors were previously shown to be important for determining the mechanism of action and the physiological role of several mammalian metallopeptidases .…”

Human DPP III – Keap1 Interactions: A Combined Experimental And Computational Study

“…[10] Extensive investigations of the biochemical properties, structure and dynamics of this protein have been conducted by our group. [11][12][13][14][15][16] An enhanced level of human DPP III activity and protein was found in endometrial carcinomas and in ovarian malignant neoplasms. [17] Moreover, a correlation between DPP III activity and the aggressiveness of ovarian primary carcinomas was shown.…”