NMR mapping of RANTES surfaces interacting with CCR5 using linked extracellular domains

Abstract

Chemokines constitute a large family of small proteins that regulate leukocyte trafficking to the site of inflammation by binding to specific cell-surface receptors belonging to the GPCR superfamily. The interactions between N-terminal (Nt-) peptides of these GPCRs and chemokines have been studied extensively using NMR spectroscopy. However, due to lower affinities of peptides representing the three extracellular loops (ECLs) of chemokine receptors to their respective chemokine ligands, information concerning these interactions is scarce. To overcome the low affinity of ECL peptides to chemokines, we linked two or three CCR5 extracellular domains by either biosynthesis in Escherichia coli or by chemical synthesis. Using such chimeras, CCR5 binding to RANTES was followed using 1H-15N-HSQC spectra to monitor titration of the chemokine with peptides corresponding to the extracellular surface of the receptor. Nt-CCR5 and ECL2 were found to be the major contributors to CCR5 binding to RANTES, creating a nearly closed ring around this protein by interacting with opposing faces of the chemokine. A RANTES positively charged surface involved in Nt-CCR5 binding resembles the positively charged surface in HIV-1 gp120 formed by the C4 and the base of the V3. The opposing surface on RANTES, composed primarily of β2-β3 hairpin residues, binds ECL2 and was found to be analogous to a surface in the crown of the gp120 V3. The chemical and biosynthetic approaches for linking GPCR surface regions discussed herein should be widely applicable to investigation of interactions of extracellular segments of chemokine receptors with their respective ligands.

Structural Basis of Receptor Sulfotyrosine Recognition by a CC Chemokine: The N-Terminal Region of CCR3 Bound to CCL11/Eotaxin-1

“…Consequently, if CCL11, CXCL8, and CXCL12 bind to their receptor N termini in the same manner as observed in their respective peptide complexes, the three chemokines would assume distinct orientations relative to the transmembrane helical bundles of their receptors, allowing them to engage different regions of the receptors and induce distinct conformational changes, as represented by the models in Figure 5C. Notably, the orientation of CCL11 shown in Figure 5C is consistent with the data of Schnur and colleagues, which suggested that the receptor ECL2 element interacts with the opposite face of the chemokine from the receptor N terminus (Schnur et al, 2013). At this stage, it would be speculative to propose the details of the distinct interactions formed by different chemokine:receptor pairs.…”

“…Previous NMR studies have shown that sulfotyrosine-containing N-terminal peptides derived from two other CC chemokine receptors, CCR2 and CCR5, also bind to the N-loop/b2-b3 regions of cognate chemokines (Duma et al, 2007;Schnur et al, 2013;Tan et al, 2013a). Moreover, the recognition residues in the CCL11:Su1617 structure are also highly conserved in CC chemokines that recognize CCR2 and CCR5 ( Figure 3E), suggesting that the interactions observed in the current structure are conserved across the CC chemokine family.…”

Interactions of the Chemokine CCL5/RANTES with Medium-Sized Chondroitin Sulfate Ligands

“…This result is consistent with previous in vitro studies that utilized sulfated peptides derived from the N-terminus of CCR5, which showed residues in both the N-loop (including R17) and the BBXB motif were perturbed by the sulfated peptide. In fact, the chemical shift perturbations that were observed for the N-loop in these studies were larger than those observed for the residues in the 40s loop (Schnur et al, 2013). Comparisons of the vMIP-II-CXCR4 complex structure and the CCR5 N-terminus-bound vMIP-II model with the current CS-bound CCL5 show the models share a good deal of similarity (Figure 9).…”

“…To investigate the role of CCL5 dimerization, we capitalized on the fact that low concentrations (< 50 µM) of E66S CCL5 exists as discrete populations of monomer and dimers at 40 °C (Schnur et al, 2013) and investigated whether CS444 is capable of promoting dimerization of E66S. In the absence of CS444, the ratio of dimer-to-monomer in a 30 µM E66S CCL5 sample is approximately 1:1 at 40 °C (Figure S4).…”

Chemokine-Receptor Interactions: Solving the Puzzle, Piece by Piece

“…More recent work also recognizes the importance of sulfated tyrosines on the receptors (Choe and Farzan, 2009). Nuclear magnetic resonance (NMR) has been a particularly useful technique in this endeavor, providing information about likely binding sites on the chemokines for the receptor peptides (Duma et al, 2007;Schnur et al, 2013) as well as specific inter-protein contacts, allowing previous NMR structure determinations for CXC subfamily chemokines CXCL8 (interleukin 8 ) and CXCL12 (SDF-1) with a peptide from their respective receptors (Skelton et al, 1999, Veldkamp et al, 2008.…”