Molecular mechanism of distinct chemokine engagement and functional divergence of the human Duffy antigen receptor.

The Duffy antigen receptor is a seven-transmembrane (7TM) protein expressed primarily at the surface of red blood cells and displays strikingly promiscuous binding to multiple inflammatory and homeostatic chemokines. It serves as the basis of the Duffy blood group system in humans and also acts as the primary attachment site for malarial parasite Plasmodium vivax and pore-forming toxins secreted by Staphylococcus aureus. Here, we comprehensively profile transducer coupling of this receptor, discover potential non-canonical signaling pathways, and determine the cryoelectron microscopy (cryo-EM) structure in complex with the chemokine CCL7. The structure reveals a distinct binding mode of chemokines, as reflected by relatively superficial binding and a partially formed orthosteric binding pocket. We also observe a dramatic shortening of TM5 and 6 on the intracellular side, which precludes the formation of the docking site for canonical signal transducers, thereby providing a possible explanation for the distinct pharmacological and functional phenotype of this receptor.

Molecular basis of anaphylatoxin binding, activation, and signaling bias at complement receptors.

The complement system is a critical part of our innate immune response, and the terminal products of this cascade, anaphylatoxins C3a and C5a, exert their physiological and pathophysiological responses primarily via two GPCRs, C3aR and C5aR1. However, the molecular mechanism of ligand recognition, activation, and signaling bias of these receptors remains mostly elusive. Here, we present nine cryo-EM structures of C3aR and C5aR1 activated by their natural and synthetic agonists, which reveal distinct binding pocket topologies of complement anaphylatoxins and provide key insights into receptor activation and transducer coupling. We also uncover the structural basis of a naturally occurring mechanism to dampen the inflammatory response of C5a via proteolytic cleavage of the terminal arginine and the G-protein signaling bias elicited by a peptide agonist of C3aR identified here. In summary, our study elucidates the innerworkings of the complement anaphylatoxin receptors and should facilitate structure-guided drug discovery to target these receptors in a spectrum of disorders.

Structural snapshots uncover a key phosphorylation motif in GPCRs driving ß-arrestin activation.

Agonist-induced GPCR phosphorylation is a key determinant for the binding and activation of ß-arrestins (ßarrs). However, it is not entirely clear how different GPCRs harboring divergent phosphorylation patterns impart converging active conformation on ßarrs leading to broadly conserved functional responses such as desensitization, endocytosis, and signaling. Here, we present multiple cryo-EM structures of activated ßarrs in complex with distinct phosphorylation patterns derived from the carboxyl terminus of different GPCRs. These structures help identify a P-X-P-P type phosphorylation motif in GPCRs that interacts with a spatially organized K-K-R-R-K-K sequence in the N-domain of ßarrs. Sequence analysis of the human GPCRome reveals the presence of this phosphorylation pattern in a large number of receptors, and its contribution in ßarr activation is demonstrated by targeted mutagenesis experiments combined with an intrabody-based conformational sensor. Taken together, our findings provide important structural insights into the ability of distinct GPCRs to activate ßarrs through a significantly conserved mechanism.

In-cellulo chemical cross-linking to visualize protein-protein interactions.

Reversible protein-protein interaction in cells is an integral and central aspect of intracellular signaling mechanisms. This allows distinct signaling cascades to become active upon stimulation with external signal resulting in cellular and physiological responses. Several distinct methods are currently available and utilized routinely to monitor protein-protein interactions including co-immunoprecipitation (co-IP). An inherent limitation associated with co-IP assay however is the inability to efficiently capture transient and short-lived interactions in cells. Chemical cross-linking of such transient interactions in cellular context using cell permeable reagents followed by co-IP overcomes this limitation, and allows a simplified approach without requiring any sophisticated instrumentation. In this chapter, we present a step-by-step protocol for monitoring protein-protein interaction by combining chemical cross-linking and co-immunoprecipitation using GPCR-ß-arrestin complex as a case example. This protocol is based on previously validated method that can potentially be adapted to capture and visualize transient protein-protein interactions in general.

Making the switch: The role of Gq in driving GRK selectivity at GPCRs.

Selective engagement of signal transducers such as G proteins and ß-arrestins with GPCRs upon stimulation with biased agonists is thought to be due to distinct receptor conformations. Kawakami et al. propose an additional mechanism whereby activation of Gq determines GPCR kinase subtype selectivity to the activated angiotensin receptor, leading to distinct binding modalities of ß-arrestins and functional outcomes.

Transmitting the Signal: Structure of the ß1-Adrenergic Receptor-Gs Protein Complex.

In this issue of Molecular Cell, Su et al. (2020) report a cryo-EM structure of the ß1-adrenergic receptor (ß1AR) in complex with a heterotrimeric Gs protein, which offers novel insights into receptor activation and provides a structural framework to better understand the transducer-coupling mechanism for adrenergic receptors.