Solution Structure of CCL19 and Identification of Overlapping CCR7 and PSGL-1 Binding Sites

Abstract

CCL19 and CCL21 are chemokines involved in the trafficking of immune cells, particularly within the lymphatic system, through activation of CCR7. Concurrent expression of PSGL-1 and CCR7 in naive T-cells enhances recruitment of these cells to secondary lymphoid organs by CCL19 and CCL21. Here the solution structure of CCL19 is reported. It contains a canonical chemokine domain. Chemical shift mapping shows the N-termini of PSGL-1 and CCR7 have overlapping binding sites for CCL19 and binding is competitive. Implications for the mechanism of PSGL-1's enhancement of resting T-cell recruitment are discussed.

Figure 1

Solution structure of CCL19. (A) Lowest energy conformer of CCL19. CCL19 has a canonical chemokine fold consisting of a flexible N-terminus and N-loop followed by an antiparallel three-stranded β-sheet, a C-terminal α-helix, and a short flexible C-terminus. Conserved disulfide bonds are shown in yellow. (B) Ensemble of 20 CCL19 structures.

Figure 2

CCL19’s binding site for the N-termini of CCR7 and PSGL-1 overlap and binding is competitive. (A) Structure of CCL19 with perturbations from the CCR7 N-terminus highlighted in magenta. (B) Perturbations from the N-terminus of PSGL-1 are mapped onto the structure of CCL19 in green. (C) A portion of an ¹⁵N-¹H HSQC spectra showing the titration of [U-¹⁵N] CCR7 2–30 C24A with CCL19 (left) followed by titration with PSGL-1 2–15 (right). Upon addition of PSGL-1 2–15 peaks from CCR7 return toward their original position or unbound state. Molar ratios are as indicated in the figure.