Structural characterization of agonist binding to protease-activated receptor 2 through mutagenesis and computational modeling

Kennedy, A. J., Ballante, F., Johansson, J. R., Milligan, G. , Sunström, L., Nordqvist, A. and Carlsson, J. (2018) Structural characterization of agonist binding to protease-activated receptor 2 through mutagenesis and computational modeling. ACS Pharmacology and Translational Science, 1(2), pp. 119-133. (doi: 10.1021/acsptsci.8b00019)

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 higher functional potency than SLIGKV. The final model may be used to understand the structural basis of PAR2 activation and in virtual screens to identify novel PAR2 agonist 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.

• Item Type: Articles
• Additional Information: This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement: 715052). The work was also supported by grants from the Science for Life Laboratory and the Swedish Research Council (2017-4676) to J.C. The computations were performed on resources provided by the Swedish National Infrastructure for Computing (SNIC) at C3SE and NSC.
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Milligan, Professor Graeme
• Authors: Kennedy, A. J., Ballante, F., Johansson, J. R., Milligan, G., Sunström, L., Nordqvist, A., and Carlsson, J.
• College/School: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Journal Name: ACS Pharmacology and Translational Science
• Publisher: American Chemical Society
• ISSN: 2575-9108
• ISSN (Online): 2575-9108
• Published Online: 16 October 2018
• Copyright Holders: Copyright © 2018 American Chemical Society
• First Published: First published in ACS Pharmacology and Translational Science 1(2): 119-133
• Publisher Policy: Reproduced in accordance with the copyright policy of the publisher