Molecular basis of CXC chemokine receptor 3 ligand multispecificity

C-X-C motif chemokine receptor 3 (CXCR3) is essential for immune cell functions and pivotal in T helper 1 cell infiltration in autoimmune and chronic inflammatory diseases and in tumor proliferation and metastasis, but the mechanisms by which the endogenous ligands CXCL9, CXCL10, and CXCL11 differentially recognize and activate CXCR3 are not fully understood. Here, we present cryo-electron microscopy structures of all three chemokine-CXCR3-G_i complexes, complemented by cell binding studies and functional mutagenesis data. We systematically compare the pharmacological and interaction profiles of CXCL9, CXCL10, and CXCL11 to rationalize their varying efficacies and potencies and to reveal the critical role of the membrane-distal CXCR3 N terminus in ligand binding and signaling. Using chimeric chemokines and molecular dynamics, we reveal the signaling plasticity of chemokine ligands and signaling determinants. Together, these insights enable us to propose a multimodal binding and activation framework that explains CXCR3 chemokine ligand multispecificity and signaling versatility and offer tools to interrogate and modulate CXCR3 biology.