Solution structure of CCL21 and identification of a putative CCR7 binding site

Abstract

CCL21 is a human chemokine that recruits normal immune cells and metastasizing tumor cells to lymph nodes through activation of the G protein-coupled receptor CCR7. The CCL21 structure solved by NMR contains a conserved chemokine domain followed by an extended, unstructured C-terminus that is not typical of most other chemokines. A sedimentation equilibrium study showed CCL21 to be monomeric. Chemical shift mapping indicates that the CCR7 N-terminus binds to the N-loop and third β-strand of CCL21's chemokine domain. Details of CCL21-receptor recognition may enable structure-based drug discovery of novel antimetastatic agents.

Figure 1

Solution structure of CCL21. (A) Ensemble of 20 CCL21 structures. CCL21 has the typical chemokine domain consisting of an unstructured N-terminus (residues 1–7, not shown for clarity) followed by the N-loop, a three-stranded β-sheet and an α-helix. CCL21 also contains an unstructured C-terminus (residues 71–111, not shown for clarity) distinct from most chemokines. The conserved disulfide bonds, between C8 and C34 and C9 and C52 in CCL21, are shown in yellow. (B) Lowest energy structure of CCL21 showing residues 8–70. (C) Heteronuclear ¹⁵N-¹H NOE values plotted versus CCL21 amino acid number. The core chemokine domain of CCL21 is structured (residues 8–68) while the N-terminus and extended C-terminus are unstructured. The small positive values in the C-terminus are near C80 and C99, which form a disulfide bond.

Figure 2

Putative CCR7 binding site. (A) Titration of CCL21 with a peptide corresponding to the N-terminus of CCR7 as monitored by ¹⁵N-¹H HSQC spectra. The spectrum of free CCL21 is shown in black with successive gray spectra indicating increasing concentrations of CCR7 peptide. CCL21 with a 5 molar excess of CCR7 peptide is shown in blue. (B) Non-linear fitting yields a K_d of 150 ± 30 µM for CCL21 and the CCR7 N-terminus. (C) Combined amide proton and nitrogen chemicals shift perturbations induced by the CCR7 peptide are plotted versus CCL21 residue number. Residues with significant perturbations whose signal ultimately broadened beyond detection (residues 12–14, 45 and 51) were given 3.5 ppm values with the last observable perturbation values for these residues shown as red bars. Residues 12, 14, and 51 were last observed at a molar ratio of 1:1.5 CCL21 to CCR7. Residues 13 and 45 were last detected at a molar ratio of 1:0.25 with each signal broadening beyond detection after the second peptide addition (1:0.5). Prolines and unobserved residues have values of 0. (D) Chemical shift mapping onto the surface of CCL21 suggests regions involved in CCR7 binding. Light blue indicates residues with chemical shift perturbations from 1.0 to 1.5 while those in blue have perturbations > 1.5.