Submolecular probing of the complement C5a receptor\u2013ligand binding reveals a cooperative two-site binding mechanism

Andra C Dumitru,R N V Krishna Deepak,Melanie Koehler,Heng Liu,Hao Fan,Cheng Zhang,David Alsteens

doi:10.1038/s42003-020-01518-8

Abstract

A current challenge to produce effective therapeutics is to accurately determine the location of the ligand-biding site and to characterize its properties. So far, the mechanisms underlying the functional activation of cell surface receptors by ligands with a complex binding mechanism remain poorly understood due to a lack of suitable nanoscopic methods to study them in their native environment. Here, we elucidated the ligand-binding mechanism of the human G protein-coupled C5a receptor (C5aR). We discovered for the first time a cooperativity between the two orthosteric binding sites. We found that the N-terminus C5aR serves as a kinetic trap, while the transmembrane domain acts as the functional site and both contributes to the overall high-affinity interaction. In particular, Asp282 plays a key role in ligand binding thermodynamics, as revealed by atomic force microscopy and steered molecular dynamics simulation. Our findings provide a new structural basis for the functional and mechanistic understanding of the GPCR family that binds large macromolecular ligands.

Highlights

A current challenge to produce effective therapeutics is to accurately determine the location of the ligand-biding site and to characterize its properties
Together with steered molecular dynamics (SMD) simulation and functional assays, our observations point toward a cooperative model in which the binding site acts as a kinetic trap, effectively boosting local ligand concentration and promoting the interaction at the functionally relevant effector site
As predominant actors in cells, G-protein-coupled receptors (GPCRs) are intensively studied as drug targets, where, in particular, C5a receptor (C5aR) has long been suggested as a new promising anti-inflammatory target

Summary

Introduction

A current challenge to produce effective therapeutics is to accurately determine the location of the ligand-biding site and to characterize its properties. We found that the N-terminus C5aR serves as a kinetic trap, while the transmembrane domain acts as the functional site and both contributes to the overall high-affinity interaction. There is no molecular evidence of the kinetic and thermodynamic contributions of the different binding sites to the overall receptor–ligand binding It is still unclear whether the effector site and binding site are acting in concerted manner or separately. Understanding this process is likely to illuminate the binding paradigm common to members of the GPCR family that bind large macromolecular ligands. Together with steered molecular dynamics (SMD) simulation and functional assays, our observations point toward a cooperative model in which the binding site acts as a kinetic trap, effectively boosting local ligand concentration and promoting the interaction at the functionally relevant effector site. This study provides new insights on the role of positive allosteric interactions from a kinetic, thermodynamic, and functional point of view

Methods

Results

Conclusion