The human CC chemokine receptor 5 (CCR5) is a G protein–coupled receptor (GPCR) that plays a major role in inflammation and is involved in cancer, HIV, and COVID-19. Despite its importance as a drug target, the molecular activation mechanism of CCR5, i.e., how chemokine agonists transduce the activation signal through the receptor, is yet unknown. Here, we report the cryo-EM structure of wild-type CCR5 in an active conformation bound to the chemokine super-agonist [6P4]CCL5 and the heterotrimeric Gi protein. The structure provides the rationale for the sequence-activity relation of agonist and antagonist chemokines. The N terminus of agonist chemokines pushes onto specific structural motifs at the bottom of the orthosteric pocket that activate the canonical GPCR microswitch network. This activation mechanism differs substantially from other CC chemokine receptors that bind chemokines with shorter N termini in a shallow binding mode involving unique sequence signatures and a specialized activation mechanism.

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The two non-visual arrestin isoforms, arrestin2 and arrestin3 recognize and bind hundreds of G protein-coupled receptors (GPCRs) with different phosphorylation patterns leading to distinct functional outcomes. The impact of phosphorylation on arrestin interactions has been well studied only for very few GPCRs. Here we have characterized the interactions between the phosphorylated CC chemokine receptor 5 (CCR5) and arrestin2. We detected several new CCR5 phosphorylation sites, which are necessary for stable complex formation with arrestin2. Crystal structures of arrestin2 in apo form and in complexes with CCR5 C-terminal phosphopeptides together with NMR spectroscopy, biochemical and functional assays revealed three phosphoresidues in a pXpp motif that are essential for the arrestin2 interactions and activation. The same phosphoresidue cluster is present in other receptors, which form stable complexes with arrestin2. We propose that the identified pXpp motif is responsible for robust arrestin2 recruitment in many GPCRs. An analysis of available sequences, structural and functional information on other GPCR-arrestin interactions suggests that a particular arrangement of phosphoresidues within the GPCR intracellular loop 3 and C-terminal tail determines arrestin2 and 3 isoform specificity. Taken together, our findings demonstrate how multi-site phosphorylation controls GPCR-arrestin interactions and provide a framework to probe the intricate details of arrestin activation and signaling.

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The human CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor (GPCR) that plays a major role in inflammation and is involved in the pathology of cancer, HIV, and COVID-19. Despite its significance as a drug target, the activation mechanism of CCR5, i.e. how chemokine agonists transduce the activation signal through the receptor, is yet unknown. Here, we report the cryo-EM structure of wild-type CCR5 in an active conformation bound to the chemokine super-agonist [6P4]CCL5 and the heterotrimeric Gi protein. The structure provides the rationale for the sequence-activity relation of agonist and antagonist chemokines. The N-terminus of agonist chemokines pushes onto an aromatic connector that transmits activation to the canonical GPCR microswitch network. This activation mechanism differs significantly from other CC chemokine receptors that bind shorter chemokines in a shallow binding mode and have unique sequence signatures and a specialized activation mechanism.One-sentence summaryThe structure of CCR5 in complex with the chemokine agonist [6P4]CCL5 and the heterotrimeric Gi protein reveals its activation mechanism