Ticks draw blood for days to weeks without eliciting inflammation ( 10), and investigation of saliva of the brown dog tick Rhipicephalus sanguineus revealed the presence of chemokine-binding proteins ( 11, – 13). Viral proteins ( 9) such as poxvirus CrmD and viral CC-chemokine inhibitor (vCCI), herpesvirus R17 and M3, and papovavirus chemokine-binding protein (CBP), bind multiple chemokines, typically via either the proximal N terminus or via the N-loop/40S loop, preventing binding to CRS1 ( 3). Several parasites and infectious agents overcome the chemokine network and consequent inflammation by producing structurally unrelated proteins that bind, in a promiscuous fashion, to multiple chemokines ( 8, 9). It explains, at least in part, the failure of targeting individual chemokines or receptors as a therapeutic strategy for inflammatory disorders ( 5, 6, 8). This phenomenon, together with the expression of a large number of chemokines at sites of inflammation ( 5) and the expression of several synergistically acting chemokine receptors on inflammatory cells ( 6, 7), renders the chemokine network robust to attack. The binding of chemokines to receptors typically involves promiscuous interactions, with several chemokines possessing the ability to bind multiple receptors and, conversely, several receptors having the ability to bind multiple chemokines ( 4). The “two-site” model has been refined more recently with the identification of further interaction sites: CRS1.5, between CRS1 and 2, which binds the conserved chemokine disulfide, and CRS0.5, at the receptor distal N terminus, which binds the β1-strand of the chemokine ( 3). CRS1 binds the proximal N terminus and N-loop/40S loop, whereas CRS2 binds the chemokine distal N terminus ( 3). Binding of chemokines to receptors occurs via chemokine recognition site 1 (CRS1), located in the extracellular N terminus of the receptor, and CRS2, located in the seven-transmembrane bundle. Certain CXC-chemokines, referred to as “ELR+,” contain a characteristic Glu-Leu-Arg motif in the N-terminal region that binds chemokine receptors CXCR1 7 and CXCR2 and activates neutrophil migration ( 2). Chemokines are structurally conserved, with a three-stranded β-sheet, an α-helical segment, and an N-terminal unstructured region ( 1) along with an N-loop between the second Cys and the β1-strand and 30S and 40S loops between the three β-strands. Their chemotactic functions are mediated by binding to a family of G-protein coupled receptors. The 45–50 mammalian chemokines are small secreted proteins that are grouped into CC, CXC, XC and CX3C classes based on the spacing between N-terminal cysteine residues. These studies provide structural and mechanistic insight into how CXC-chemokine–binding tick EVAs achieve class specificity but also engage in promiscuous binding. The solvent-accessible surfaces of the knottin scaffold segments have distinctive shape and charge, which we suggest drives chemokine-binding specificity. Swapping segments also transferred chemokine-binding activity, resulting in a hybrid EVA with dual CXCL10- and CXCL8-binding activities. 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. 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. 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 XC-motif chemokine ligand 10 (CXCL10) but, surprisingly, not CXCL8. Using yeast surface display, we identified and characterized 27 novel CXC-chemokine–binding evasins homologous to EVA3 and defined two functional classes. 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. Tick evasins (EVAs) bind either CC- or CXC-chemokines by a poorly understood promiscuous or “one-to-many” mechanism to neutralize inflammation.
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