Structural Characterization of Agonist Binding to Protease-Activated Receptor 2 through Mutagenesis and Computational Modeling

Abstract

Protease-activated receptor 2 (PAR2) is a G-protein-coupled receptor that is activated by proteolytic cleavage of its N-terminus. The unmasked N-terminal peptide then binds to the transmembrane bundle, leading to activation of intracellular signaling pathways associated with inflammation and cancer. Recently determined crystal structures have revealed binding sites of PAR2 antagonists, but the binding mode of the peptide agonist remains unknown. In order to generate a model of PAR2 in complex with peptide SLIGKV, corresponding to the trypsin-exposed tethered ligand, the orthosteric binding site was probed by iterative combinations of receptor mutagenesis, agonist ligand modifications, and data-driven structural modeling. Flexible-receptor docking identified a conserved binding mode for agonists related to the endogenous ligand that was consistent with the experimental data and allowed synthesis of a novel peptide (1-benzyl-1H[1,2,3]triazole-4-yl-LIGKV) with functional potency higher than that of SLIGKV. The final model may be used to understand the structural basis of PAR2 activation and in virtual screens to identify novel agonists and competitive antagonists. The combined experimental and computational approach to characterize agonist binding to PAR2 can be extended to study the many other G protein-coupled receptors that recognize peptides or proteins.

Protease-activated receptor-2 ligands reveal orthosteric and allosteric mechanisms of receptor inhibition

“…AZ2429 was prepared using Ligprep 43 . Docking of AZ2429 was performed using Glide SP 44 to a refined PAR2 model previously described 26 .…”

“…However, the definition of the orthosteric site of the tethered ligand remains elusive due to the lack of an agonist-bound PAR2 crystal structure. More recently, with the AZ8838-bound structure as a starting point, we used an extensive combinatorial approach of site-directed mutagenesis and computational modelling to propose the putative orthosteric site 26 .…”

The development of proteinase-activated receptor-2 modulators and the challenges involved

“…This The non-peptide AZ3451 (PDB ID:5NDZ) occupies the allosteric pocket. While original data suggests AZ8838 occupies allosteric site [73,74], recent novel agonist-bound models propose AZ8838 binds to the orthosteric pocket [75] remains to be characterised. Visuals created using Mol* [80] with PDB IDs for 5NDZ, 5NDD and 5NJ6 [73].…”

“… The non-peptide AZ3451 (PDB ID:5NDZ) occupies the allosteric pocket. While original data suggests AZ8838 occupies allosteric site [ 73 , 74 ], recent novel agonist-bound models propose AZ8838 binds to the orthosteric pocket [ 75 ] (PAR2, PDB ID:5NDD). PAR2-Fab3949 (PDB ID: 5NJ6) engagement on the extracellular interface is presented.…”

“…The authors demonstrated that the antagonist AZ8838 binds in a fully occluded pocket near the extracellular surface. While the study by Cheng et al in 2017 proposed AZ8838 binding to an allosteric site [ 73 , 74 ], further investigation by Kennedy et al in 2018 using novel SLIGKV agonist binding models of PAR2 revealed AZ8838 occupancy in the orthosteric site [ 75 ]. Thus, AZ8838 is now proposed to serve as a competitive PAR2 antagonist.…”