Decoding the structural events in substrate‐gating mechanism of eukaryotic prolyl oligopeptidase using normal mode analysis and molecular dynamics simulations

Abstract

Prolyl oligopeptidase (POP) is a serine protease, unique for its ability to cleave various small oligopeptides shorter than 30 amino acids. POP is an important drug target since it is implicated in various neurological disorders. Although there is structural evidence that bacterial POPs undergo huge interdomain movements and acquire an "open" state in the substrate-unbound form, hitherto, no crystal structure is available in the substrate-unbound domain-open form of eukaryotic POPs. Indeed, there is no difference between the substrate-unbound/bound states of eukaryotic POPs. This raises unanswered questions about whether difference in the substrate access pathway exists between bacterial and eukaryotic POPs. Here, we have used normal mode analysis and molecular dynamics to unravel the mechanism of substrate entry in mammalian POPs, which has been debated until now. Motions observed using normal modes of porcine and bacterial POPs were analyzed and compared, augmented by molecular dynamics of these proteins. Identical to bacterial POPs, interdomain opening was found to be the possible pathway for the substrate-gating in mammals as well. On the basis of our analyses and evidences, a mechanistic model of substrate entry in POPs has been proposed. Up-down movement of N-terminal hydrolase domain resulted in twisting motion of two domains, followed by the conformational changes of interdomain loop regions, which facilitate interdomain opening. Similar to bacterial POPs, an open form of porcine POP is also proposed with domain-closing motion. This work has direct implications for the development of novel inhibitors of mammalian POPs to understand the etiology of various neurological diseases.

Dynamics and ligand-induced conformational changes in human prolyl oligopeptidase analyzed by hydrogen/deuterium exchange mass spectrometry

“…Both induced fit and conformational selection are consistent with substrate hydrolysis kinetic studies of PREP, showing that the experimental kinetic parameters are substrate-dependent and that a physical rather than a chemical step is rate determining 13 . Since functionally essential residues in the substrate binding pocket and the inter-domain interface are conserved between species, domain movement may be common in the catalytic cycle of all PREPs 14 .…”

Preparation, Characterization, and Oxygenase Activity of a Photocatalytic Artificial Enzyme

“…As in our initial report of artificial enzyme formation using SPAAC, [11] an amber codon was introduced into the POP gene [15] to replace the catalytically active serine (S477) with a Z residue (Z477). Four alanine mutations (E104A, F146A, K199A, D202A) were introduced to open a small pore in the POP structure [16] to allow cofactor entry into the active site. A codon optimized POP gene was used as a template for genetic manipulation, and the resulting scaffold, POP-ZA 4 , was expressed in high yield in E. coli with essentially quantitative Z incorporation.…”

Computational Analysis of the Domain Architecture and Substrate-Gating Mechanism of Prolyl Oligopeptidases from Shewanella woodyi and Identification of Probable Lead Molecules

“…Various efforts such as sequence search method and true homologue collection method have been made with a single query sequence to choose the ''true'' hits. These sequence search methods enabled the identification of possible true homologues which cannot be picked by direct BLAST search [6].…”

“…The active site is hidden in a large cavity formed by the interface of propeller and hydrolase domain [5]. The molecular dynamics simulation studies have suggested that opening of two domains is the only possible pathway for the substrate entry in POPs [6]. In order to understand the role of POP in biological and disease processes, both synthetic and natural ligands have been tested [7].…”

“…In the presence of inhibitor, two domains will be tightly bound to each other acting as gating filter for substrate to enter into the active site known as inhibitor-bound form forming a ''closed'' state [6].…”