Structure-guided engineering of tick evasins for targeting chemokines in inflammatory diseases

Abstract

As natural chemokine inhibitors, evasin proteins produced in tick saliva are potential therapeutic agents for numerous inflammatory diseases. Engineering evasins to block the desired chemokines and avoid off-target side effects requires structural understanding of their target selectivity. Structures of the class A evasin EVA-P974 bound to human CC chemokine ligands 7 and 17 (CCL7 and CCL17) and to a CCL8-CCL7 chimera reveal that the specificity of class A evasins for chemokines of the CC subfamily is defined by conserved, rigid backbone–backbone interactions, whereas the preference for a subset of CC chemokines is controlled by side-chain interactions at four hotspots in flexible structural elements. Hotspot mutations alter target preference, enabling inhibition of selected chemokines. The structure of an engineered EVA-P974 bound to CCL2 reveals an underlying molecular mechanism of EVA-P974 target preference. These results provide a structure-based framework for engineering evasins as targeted antiinflammatory therapeutics.

Swapping N-terminal regions among tick evasins reveals cooperative interactions influencing chemokine binding and selectivity

“…We compared the chemokine-binding selectivity of EVA-R to those of three other well-characterized class A evasins. EVA-1 has been reported to bind to only three of 24 CC chemokines tested, whereas EVA-4 and EVA-P974 (referred to hereafter as EVA-P) have been reported to bind to 17 and 14 CC chemokines, respectively ( 16 , 18 , 22 ). We expressed these evasins using HEK293 cells, purified them, and verified their chemokine binding selectivity using SPR ( Table 2 , Figs.…”

“…S2 ), but unlikely to influence chemokine binding ( 13 , 15 , 24 ). Previous studies have shown that residues within the N-terminal regions (before the first conserved Cys residue) of EVA-1 and EVA-P play roles in chemokine binding ( 16 , 18 ). The N-terminal regions of all four evasins in the present study contain one or two predicted tyrosine sulfation sites.…”

“…( 20 ) showed that replacing the N terminus and the first β-sheet of EVA-1 with those of EVA-P972 caused a substantial change in chemokine selectivity. More recently, we have shown that truncations and point mutations in the N-terminal region of EVA-P alter the chemokine selectivity profile of this evasin ( 16 ).…”

“…Within each class, each evasin exhibits a unique spectrum of chemokine binding selectivity, ranging from quite high selectivity (∼5 chemokine targets) to broad spectrum activity (>15 chemokine targets) ( 13 , 14 , 15 ). Here, we focus on class A evasins, for which the structural basis of chemokine recognition is beginning to be understood ( 16 , 17 , 18 ). Structures of EVA-1 bound to CCL3 ( 18 ) and EVA-P974 bound to CCL7, CCL17, and a CCL8-CCL7 chimera, supported by mutational data ( 16 ), showed that the specificity of class A evasins for CC chemokines (rather than other chemokine subfamilies) is governed by structurally constrained backbone-backbone hydrogen bonds.…”

“…Here, we focus on class A evasins, for which the structural basis of chemokine recognition is beginning to be understood ( 16 , 17 , 18 ). Structures of EVA-1 bound to CCL3 ( 18 ) and EVA-P974 bound to CCL7, CCL17, and a CCL8-CCL7 chimera, supported by mutational data ( 16 ), showed that the specificity of class A evasins for CC chemokines (rather than other chemokine subfamilies) is governed by structurally constrained backbone-backbone hydrogen bonds. In contrast, binding preference among CC chemokines is largely controlled by the interactions of side chains in the flexible N- and C-terminal regions of the evasin.…”