Identification and mechanism of G protein-biased ligands for chemokine receptor CCR1

Abstract

Biased signaling of G protein-coupled receptors describes an ability of different ligands that preferentially activate an alternative downstream signaling pathway. In this work, we identified and characterized different N-terminal truncations of endogenous chemokine CCL15 as balanced or biased agonists targeting CCR1, and presented three cryogenic-electron microscopy structures of the CCR1–Gi complex in the ligand-free form or bound to different CCL15 truncations with a resolution of 2.6–2.9 Å, illustrating the structural basis of natural biased signaling that initiates an inflammation response. Complemented with pharmacological and computational studies, these structures revealed it was the conformational change of Tyr291 (Y2917.43) in CCR1 that triggered its polar network rearrangement in the orthosteric binding pocket and allosterically regulated the activation of β-arrestin signaling. Our structure of CCL15-bound CCR1 also exhibited a critical site for ligand binding distinct from many other chemokine–receptor complexes, providing new insights into the mode of chemokine recognition.

Molecular insights into ligand recognition and activation of chemokine receptors CCR2 and CCR3

“…The most N-terminal residues of CCL2 and CCL20 both function as a determinant in the chemokine recognition and activation of their receptors 11 . However, the ligands of CCR1 and CCR5 display diversity in the N-terminal segment proceeding the first two cysteines, but exhibited evident similarity within the core region 12 , 13 .…”

“…In this study, the dose-response curve of cAMP accumulation was performed as previously described 13 . For the measurement of constitutive activity, we fused a FLAG-tag into the N-terminus of receptors, and cloned it into pcDNA3.1 plasmids.…”

“…Particle selections for two-dimensional (2D) and 3D classifications were performed on a binned dataset using RELION-3.0-beta2 43 . For the CCL2–CCR2–G i complex, 1,899,329 particles yielded by automated particle picking were subjected to 2D classification, and two rounds of 3D classification using the CCL15–CCR1–G i complex low-pass filtered map as an initial reference model, resulting in a well-defined subset with 529,703 particles 13 . The selected subsets were subsequently subjected to 3D classification with a mask on the receptor or receptor–G i complex, respectively.…”

“…Among them, the CC chemokine receptors form the largest subfamily of chemokine receptors and exhibit bidirectional promiscuity with their ligands: some chemokines can activate more than one subtypes of chemokine receptors, and many chemokine receptors can be activated by more than one chemokine 7 , 8 . Although significant progress has been made in structural studies of chemokine receptors in their active states, the molecular mechanisms underlying the activation and ligand recognition of CC chemokine receptors remain elusive 9 – 13 .…”

“…So far, only a few three-dimensional (3D) structures of CC chemokine receptors with their endogenous ligands have been determined: CCR1 in complex with CCL15; CCR5 in complex with CCL3 or CCL5; and CCR6 in complex with CCL20 11 – 13 . Although the receptors mentioned above belong to the same subfamily of chemokine receptors, the molecular mechanisms underlying the chemokine recognition, constitutive, and ligand-induced activation seems variable, which forms a complex regulatory network for the fine-tuning of chemokine-induced physiological responses.…”