Structural basis of chemokine sequestration by a tick chemokine binding protein: the crystal structure of the complex between Evasin-1 and CCL3

Abstract

Background: Chemokines are a subset of cytokines responsible for controlling the cellular migration of inflammatory cells through interaction with seven transmembrane G protein-coupled receptors. The blocking of a chemokine-receptor interaction results in a reduced inflammatory response, and represents a possible anti-inflammatory strategy, a strategy that is already employed by some virus and parasites. Anti-chemokine activity has been described in the extracts of tick salivary glands, and we have recently described the cloning and characterization of such chemokine binding proteins from the salivary glands, which we have named Evasins.

Methodology/principal findings: We have solved the structure of Evasin-1, a very small and highly selective chemokine-binding protein, by x-ray crystallography and report that the structure is novel, with no obvious similarity to the previously described structures of viral chemokine binding proteins. Moreover it does not possess a known fold. We have also solved the structure of the complex of Evasin-1 and its high affinity ligand, CCL3. The complex is a 1:1 heterodimer in which the N-terminal region of CCL3 forms numerous contacts with Evasin-1, including prominent pi-pi interactions between residues Trp89 and Phe14 of the binding protein and Phe29 and Phe13 of the chemokine.

Conclusions/significance: However, these interactions do not appear to be crucial for the selectivity of the binding protein, since these residues are found in CCL5, which is not a ligand for Evasin-1. The selectivity of the interaction would appear to lie in the N-terminal residues of the chemokine, which form the "address" whereas the hydrophobic interactions in the rest of the complex would serve primarily to stabilize the complex. A thorough understanding of the binding mode of this small protein, and its other family members, could be very informative in the design of potent neutralizing molecules of pro-inflammatory mediators of the immune system, such as chemokines.

Figure 1. Overall structure of Evasin-1.

A stereo view of the overall structure of the non-glycosylated form of Evasin-1 is presented.

Figure 2. Secondary structure of Evasin-1.

The secondary structure, disulfide bridges, and glycosylation sites of Evasin-1 are shown.

Figure 3. Stereo diagram of the complex between Evasin-1 and CCL3.

The Evasin-1 is colored in cyan and CCL3 in green.

Figure 4. Stereo diagram of the comparison of unbound CCL3 with CCL3 bound to Evasin-1.

The unbound form of CCL3 is shown in red, the bound form displayed in green.

Figure 5. Close-up of the interactions between Evasin-1 and CCL3.

The Evasin-1 is colored in cyan and CCL3 in green. (A) Interaction between the Thr16-Ser17-Arg18 loop of CCL3 with Evasin-1 (B) Interaction between Phe13 of CCL3 and Phe14 of Evasin-1 (C) Interaction between Phe29 of CCL3 and Trp89 of Evasin-1 (D) 2Fo-fc electron density map, contoured at 1.5σ of the interaction of the N-terminal region of CCL3 with Evasin-1.

Figure 6. Electrostatic surface complementarity between Evasin-1 and CCL3.

(A) The CCL3 molecule is displayed as a cyan-colored ribbon, while the Evasin-1 is displayed as a molecular surface colored by surface electrostatic potential. (B) The complex in (A) is rotated 180° along a central vertical axis and the Evasin-1 is displayed as a green ribbon and the CCL3 molecule as a molecular surface colored by surface electrostatic potential.

Figure 7. Stereo diagram comparing the structure of the complexes of CC chemokines with different CC chemokine binding proteins.

(A) Ribbon diagram of the complex of CCL3 with Evasin-1. CCL3 is displayed in cyan and Evasin-1 in green. (B) Ribbon diagram of the complex of CCL4 with vCCI. CCL4 is displayed in cyan and vCCI in violet, (C) Ribbon diagram of the complex of CCL2 with M3 decoy receptor. CCL4 in cyan and the M3 decoy receptor in mauve.

Figure 8. Amino acid sequence alignments.

(A) Alignment of mammalian CCL3 sequences. Fully conserved residues are background colored in blue, highly conserved (>80% identity amongst the species shown) in dark grey, and poorly conserved (>60% identity) in light grey. (B) Alignment of chemokines towards CCL3. The blue background identifies amino acids that are identical to CCL3. (C) Amino acid sequences of the chemokine chimera.

Figure 9. The N terminus of CCL3 is involved in the selectivity of Evasin-1 binding.

Upper panels: sensograms obtained for binding experiments, lower panels: kinetic parameters relative to binding experiments. A) Chemokine binding to immobilized Evasin-1. Sensogram corresponding to CCL3/CCL5 (green) shows similar binding properties to CCL3 (red) and Δ4CCL3 (cyan); CCL5/CCL3 (light green), and CCL5 (blue) are unable to bind, as is CXCL8. nd  =  not determined; the affinity of the chemokine was too low for accurate measurement. B) Evasin-1 WT (Black) or F14A W89A (brown) to immobilized CCL3.