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

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. Dumitru et al. probe the ligand-binding mechanism and activation of the human G protein-coupled C5a receptor (C5aR) and discover a cooperativity between the two orthosteric binding sites. Their findings, probing the dynamic aspects of receptor-ligand interaction, are valuable to develop a better understanding of GPCR activation and signalling.