Sulfopeptide probes of the CXCR4/CXCL12 interface reveal oligomer-specific contacts and chemokine allostery

Abstract

Tyrosine sulfation is a post-translational modification that enhances protein-protein interactions and may identify druggable sites in the extracellular space. The G protein-coupled receptor CXCR4 is a prototypical example with three potential sulfation sites at positions 7, 12, and 21. Each receptor sulfotyrosine participates in specific contacts with its chemokine ligand in the structure of a soluble, dimeric CXCL12:CXCR4(1-38) complex, but their relative importance for CXCR4 binding and activation by the monomeric chemokine remains undefined. NMR titrations with short sulfopeptides showed that the tyrosine motifs of CXCR4 varied widely in their contributions to CXCL12 binding affinity and site specificity. Whereas the Tyr21 sulfopeptide bound the same site as in previously solved structures, the Tyr7 and Tyr12 sulfopeptides interacted nonspecifically. Surprisingly, the unsulfated Tyr7 peptide occupied a hydrophobic site on the CXCL12 monomer that is inaccessible in the CXCL12 dimer. Functional analysis of CXCR4 mutants validated the relative importance of individual CXCR4 sulfotyrosine modifications (Tyr21 > Tyr12 > Tyr7) for CXCL12 binding and receptor activation. Biophysical measurements also revealed a cooperative relationship between sulfopeptide binding at the Tyr21 site and CXCL12 dimerization, the first example of allosteric behavior in a chemokine. Future ligands that occupy the sTyr21 recognition site may act as both competitive inhibitors of receptor binding and allosteric modulators of chemokine function. Together, our data suggests that sulfation does not ubiquitously enhance complex affinity and that distinct patterns of tyrosine sulfation could encode oligomer selectivity, implying another layer of regulation for chemokine signaling.

Figure 1. CXCL12₁ and CXCL12₂ have discrete CXCR4_1–38 binding sites

(A) The CXCL12 NMR structure (PDB ID 1SDF) solved in acetate (pH 4.9) was used to identify and model the I55C/L58C mutations for disulfide formation (yellow). Dimerization is inhibited when the helix is constrained to an acute angle relative to the β-sheet (33). Chemical shift perturbations (orange) produced by CXCR4_1–3 mapped onto (B) CXCL12₁ (PDB ID 1SDF) and (C) CXCL12₂ (PDB ID 2K01). Chemical shift perturbations unique to CXCL12₁ are highlighted in ruby. Both structures are rotated 180° relative to their respective ribbon representations. (D) CXCR4_1–38 induced chemical shift perturbations fitted to a quadratic binding equation resulted in CXCL12₁ and CXCL12₂ affinities of 3.5 ± 0.1 and 0.9 ± 0.3 µM, respectively.

Figure 2. Chemical shift perturbations of CXCR4 sulfopeptides designed from the extracellular N-terminus

(A) The residues corresponding to the sTyr7 (cyan), sTyr12 (wheat), and sTyr21 (green) heptapeptides are indicated on the CXCR4 N-terminus amino acid sequence. The previously defined positions of sTyr7 (B), sTyr12 (C), and sTyr21 (D) heptapeptides are reproduced from the CXCL12₂:CXCR4_1–38 NMR structure (PDB ID 2K05). Chemical shift perturbations induced by sTyr7 (E, left two panels), sTyr12 (F, left two panels), and sTyr21 (G, left two panels) sulfopeptides map to the Tyr21 pocket on CXCL12₂ (PDB 2K05). In contrast, the chemical shift perturbations identify distinct binding sites for the sTyr7 (E, right two panels) and sTyr21 (F, right two panels) sulfopeptides on CXCL12₁ (PDB 2K05). Non-specific binding was observed for the sTyr12 sulfopeptide (G, right two panels).

Figure 3. Sulfation of Tyr21 improves sulfopeptide binding affinity and modulates full-length receptor activity

(A) The largest chemical shift perturbations (orange) are consistent with the putative sTyr21 sulfopeptide (green) binding site on CXCL12₂ (PDB ID 2K05). (B) Chemical shift perturbations induced by sulfated and unsulfated peptides were fitted to a quadratic binding equation to yield K_d values. The sTyr21 sulfopeptide (circles) bound CXCL12_WT with K_d = 1.8 ± 0.2 mM (black), CXCL12₁ with K_d = 1.6 ± 0.2 mM (blue) and CXCL12₂ with K_d = 211 ± 23 µM (red). The Tyr21 peptide (triangles) bound CXCL12_WT with K_d = 2.7 ± 0.5 mM (black), CXCL12₁ with K_d = 1.5 ± 0.4 mM (blue) and CXCL12₂ with K_d = 831 ± 137 µM (red). (C) The calcium response of FLAG-tagged CXCR4 variants was measured as a function of CXCL12_WT concentration. Data are representative of two experiments each performed with three replicates. (D) Four parameter fits yielded each CXCR4 variants EC₅₀ and maximum calcium response. CXCR4 variants with EC₅₀ or maximum calcium response values more than three standard deviations from the mean CXCR4_WT quantities are indicated with an asterisk.

Figure 4. The sTyr21 binding site is allosterically linked to CXCL12 dimerization

(A) Intrinsic tryptophan fluorescence was used to calculate the CXCL12_WT dimerization affinity alone (circles; K_d = 15.1 ± 0.4 mM), in the presence of 50 mM sulfotyrosine (squares; K_d = 8.9 ± 1.8 mM), or in the presence of 3 mM sTyr21 sulfopeptide (diamonds; K_d = 2.6 ± 0.4 mM). (B) FP and NMR derived binding affinities were used to produce a thermodynamic cycle, which illustrates that CXCL12 dimerization and sTyr21 sulfopeptide binding are coupled. After one ligand has bound, the affinity for the second ligand is enhanced with a cooperativity factor (c).