The chemokine X-factor: Structure-function analysis of the CXC motif at CXCR4 and ACKR3

Abstract

The human chemokine family consists of 46 protein ligands that induce chemotactic cell migration by activating a family of 23 G protein-coupled receptors. The two major chemokine subfamilies, CC and CXC, bind distinct receptor subsets. A sequence motif defining these families, the X position in the CXC motif, is not predicted to make significant contacts with the receptor, but instead links structural elements associated with binding and activation. Here we use comparative analysis of chemokine NMR structures, structural modeling, and molecular dynamic simulations that suggested the X position reorients the chemokine N-terminus. Using CXCL12 as a model CXC chemokine, deletion of the X residue (P10) had little to no impact on the folded chemokine structure but diminished CXCR4 agonist activity as measured by ERK phosphorylation, chemotaxis, and Gi/o-mediated cAMP inhibition. Functional impairment was attributed to over 100-fold loss of CXCR4 binding affinity. Binding to the other CXCL12 receptor, ACKR3, was diminished nearly 500-fold. Deletion of P10 had little effect on CXCL12 binding to the CXCR4 N-terminus, a major component of the chemokine-GPCR interface. Replacement of the X residue with the most frequent amino acid at this position (P10Q) had an intermediate effect between wild-type and P10del in each assay, with ACKR3 having a higher tolerance for this mutation. This work shows that the X residue helps to position the CXCL12 N-terminus for optimal docking into the orthosteric pocket of CXCR4 and suggests that the CC/CXC motif contributes directly to receptor selectivity by orienting the chemokine N-terminus in a subfamily-specific direction.

The chemokines CXCL8 and CXCL12: molecular and functional properties, role in disease and efforts towards pharmacological intervention

“…Moreover, within CXCL12, Pro10 (the “X” in the CXC motif) was found to be crucial to exert efficient CXCR4 binding and agonist activity whereas deletion of this Pro residue had negligible effect on binding to the CXCR4 NH 2 -terminus only. Probably, the Pro10 residue optimally positions the CXCL12 NH 2 -terminus for efficient docking into the receptor pocket and activation of CXCR4 [ 225 ].…”

Gene Structure Analysis of Chemokines and Their Receptors in Allotetraploid Frog, Xenopus laevis

“…Among cxcl8 homeologous genes, cxcl8b.1 were distinct in sequence homology (68%) and expression patterns, with L dominant in embryos and S dominant in tissues ( Table 1 , Figures 5 , 6 ). Since the X residue of the CXC motif has been noted for altering the binding ability to the receptor ( Wedemeyer et al, 2020 ), we examined that, of the cxcl8 chemokine family, four cxcl8 homologs between X. laevis and X. tropicalis were conserved as CQC in cxcl8b.2 and cxcl2 , and CLC in cxcl8a.2 and cxcl8a.1 homologous genes. However, cxcl8b.1 homologs presented unique sequences as CKC in cxcl8b.1 , CRC in cxcl8b.1.L , and CQC in cxcl8b.1.S , respectively.…”

The Chemokine, CCL20, and Its Receptor, CCR6, in the Pathogenesis and Treatment of Psoriasis and Psoriatic Arthritis

“…Most chemokines have an N-terminal tail of 8 or more residues, with an average length of 10 residues (Figure 1A), 40 and alterations in the length or amino acid composition reduce or abrogate chemokine activity. 41 In contrast, the CCL20 N-terminus consists of only five amino acids (NH2-ASNFD CC ) (Figure 1B).…”