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.

Structural basis of chemokine recognition by the class A3 tick evasin EVA‐ACA1001

“…Comparison of the EVA-ACA1001:CCL16 complex to previously reported chemokine-bound structures of the class A1 evasins EVA-1 (Dias et al, 2009) and EVA-P974 (Bhusal et al, 2022) and the class A3 evasin EVA-AAM1001 (Devkota et al, 2023) reveals that the structural basis of CC motif recognition is conserved across all class A1 and A3 evasins. Specifically, in all cases, the chemokine CC motif forms at least four backbone-backbone hydrogen bonds with the evasin β1 strand.…”

“…In contrast to the conserved role of the evasin β1 strand in CC motif recognition, the evasin N‐ and C‐termini play different roles in class A3 compared to class A1 evasins. In the class A3 evasins EVA‐ACA1001 and EVA‐AAM1001, the complete removal of the N‐ or C‐terminal residues had minimal effects on chemokine binding (Devkota et al, 2023). On the other hand, similar truncations of the class A1 evasin EVA‐P974 resulted in a loss of binding to most target chemokines (Bhusal et al, 2022).…”

“…Evasins belonging to class A bind exclusively to CC chemokines, whereas those belonging to class B bind exclusively to CXC chemokines. Recently, class A has been subclassified into classes A1, A2, and A3 according to the number of conserved cysteines (Devkota et al, 2023). Class A1 evasins such as EVA‐1, EVA‐4, and EVA‐P974 (Bhusal et al, 2022; Denisov et al, 2020; Dias et al, 2009) possess eight cysteines that form four conserved disulfide bonds, whereas class A2 evasins have six cysteines and lack one of the disulfide bonds (Bhattacharya & Nuttall, 2021).…”

“…Class A1 evasins such as EVA‐1, EVA‐4, and EVA‐P974 (Bhusal et al, 2022; Denisov et al, 2020; Dias et al, 2009) possess eight cysteines that form four conserved disulfide bonds, whereas class A2 evasins have six cysteines and lack one of the disulfide bonds (Bhattacharya & Nuttall, 2021). We recently described class A3 evasins, which have 10 cysteines and an additional disulfide, and reported chemokine‐bound structures of the class A3 evasin EVA‐AAM1001 (Devkota et al, 2023). We found that, unlike the recognition mechanism of class A1 evasins, the N‐ and C‐termini of EVA‐AAM1001 make minimal contribution to chemokine binding affinity; instead, EVA‐AAM1001 utilizes a distinct hydrophobic pocket to interact with the CC + 1 residue of target CC chemokines (Devkota et al, 2023).…”

“…We recently described class A3 evasins, which have 10 cysteines and an additional disulfide, and reported chemokine‐bound structures of the class A3 evasin EVA‐AAM1001 (Devkota et al, 2023). We found that, unlike the recognition mechanism of class A1 evasins, the N‐ and C‐termini of EVA‐AAM1001 make minimal contribution to chemokine binding affinity; instead, EVA‐AAM1001 utilizes a distinct hydrophobic pocket to interact with the CC + 1 residue of target CC chemokines (Devkota et al, 2023). We now describe a structure–function analysis of EVA‐ACA1001, a class A3 evasin from Amblyomma cajennense (Cayenne tick).…”