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The Duffy antigen receptor, also known as FY glycoprotein or CD234, is a seven transmembrane protein expressed primarily at the surface of red blood cells, which displays promiscuous binding to multiple chemokines. Not only does it serve as the basis of the Duffy blood group system but it also acts as the primary attachment site for malarial parasite Plasmodium vivax on erythrocytes and as one of the nucleating receptors for the pore forming toxins secreted by Staphylococcus aureus. Despite a predicted 7TM architecture and efficient binding to a spectrum of chemokines, it fails to exhibit canonical second messenger response such as calcium release, likely due to a lack of G protein coupling. Unlike prototypical GPCRs and β-arrestin-biased atypical chemokine receptors, the Duffy antigen receptor also appears to lack β-arrestin binding, making it an enigmatic 7TM chemokine receptor. In order to decipher the molecular mechanism of this intriguing functional divergence exhibited by the Duffy antigen receptor, we have determined its cryo-EM structure in complex with a C-C type chemokine, CCL7. The structure reveals a relatively superficial binding mode of CCL7, with the N-terminus of the receptor serving as the key interaction interface, and a partially formed orthosteric binding pocket lacking the second site for chemokine recognition compared to prototypical chemokine receptors. The structural framework allows us to employ HDX-MS approach to uncover ligand-induced structural changes in the receptor and draw important insights into the promiscuous nature of chemokine binding. Interestingly, we also observe a dramatic shortening of TM5 and 6 on the intracellular side, compared to prototypical GPCRs, which precludes the coupling of canonical signal-transducers namely G proteins, GRKs and β-arrestins, as demonstrated through extensive cellular assays. Taken together, our study uncovers a previously unknown structural mechanism that imparts unique functional divergence on the 7TM fold encoded in the Duffy antigen receptor while maintaining its scavenging function and should facilitate the designing of novel therapeutics targeting this receptor.

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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.