Structural basis for chemokine recognition by a G protein–coupled receptor and implications for receptor activation

Abstract

Chemokines orchestrate cell migration for development, immune surveillance, and disease by binding to cell surface heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs). The array of interactions between the nearly 50 chemokines and their 20 GPCR targets generates an extensive signaling network to which promiscuity and biased agonism add further complexity. The receptor CXCR4 recognizes both monomeric and dimeric forms of the chemokine CXCL12, which is a distinct example of ligand bias in the chemokine family. We demonstrated that a constitutively monomeric CXCL12 variant reproduced the G protein–dependent and β-arrestin–dependent responses that are associated with normal CXCR4 signaling and lead to cell migration. In addition, monomeric CXCL12 made specific contacts with CXCR4 that are not present in the structure of the receptor in complex with a dimeric form of CXCL12, a biased agonist that stimulates only G protein–dependent signaling. We produced an experimentally validated model of an agonist-bound chemokine receptor that merged a nuclear magnetic resonance–based structure of monomeric CXCL12 bound to the amino terminus of CXCR4 with a crystal structure of the transmembrane domains of CXCR4. The large CXCL12:CXCR4 protein-protein interface revealed by this structure identified previously uncharacterized functional interactions that fall outside of the classical “two-site model” for chemokine-receptor recognition. Our model suggests a mechanistic hypothesis for how interactions on the extracellular face of the receptor may stimulate the conformational changes required for chemokine receptor-mediated signal transduction.

The solution structure of monomeric CCL5 in complex with a doubly sulfated N‐terminal segment of CCR5

“…NOESY and TOCSY spectra were recorded with 13 C and 15 N filters in both F1 and F2 dimensions, to eliminate contributions of CCL5(P9S) and to assign the resonances of the Nt-CCR5(1-27) peptide, under conditions detailed above. Nearly complete backbone and side-chain assignments were obtained for the receptor N-terminal peptide.…”

“…The generality of the above types of chemokine receptor contacts is also seen in the CXC family. Despite sequence and structural divergence, the chemokines of the Nt‐CXCR1:CXCL8 and both Nt‐CXCR4:CXCL12 complexes solved previously have an arginine at position 47 interacting either with a tyrosine residue of their respective receptors— LD CXCR4‐NT sY21, LM CXCR4‐NT Y21, and CXCR1‐NT Y2—or an immediately adjacent aspartate (equivalent to CCR5 D11). Although, in these experiments, only the peptide used to determine the structure of Nt‐CXCR4 bound to dimeric (LD) CXCL12 was sulfated, CXCR1‐NT Y2 is flanked by negatively charged amino acids, a motif used to predict sulfation sites .…”

“…Based on the backbone and side-chain assignments of CCL5(P9S) in the presence of excess doubly sulfated Nt-CCR5(1-27), we obtained a total of 1385 intramolecular NOEs from 15 N-separated NOESY and 13 C-separated NOESY spectra using CYANA [27]. Of these, 199 belonged to Nt-CCR5(1-27), while the remaining 1186 belonged to CCL5(P9S).…”

Characterization of a chimeric chemokine as a specific ligand for ACKR3

“…In solution, CXCL12 can form dimers depending on the environmental conditions, such as pH and heparin . Monomeric and dimeric forms of CXCL12 were shown to bind and to induce signal bias on CXCR4, where dimeric CXCL12 poorly recruits arrestin and essentially does not stimulate cell migration . ACKR3 binds preferentially monomeric CXCL12 with about 10‐fold higher affinity than CXCR4 (apparent K d for ACKR3 ∼ 0.3 nM) .…”

Decoding the chemotactic signal

“…Recent structural studies of chemokine–receptor interactions demonstrate an enormous ligand–receptor interface, spanning as much as 1700 Å 2 . Importantly, other recent studies suggest that differences in chemokine‐receptor interactions far from the traditional receptor binding pocket influence how a single receptor can mediate alternative functional responses . In one example, CXCL12 provokes and arrests chemotaxis in a monomeric and dimeric form, respectively, and structures of CXCL12 bound to its receptor CXCR4 in both forms demonstrate mutually exclusive interactions that may influence CXCL12's opposing effects on chemotaxis .…”