Structural insights into ligand-recognition, activation, and signaling-bias at the complement C5a receptor, C5aR1

Abstract

Activation of the complement cascade is a critical part of our innate immune response against invading pathogens, and it operates in a concerted fashion with the antibodies and phagocytic cells towards the clearance of pathogens. The complement peptide C5a, generated during the activation of complement cascade, is a potent inflammatory molecule, and increased levels of C5a are implicated in multiple inflammatory disorders including the advanced stages of COVID-19 pathophysiology. The proximal step in C5a-mediated cellular and physiological responses is its interaction with two different seven transmembrane receptors (7TMRs) known as C5aR1 and C5aR2. Despite a large body of functional data on C5a-C5aR1 interaction, direct visualization of their interaction at high-resolution is still lacking, and it represents a significant knowledge gap in our current understanding of complement receptor activation and signaling. Here, we present cryo-EM structures of C5aR1 activated by its natural agonist C5a, and a G-protein-biased synthetic peptide ligand C5apep, in complex with heterotrimeric G-proteins. The C5a-C5aR1 structure reveals the ligand binding interface involving the N-terminus and extracellular loops of the receptor, and we observe that C5a exhibits a significant conformational change upon its interaction with the receptor compared to the basal conformation. On the other hand, the structural details of C5apep-C5aR1 complex provide a molecular basis to rationalize the ability of peptides, designed based on the carboxyl-terminus sequence of C5a, to act as potent agonists of the receptor, and also the mechanism underlying their biased agonism. In addition, these structural snapshots also reveal activation-associated conformational changes in C5aR1 including outward movement of TM6 and a dramatic rotation of helix 8, and the interaction interface for G-protein-coupling. In summary, this study provides previously lacking molecular basis for the complement C5a recognition and activation of C5aR1, and it should facilitate structure-based discovery of novel lead molecules to target C5aR1 in inflammatory disorders.

Structural insights into agonist-binding and activation of the human complement C3a receptor

“…In addition, we uncover the molecular mechanism that allows the carboxyl-terminal peptide fragments of C3a and C5a to bind and activate C3aR. Taken together with our accompanying manuscript 37 , we have visualized the structural basis of ligandrecognition and activation of two of the three complement receptors, which paves the way for structure-guided ligand discovery with subtype selective pharmacology and therapeutic potential in inflammatory conditions. Finally, similar to C5aR1, C3aR also exhibits efficient interaction with βarrs [9][10][11] , and therefore, it would be interesting to visualize C3a-C3aR-βarr complexes to better understand the details of transducer-coupling in the complement receptor system.…”

“…We have recently determined the structure of C5a-C5aR1-Gαoβ1γ2 complex by cryo-EM 37 . In order to identify potential commonalities and differences between C3a and C5a binding to their respective receptors, we compared the overall structures and agonist-binding modes in C3a-C3aR and C5a-C5aR1.…”

A genetically-encoded nanobody sensor reveals conformational diversity in β-arrestins orchestrated by distinct seven transmembrane receptors

“…NanoBiT assay to monitor GPCR-βarr interaction and Ib32/Ib30 reactivity was carried out following the previously described protocols 13,20,27,31,[45][46][47]51 . For the Ib32 reactivity assay described here for the first time, various combinations of LgBiT and SmBiT tagged at the Nterminus and C-terminus of ꞵarr1/2 and Ib32 were screened to obtain the optimal combination.…”

“…14486). C5a, C3a, CCL7, CXCL8, CXCL11, and CXCL12 were purified from the E. coli BL21(DE3) cells following the protocols described previously [49][50][51] .…”