Oligomeric structure of the chemokine CCL5/RANTES from NMR, MS, and SAXS data

Abstract

CCL5 (RANTES) is a proinflammatory chemokine known to activate leukocytes through its receptor, CCR5. Although the monomeric form of CCL5 is sufficient to cause cell migration in vitro, CCL5's propensity for aggregation is essential for migration in vivo, T cell activation and apoptosis, and HIV entry into cells. However, there is currently no structural information on CCL5 oligomers larger than the canonical CC chemokine dimer. In this study the solution structure of a CCL5 oligomer was investigated using an integrated approach, including NMR residual dipolar couplings to determine allowed relative orientations of the component monomers, SAXS to restrict overall shape, and hydroxyl radical footprinting and NMR cross-saturation experiments to identify interface residues. The resulting model of the CCL5 oligomer provides a basis for explaining the disaggregating effect of E66 and E26 mutations and suggests mechanisms by which glycosaminoglycan binding may promote oligomer formation and facilitate cell migration in vivo.

Figure 1

A) Sauson-Flamsteed projection plot of alignment frame orientations for WT CCL5 aligned in both positively charged and neutral polyacrylamide gel. The X-axes of the two frames share a common orientation. B) Orientation of the alignment tensor relative to the CCL5 dimer. The X-axis of the principal axes of the alignment tensor is shown in red, the Y-axis is shown in blue and the Z-axis is in orange. The golden arrow indicates the orientation of the symmetry axis from the dimer crystal structure. (see also Figure S2 and S3)

Figure 2

A) Fitting to SAXS scattering data for model from grid point 19×13, χ=1.13, 40% of the protein is assumed in be in the tetramer form. B) Contour map of the SAXS fitting χ values of the models generated on the grid. Colors ranging from blue to yellow represent high to low χ values. (see also Figure S4)

Figure 3

A) Contour maps of the combined residue pair and SAXS scores for each model on the grid. B) Model from grid point 19×13, which is representative of the models from the group with the best (orange region) combined score. (see also Figure S5)

Figure 4

A) Plot of residue specific cross saturation-induced amide proton signal intensity changes for WT and E66S CCL5. B) Surface plot of the CCL5 tetramer model with residues identified as being specifically perturbed in the wild type (residues 26 to 29, 33, 34, 66 and 67) colored in orange and the dimer interface (residues 6 to 10) colored in red.

Figure 5

A) Plot of residue specific hydroxyl radical modification percentage for WT and E66S CCL5. The degree of modification is analyzed at the peptide level, and the major sites of oxidation are identified at the residue level for each peptide. Residues identified as being protected from modification are indicated. B) Surface plot of the CCL5 tetramer with residues identified as being in the tetrameric interface (residues 26 to 29, 41, 61, 62, 67, 68,) colored orange. (see also Figure S6)

Figure 6

A) Native spray mass spectrum of WT CCL5 (10 μM) at pH 4.5. Even-numbered oligomers from dimer to octamer are observed, indicating that the oligomer is built from a concatenation of dimer substructures. B) Surface plot of the CCL5 octamer model with residues perturbed by GAGs (residues 44–48, 55, 56) shown in red and residues known to contact CCR5 at pH 6 (residues 16, 17, 21, 23) shown in blue. The N-terminus of CCL5, which is both perturbed by GAGs and known to bind to CCR5 in CCL5 monomer is colored yellow. C) Electrostatic potential plot of the octamer surface showing large patches of basic regions (blue) throughout the protein.

Figure 7

A) Details of the inter-dimer hydrophobic interactions in model 19×13. The hydrophobic interface is formed by Y27, F28, I62 and L65. B) Electrostatic interactions at the dimer-dimer interface. K25, E26, E66 & R44 can form pairs of electrostatic bonds. C) Ribbon representations of the proposed CCL5 tetramer (red) and the MIP1α tetramer (blue). A single dimer unit from each tetramer is arranged in identical orientation.