The loops facing the active site of prolyl oligopeptidase are crucial components in substrate gating and specificity

Abstract

Prolyl oligopeptidase (POP) has emerged as a drug target for neurological diseases. A flexible loop structure comprising loop A (res. 189-209) and loop B (res. 577-608) at the domain interface is implicated in substrate entry to the active site. Here we determined the kinetic and structural properties of POP with mutations in loop A, loop B and in two additional flexible loops. POP lacking loop A proved to be an inefficient enzyme as did POP with a mutation in loop B (T590C). Both constructs displayed an altered substrate preference profile. Ligand binding become markedly degraded. Conversely, the T202C mutation increased the flexibility of loop A, enhancing the catalytic efficiency beyond that of the native enzyme. The T590C mutation in loop B increased the preference for shorter peptides, indicating a role in substrate gating. Loop A and the His loop housing the catalytic histidine are disordered in the H680A mutant crystal structure, implying coordinated structural dynamics of these loops. A 17-mer peptide could not inhibit variants possessing malfunctioning loop A. This substrate may bind non-productively to an exosite involving loop A or to an open enzyme form. Biophysical studies suggest that mammalian POP resides in a predominantly closed conformational state, especially at physiological conditions. The flexible loop A, loop B and His loop system at the active site is the main regulator of substrate gating and specificity and represents a new inhibitor target.

Prolyl Oligopeptidase Enhances α-Synuclein Dimerization via Direct Protein-Protein Interaction

“…Previously, it was demonstrated that PREP adopts three distinct conformations, a compact monomeric form, an open monomeric form, and an oligomeric form, and that KYP-2047 has a prominent effect on the conformational state of PREP (29,52,53). Our data confirm that in the presence of KYP-2047, the majority of PREP oligomers adopt the compact monomeric form.…”

“…In living cells, KYP-2047 does not abolish the physical interaction between aSyn and PREP, as we hypothesized earlier (22), but changes its characteristics so that the stimulating effect on aSyn oligomerization is decreased. These observations are in line with earlier studies that indicated that PREP acts on the nucleation process of aSyn aggregation and fibril formation and that the conformational state of PREP is important for its functions inside the cell (14,29,54). However, whether the increased PCA signal between PREP and aSyn in KYP-2047-treated cells reflects higher affinity of inhibitor-bound monomeric PREP toward aSyn remains to be determined in future studies.…”

“…Inactive PREP Mutant-It has been previously shown that KYP-2047 binding modifies conformation of PREP (29). Our data suggested that KYP-2047 binds both to PREP and PREP(S554A) but showed a decrease in aSyn reporter dimerization in PCA only when incubated with active PREP.…”

“…One possibility could be the access loop structures of PREP as suggested earlier in the literature (29,55). These structures contain three loops (A, B, and C), of which loop A seems to be the most important for substrate gating, and it changes its conformation to a substrateinaccessible position in the presence of an inhibitor (55).…”

“…Native Polyacrylamide Gel Electrophoresis (PAGE)-Native-PAGE was carried out as described earlier in Szeltner et al (29) by leaving out SDS from the PAGE-gel, sample buffer (BioRad), and Tris-glycine running buffer. 6 g of purified recombinant PREP or PREP(S554A) proteins were incubated with a 30-fold molar excess of KYP-2047 or vehicle (0.15% DMSO in PBS) for 30 min at room temperature.…”