Structural basis of ligand interaction with atypical chemokine receptor 3

Martin Gustavsson,Thomas J Schall,Ruben Abagyan,Sichun Yang,Mark R Chance,Tracy M Handel,Noortje Van Gils,Irina Kufareva,Penglie Zhang,Bryan S Stephens,Liwen Wang

doi:10.1038/ncomms14135

Abstract

Chemokines drive cell migration through their interactions with seven-transmembrane (7TM) chemokine receptors on cell surfaces. The atypical chemokine receptor 3 (ACKR3) binds chemokines CXCL11 and CXCL12 and signals exclusively through β-arrestin-mediated pathways, without activating canonical G-protein signalling. This receptor is upregulated in numerous cancers making it a potential drug target. Here we collected over 100 distinct structural probes from radiolytic footprinting, disulfide trapping, and mutagenesis to map the structures of ACKR3:CXCL12 and ACKR3:small-molecule complexes, including dynamic regions that proved unresolvable by X-ray crystallography in homologous receptors. The data are integrated with molecular modelling to produce complete and cohesive experimentally driven models that confirm and expand on the existing knowledge of the architecture of receptor:chemokine and receptor:small-molecule complexes. Additionally, we detected and characterized ligand-induced conformational changes in the transmembrane and intracellular regions of ACKR3 that elucidate fundamental structural elements of agonism in this atypical receptor.

Highlights

Chemokines drive cell migration through their interactions with seven-transmembrane (7TM) chemokine receptors on cell surfaces
atypical chemokine receptor 3 (ACKR3) fused to Renilla luciferase 3 (ACKR3-Rluc3) was transiently transfected into HEK293 cells stably expressing b-arrestin-2 fused to green fluorescent protein 10 (GFP10) and recruitment of b-arrestin-2 was measured as an increase in bioluminescence resonance energy transfer (BRET) after stimulation with ligand
In this study, we investigate the structural basis of the interaction between ACKR3 and its endogenous chemokine agonist, CXCL12, as well as with the small-molecule partial agonist, CCX777, using a synergistic combination of radiolytic footprinting, disulfide trapping, mutagenesis, functional experiments and molecular modelling

Summary

Introduction

Chemokines drive cell migration through their interactions with seven-transmembrane (7TM) chemokine receptors on cell surfaces. The atypical chemokine receptor 3 (ACKR3) binds chemokines CXCL11 and CXCL12 and signals exclusively through b-arrestin-mediated pathways, without activating canonical G-protein signalling This receptor is upregulated in numerous cancers making it a potential drug target. In addition to its unusual signalling properties, ACKR3 acts as a scavenger of extracellular CXCL12 to establish chemokine levels that maintain cellular responsiveness by preventing excessive desensitization and downregulation of CXCR4 In this context, ACKR3-expressing cells in a primary breast tumor enhanced the metastasis of CXCR4-expressing breast cancer cells[9]. Pharmacological inhibition of ACKR3 has been shown to cause pronounced increases in plasma CXCL12 levels[9] with associated impairment of leukocyte migration towards CXCL12, likely because of CXCR4 downregulation These and other studies suggest that ACKR3 may be a good therapeutic target for cancer[12]. We utilize experimentally guided homology modelling to produce models of ACKR3:CXCL12 and ACKR3:CCX777 complexes that reveal insights into receptor:ligand recognition and may guide drug discovery efforts

Methods

Results

Conclusion