Engineering Broad-spectrum Inhibitors of Inflammatory Chemokines from a New Family of Tick Evasins

Abstract Chemokines, the key regulators of leukocyte trafficking, are attractive targets for anti-inflammatory therapy. Evasins are anti-inflammatory, chemokine-binding proteins found in tick saliva, with important therapeutic potential. However, therapeutic application of evasins will require manipulation of their chemokine target selectivity. Here we describe a new family of evasins, class A3 evasins, that is unique to the tick genus Amblyomma and distinguished from “classical” class A1 evasins by an additional disulfide-bonded pair of cysteine residues near the chemokine recognition interface. The class A3 evasin EVA-AAM1001 (EVA-A) bound to CC chemokines and inhibited their receptor activation. However, unlike class A1 evasins, EVA-A did not utilise N- and C-terminal regions to differentiate chemokine targets. Instead, structures of EVA-A bound to four chemokines revealed a deep hydrophobic pocket, unique to class A3 evasins, that interacts with the residue immediately following the CC motif of the chemokine (the “CC+1” residue). The preference of EVA-A for chemokines with aliphatic CC+1 residues results from negative selection against binding of aromatic CC+1 residues into this pocket. Consequently, mutations to alleviate this negative selection yielded broad-spectrum chemokine inhibitors. This study illustrates that class A3 evasins are an excellent platform for engineering proteins with targeted chemokine binding selectivity for applications in research, diagnosis or anti-inflammatory therapy.