These results suggest a different interaction mechanism between this atypical receptor and its ligands and illustrate its strong propensity to activation.

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The chemokine receptor CXCR4 interacts with a single endogenous chemokine, CXCL12, and regulates a wide variety of physiological and pathological processes including inflammation and metastasis development. CXCR4 also binds the HIV-1 envelope glycoprotein, gp120, resulting in viral entry into host cells. Therefore, CXCR4 and its ligands represent valuable drug targets. In this study, we investigated the inhibitory properties of synthetic peptides derived from CXCR4 extracellular loops (ECL1-X4, ECL2-X4 and ECL3-X4) towards HIV-1 infection and CXCL12-mediated receptor activation. Among these peptides, ECL1-X4 displayed anti-HIV-1 activity against X4, R5/X4 and R5 viruses (IC50=24 to 76μM) in cell viability assay without impairing physiological CXCR4-CXCL12 signalling. In contrast, ECL2-X4 only inhibited X4 and R5/X4 strains, interfering with HIV-entry into cells. At the same time, ECL2-X4 strongly and specifically interacted with CXCL12, blocking its binding to CXCR4 and its second receptor, CXCR7 (IC50=20 and 100μM). Further analysis using mutated and truncated peptides showed that ECL2 of CXCR4 forms multiple contacts with the gp120 protein and the N-terminus of CXCL12. Chemokine neutralisation was mainly driven by four aspartates and the C-terminal residues of ECL2-X4. These results demonstrate that ECL2 represents an important structural determinant in CXCR4 activation. We identified the putative site for the binding of CXCL12 N-terminus and provided new structural elements to explain the recognition of gp120 and dimeric CXCR4 ligands.

The atypical chemokine receptor ACKR3/CXCR7 plays crucial roles in numerous physiological processes but also in viral infection and cancer. ACKR3 shows strong propensity for activation and, unlike classical chemokine receptors, can respond to chemokines from both the CXC and CC families as well as to the endogenous peptides BAM22 and adrenomedullin. Moreover, despite belonging to the G protein coupled receptor family, its function appears to be mainly dependent on β-arrestin. ACKR3 has also been shown to continuously cycle between the plasma membrane and the endosomal compartments, suggesting a possible role as a scavenging receptor. So far, the molecular basis accounting for these atypical binding and signalling properties remains elusive. Noteworthy, ACKR3 extracellular domains bear three disulphide bridges. Two of them lie on top of the two main binding subpockets and are conserved among chemokine receptors, and one, specific to ACKR3, forms an intra-N terminus four-residue-loop of so far unknown function. Here, by mutational and functional studies, we examined the impact of the different disulphide bridges for ACKR3 folding, ligand binding and activation. We showed that, in contrast to most classical chemokine receptors, none of the extracellular disulphide bridges was essential for ACKR3 function. However, the disruption of the unique ACKR3 N-terminal loop drastically reduced the binding of CC chemokines whereas it only had a mild impact on CXC chemokine binding. Mutagenesis also uncovered that chemokine and endogenous non-chemokine ligands interact and activate ACKR3 according to distinct binding modes characterized by different transmembrane domain subpocket occupancy and N-terminal loop contribution, with BAM22 mimicking the binding mode of CC chemokine N terminus.

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