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

Alexandra Tsirigotaki,Roos Van Elzen,Pieter Van Der Veken,Anne-Marie Lambeir,Anastassios Economou

doi:10.1038/s41598-017-02550-1

Alexandra Tsirigotaki, Roos Van Elzen + Show 3 more

Open Access

https://doi.org/10.1038/s41598-017-02550-1

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Abstract

Prolyl oligopeptidase (PREP) is conserved in many organisms across life. It is involved in numerous processes including brain function and neuropathology, that require more than its strict proteolytic role. It consists of a seven-bladed β-propeller juxtaposed to a catalytic α/β-hydrolase domain. The conformational dynamics of PREP involved in domain motions and the gating mechanism that allows substrate accessibility remain elusive. Here we used Hydrogen Deuterium eXchange Mass Spectrometry (HDX-MS) to derive the first near-residue resolution analysis of global PREP dynamics in the presence or absence of inhibitor bound in the active site. Clear roles are revealed for parts that would be critical for the activation mechanism. In the free state, the inter-domain interface is loose, providing access to the catalytic site. Inhibitor binding “locks” the two domains together exploiting prominent interactions between the loop of the first β-propeller blade and its proximal helix from the α/β-hydrolase domain. Loop A, thought to drive gating, is partially stabilized but remains flexible and dynamic. These findings provide a conformational guide for further dissection of the gating mechanism of PREP, that would impact drug development. Moreover, they offer a structural framework against which to study proteolysis-independent interactions with disordered proteins like α-synuclein involved in neurodegenerative disease.

Highlights

Prolyl oligopeptidase (PREP, EC 3.4.21.26) is a proline-specific serine endopeptidase, present in many organisms from all kingdoms of life[1]
Based on several different structures of the Aeromonas punctata PREP, an induced fit mechanism was proposed where PREP is in an open conformation, exposing the internal cavity to the solvent, and the catalytic site is assembled upon substrate binding leading to a closed conformation similar to the ones previously determined[10]
PREP may exist in equilibrium between open and closed states related to its gating mechanism[12, 19]

Summary

Introduction

Prolyl oligopeptidase (PREP, EC 3.4.21.26) is a proline-specific serine endopeptidase, present in many organisms from all kingdoms of life[1]. All mammalian PREP structures determined so far, in the free or inhibitor-bound states, are in a closed conformation in which the catalytic triad and the inhibitor/substrate binding site are buried in the inter-domain interface, surrounded by an extended network of hydrophobic contacts, hydrogen bonds and salt bridges between loops and turns from both domains. In this closed state PREP has a fairly substantial internal cavity that connects to external solvent by a narrow pore (~4 Å) in the β-propeller domain core[7,8,9], of insufficient width for substrate entry. 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 PREPs14

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