Structural basis of CXCR4 sulfotyrosine recognition by the chemokine SDF-1/CXCL12 - PubMed (original) (raw)

Structural basis of CXCR4 sulfotyrosine recognition by the chemokine SDF-1/CXCL12

Christopher T Veldkamp et al. Sci Signal. 2008.

Abstract

Stem cell homing and breast cancer metastasis are orchestrated by the chemokine stromal cell-derived factor 1 (SDF-1) and its receptor CXCR4. Here, we report the nuclear magnetic resonance structure of a constitutively dimeric SDF-1 in complex with a CXCR4 fragment that contains three sulfotyrosine residues important for a high-affinity ligand-receptor interaction. CXCR4 bridged the SDF-1 dimer interface so that sulfotyrosines sTyr7 and sTyr12 of CXCR4 occupied positively charged clefts on opposing chemokine subunits. Dimeric SDF-1 induced intracellular Ca2+ mobilization but had no chemotactic activity; instead, it prevented native SDF-1-induced chemotaxis, suggesting that it acted as a potent partial agonist. Our work elucidates the structural basis for sulfotyrosine recognition in the chemokine-receptor interaction and suggests a strategy for CXCR4-targeted drug development.

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Figures

Fig. 1

Fig. 1

The NMR structure of disulfide-locked SDF12. (A) The amino acid sequence of SDF12 with the conserved intramolecular disulfide bonds (black lines) and the engineered intermolecular disulfide bonds (red lines) illustrated. (B) SDS-PAGE of SDF-1 and SDF12 treated with or without dithiothreitol (DTT). SDF-1 and SDF12 migrate near the monomeric molecular weight of 8 kD when treated with DTT. In contrast, whereas SDF12 migrates as a dimer, SDF-1 migrates as a monomer in the absence of DTT. (C) Translational diffusion measurements of SDF12 indicate that SDF12 is dimeric. Diffusion coefficients (Ds) of wild-type SDF-1 (black circles) in 20 mM sodium phosphate at pH 7.4 plotted against chemokine concentration (22). Nonlinear fitting of the Ds values of SDF-1 indicates a dimer dissociation Kd of 120 µM with a pure monomer Ds value of ~ 1.6 (×10−6 cm2s−1) and a dimer value of ~1.0 (×10−6 cm2s−1) [data from Veldkamp et al. (22)]. Ds values for 10, 50, and 150 µM SDF12 (red triangles) range from 1.08–1.09 (×10−6 cm2s−1) consistent with those expected for SDF-1 in the dimeric state (data from this study). (D) Ensemble of 20 NMR solution structures of SDF12 (gray and tan) superimposed on the crystal structure of dimeric wild-type SDF-1 (blue, PDB ID 2J7Z) with an α-carbon RMSD of 1.2 Å for residues 9–66. Intermolecular Cys36-Cys65 disulfide bonds are shown in yellow. Flexible N-terminal residues of SDF-1 (–8) are omitted for clarity. Refinement statistics for the SDF12 structure ensemble are given in table S1.

Fig. 2

Fig. 2

The N-terminus of CXCR4 binds to SDF12. (A) 15N-1H HSQC spectra of 25 µM [U-15N]-SDF12 alone (black contours) and after the addition of 100 µM p38 peptide (green contours). (B) Combined 15N-1H chemical shift perturbations plotted against SDF12 residue number. Secondary structure elements are indicated and regions involved in the dimer interface are highlighted in orange. Missing values correspond to proline residues (sequence positions , , , and 53) or amino acid residues not observed in the 15N-1H HSQC spectra. (C) Chemical shift mapping on the SDF12 structure. Green surface highlighting corresponds to shift perturbations > 0.25 in (B).

Fig. 3

Fig. 3

Structures of SDF12 dimers bound to the N-terminal domain of CXCR4. (A) N-terminal peptides corresponding to the first 38 amino acids of CXCR4 are illustrated. The sequence for p38 is identical to that of CXCR4 except for a Gly-Ser dipeptide on the N-terminus, which results from a cloning artifact, and a Cys28 → Ala28 mutation to prevent oxidative peptide dimer formation. The sulfated peptides are identical to p38 except for the inclusion of sulfotyrosine at position 21 for p38-sY1 and at 7, 12, and 21 for p38-sY3. (B) Representative intermolecular NOEs for the SDF12:p38-sY1 complex. Strips from 3D F1-13C-fliltered/F3-13C-edited NOESY-HSQC spectra acquired from a complex containing [U-15N,13C]-SDF12 and unlabeled p38-sY1 (left) and a complex containing [U-15N,13C]-p38-sY1 and unlabeled SDF12 (right) contain equivalent NOEs between the methyl group of Val18 of SDF12 and sTyr21 1Hδ of p38-sY1. Ensembles of the 20 lowest energy conformers for the SDF12:p38 (C), SDF12:p38-sY1 (D), and SDF12:p38-sY3 (E) complexes. SDF12 is shown in gray and the CXCR4 N-termini are orange. Sulfotyrosine residues in N-termini of CXCR4 are shown in red.

Fig. 4

Fig. 4

Recognition of sulfotyrosines by SDF12. (A) NMR structure of SDF12 bound to p38-sY3. Individual subunits of the symmetric SDF12 dimer are shown in tan and white with symmetry-related p38-sY3 peptides in blue and orange. Chemical shift perturbations greater than 0.25 ppm (Fig. 2C) are highlighted in green on the surface of SDF12. Flexible regions of SDF12 (residues –8) and p38-sY3 (residues –38) are omitted for clarity. Sulfotyrosine side chains are shown in a ball-and-stick representation. In panels B–D, basic residues in SDF12 that pair with CXCR4 sulfotyrosines are shown in blue and SDF12 residues with NOEs to the sulfotyrosines are shown in green. (B) The sTyr7 residue of CXCR4 binds to SDF12 near Arg20 and makes NOE contacts with Val23. (C) The sTyr12 residue of CXCR4 occupies a cleft bounded by residues Lys27, Pro10, and Leu29 of SDF12. (D) The sTyr21 residue of CXCR4 pairs with Arg47 of SDF12 and makes NOE contacts with Val18 and Val49.

Fig. 5

Fig. 5

Amino acid substitutions in native SDF-1 corroborate the CXCR4 N-terminal binding site. One subunit of the SDF12 dimer and one p38-sY3 molecule from the SDF12:p38-sY3 complex solved by NMR represent a model for the equivalent 1:1 complex. Front (A) and back (B) views of the SDF-1 surface are highlighted to indicate the location and functional impact of amino acid substitutions in the wild-type SDF-1 sequence. Substitutions at the sTyr12- and sTyr21-binding sites (red) showed increased EC50 values for Ca2+ mobilization, whereas substitutions away from the CXCR4-binding site (cyan) showed no change in their EC50 values. A binding site for sTyr7 is not defined in this model because sTyr7 binds to the opposing SDF-1 subunit in the SDF12:p38-sY3 structure.

Fig. 6

Fig. 6

Dimeric SDF12 induces CXCR4-mediated Ca2+ mobilization but inhibits chemotaxis to wild-type SDF-1. (A) Ca2+ mobilization in THP-1 cells loaded with Fluo-3 indicates robust dose-dependant activation of CXCR4 by wild-type SDF-1 (●, EC50 = 3.6 nM) and SDF12 (▲, EC50 = 12.9 nM) (Data from Veldkamp et al. (53) and this study, respectively). (B) Wild-type SDF-1 induces the chemotaxis of THP-1 cells in a biphasic, concentration-dependent manner with a maximal migratory response at ~30 nM SDF-1. In contrast, SDF12 does not induce chemotaxis of THP-1 cells at any concentration from 1–1,000 nM. (C) Chemotaxis of THP-1 cells induced by 10 nM wild-type SDF-1 is inhibited by SDF12 (IC50 ~ 4 nM). (D) Wild-type SDF-1 and the dimerization-impaired His25 → Arg25 variant [SDF1(H25R)] induce chemotaxis of THP-1 cells equally well at low concentrations (0.1–10 nM). SDF1(H25R) remains monomeric at higher concentrations than does wild-type SDF-1 and induces chemotaxis over a broader range of concentrations. (E) Monomeric SDF-1 generates the full range of cellular responses to CXCR4 activation, whereas dimeric SDF1 is a partial agonist of CXCR4 that fails to induce chemotaxis. Loss of migration could be a consequence of aberrant CXCR4 trafficking.

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