Chemokines function via G-protein coupled receptors in a robust network to recruit immune cells to sites of inflammation. Due to the complexity of this network, targeting single chemokines or receptors has not been successful in inflammatory disease. Dog tick saliva contains polyvalent CC-chemokine binding peptides termed evasins 1 and 4, that efficiently disrupt the chemokine network in models of inflammatory disease. Here we develop yeast surface display as a tool for functionally identifying evasins, and use it to identify 10 novel polyvalent CC-chemokine binding evasin-like peptides from salivary transcriptomes of eight tick species in Rhipicephalus and Amblyomma genera. These evasins have unique binding profiles compared to evasins 1 and 4, targeting CCL2 and CCL13 in addition to other CC-chemokines. Evasin binding leads to neutralisation of chemokine function including that of complex chemokine mixtures, suggesting therapeutic efficacy in inflammatory disease. We propose that yeast surface display is a powerful approach to mine potential therapeutics from inter-species protein interactions that have arisen during evolution of parasitism in ticks.

Tick evasins (EVAs) bind either CC- or CXC-chemokines by a poorly understood promiscuous or “one-to-many” mechanism to neutralize inflammation. Because EVAs potently inhibit inflammation in many preclinical models, highlighting their potential as biological therapeutics for inflammatory diseases, we sought to further unravel the CXC-chemokine–EVA interactions. Using yeast surface display, we identified and characterized 27 novel CXC-chemokine–binding evasins homologous to EVA3 and defined two functional classes. The first, which included EVA3, exclusively bound ELR + CXC-chemokines, whereas the second class bound both ELR + and ELR − CXC-chemokines, in several cases including C X C-motif chemokine ligand 10 (CXCL10) but, surprisingly, not CXCL8. The X-ray crystal structure of EVA3 at a resolution of 1.79 Å revealed a single antiparallel β-sheet with six conserved cysteine residues forming a disulfide-bonded knottin scaffold that creates a contiguous solvent-accessible surface. Swapping analyses identified distinct knottin scaffold segments necessary for different CXC-chemokine–binding activities, implying that differential ligand positioning, at least in part, plays a role in promiscuous binding. Swapping segments also transferred chemokine-binding activity, resulting in a hybrid EVA with dual CXCL10- and CXCL8-binding activities. The solvent-accessible surfaces of the knottin scaffold segments have distinctive shape and charge, which we suggest drives chemokine-binding specificity. These studies provide structural and mechanistic insight into how CXC-chemokine–binding tick EVAs achieve class specificity but also engage in promiscuous binding.

Tick chemokine-binding proteins (evasins) are an emerging class of biologicals that target multiple chemokines and show anti-inflammatory activities in preclinical disease models. Using yeast surface display, we identified a CCL8-binding evasin, P672, from the tick Rhipicephalus pulchellus. We found that P672 binds CCL8 and eight other CC-class chemokines with a Kd < 10 nm and four other CC chemokines with a Kd between 10 and 100 nm and neutralizes CCL3, CCL3L1, and CCL8 with an IC50 < 10 nm. The CC chemokine–binding profile was distinct from that of evasin 1 (EVA1), which does not bind CCL8. We also show that P672's binding activity can be markedly modulated by the location of a StrepII-His purification tag. Combining native MS and bottom-up proteomics, we further demonstrated that P672 is glycosylated and forms a 1:1 complex with CCL8, disrupting CCL8 homodimerization. Homology modeling of P672 using the crystal structure of the EVA1 and CCL3 complex as template suggested that 44 N-terminal residues of P672 form most of the contacts with CCL8. Replacing the 29 N-terminal residues of EVA1 with the 44 N-terminal residues of P672 enabled this hybrid evasin to bind and neutralize CCL8, indicating that the CCL8-binding properties of P672 reside, in part, in its N-terminal residues. This study shows that the function of certain tick evasins can be manipulated simply by adding a tag. We conclude that homology modeling helps identify regions with transportable chemokine-binding functions within evasins, which can be used to construct hybrid evasins with altered properties.

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