Abstract
Tyrosine sulfation, a post-translational modification found on many chemokine receptors, typically increases receptor affinity for the chemokine ligand. A previous bioinformatics analysis suggested that a sulfotyrosine (sY)-binding site on the surface of the chemokine CXCL12 may be conserved throughout the chemokine family. However, the extent to which receptor tyrosine sulfation contributes to chemokine binding has been examined in only a few instances. Computational solvent mapping correctly identified the conserved sulfotyrosine-binding sites on CXCL12 and CCL21 detected by nuclear magnetic resonance (NMR) spectroscopy, demonstrating its utility for hot spot analysis in the chemokine family. In this study, we analyzed five chemokines that bind to CXCR2, a subset of which also bind to CXCR1, to identify hot spots that could participate in receptor binding. A cleft containing the predicted sulfotyrosine-binding pocket was identified as a principal hot spot for ligand binding on the structures of CXCL1, CXCL2, CXCL7, and CXCL8, but not CXCL5. Sulfotyrosine titrations monitored via NMR spectroscopy showed specific binding to CXCL8, but not to CXCL5, which is consistent with the predictions from the computational solvent mapping. The lack of CXCL5–sulfotyrosine interaction and the presence of CXCL8–sulfotyrosine binding suggests a role for receptor post-translational modifications regulating ligand selectivity.
Highlights
Chemokines comprise a family of approximately 50 small globular proteins that coordinate the migration of immune cells along an increasing chemokine concentration gradient by activating specific G protein-coupled receptors (GPCRs) expressed on the surface of responding cells
Consistent with the computational hot spot analysis, we observed specific binding of sulfotyrosine to CXCL8, but not CXCL5, as monitored by 2D nuclear magnetic resonance (NMR). These findings suggest that receptor recognition by CXCL5 may differ from the other CXCR1/2 chemokine ligands, and that sulfotyrosine may help CXCR1 discriminate between CXCL5 and CXCL8
We analyzed each of the CXCR2 chemokine ligands for which a solved 3D structure was available, including all members of the NMR ensemble where applicable
Summary
Chemokines comprise a family of approximately 50 small globular proteins that coordinate the migration of immune cells along an increasing chemokine concentration gradient by activating specific G protein-coupled receptors (GPCRs) expressed on the surface of responding cells. Sulfotyrosine binding to CXCL12 induced chemical shifts in a subset of the residues that were perturbed upon the binding of CXCR4-derived sulfopeptides [15] Based on their location in the N-loop/β3 cleft, we concluded that the amino acid probe bound at or near the location of sulfotyrosine 21 in the structure of a CXCR4 sulfopeptide bound to CXCL12 [12]. Consistent with the computational hot spot analysis, we observed specific binding of sulfotyrosine to CXCL8, but not CXCL5, as monitored by 2D NMR These findings suggest that receptor recognition by CXCL5 may differ from the other CXCR1/2 chemokine ligands, and that sulfotyrosine may help CXCR1 discriminate between CXCL5 and CXCL8
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