Active‐Site‐Directed Inhibitors of Prolyl Oligopeptidase Abolish Its Conformational Dynamics

Abstract

Deciphering conformational dynamics is crucial for understanding the biological functions of proteins and for designing compounds targeting them. In particular, providing an accurate description of µs-ms motions opens up the opportunity for regulating protein-protein interactions (PPIs) by modulating dynamics of one interacting partner. Here we analyzed the conformational dynamics of prolyl oligopeptidase (POP) and the effects of active site-directed inhibitors on the dynamics. For this purpose, we used an integrated structural biology approach based on NMR spectroscopy and SAXS experiments complemented by MD simulations. Our results found that POP is in a slow equilibrium in solution between open and closed conformations, and that inhibitors effectively prevented this equilibrium by stabilizing the enzyme in a closed conformation.

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

“…These are clustered in one side of the PREP interface, spanning from the blade 1 loop of the β-propeller and the His-loop of the α/β hydrolase domain to the α/β hydrolase helix-turn-helix containing Glu512 (Figs 4 and S5). The observed differences also agree with indole and methyl-labeled NMR studies, respectively showing that the inhibitor strongly affects Trp514 and methioninyl residues 235, 581 and 583 11, 12 , though HDX-MS provided a complete view of the inhibitor effect.…”

“…We now show that the previously unsuspected, His-loop connected α-helix and the blade 1 loop, are major regulatory elements involved in inter-domain closure that stabilizes the hinge out . Whether an induced fit mechanism 10 or conformational selection 12 is involved in gating cannot be concluded from our findings. The high content of fast-exchanging inter-domain loops results in poor reporting of transient closed states in the free enzyme.…”

“…PREP may exist in equilibrium between open and closed states related to its gating mechanism 12, 19 . Native gel electrophoresis revealed that the free enzyme populates at least two conformational species that migrate distinctly (Fig.…”

“…NMR and SAXS experiments have proposed that the free enzyme populates an open and closed conformation (approximately 55% open, 45% closed), an equilibrium that is shifted towards the closed conformation when inhibitor is bound 12 . While the closed state is well understood and structurally resolved, the open conformation is elusive.…”

“…On the other hand, there is experimental evidence that mammalian free PREP is distributed between different conformations and that ligand binding shifts this equilibrium to a single state, i.e. conformational selection 11, 12 . 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 .…”