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.

Keywords: chemokines; inflammatory diseases; protein engineering; tick evasins.

Fig. 1.

EVA-P974 binds and inhibits multiple CC chemokines. (A) Affinities of EVA-P974 for CC chemokines, measured using SPR. (B) Structure of EVA-P974 (gray: N terminus, red: first β-sheet, cyan: C terminus, blue: Cys residues as sticks) bound to CCL7 (green: Cys residues, yellow sticks), with key recognition regions circled and labeled. (C) Structure of EVA-P974 (shown as in B) bound to CCL17 (pink: Cys residues, yellow sticks). (D) EVA-P974 inhibits activation of chemokine receptors by their cognate CC chemokines.

Fig. 2.

Conserved backbone–backbone interactions dictate specificity of class A evasins for CC chemokines. (A) Sequence alignment of CCL7 and the CCL7 mutant, C(A)CL7, with Ala inserted in the CC motif. (B) Differential scanning fluorometry (DSF) traces show that CCL7 (green) and C(A)CL7 (orange) undergo cooperative thermal unfolding. (C) SPR traces show that CCL7 (green) binds to EVA-P974, whereas C(A)CL7 (orange) does not. (D) EVA-P974 (cyan sticks) recognizes the CC motif of CCL7 (lime green sticks) but sterically clashes with the CXC motif of CXCL8 (thin magenta sticks); the indicated distance, d, is defined as the distance from the line between the C_α atom of the third conserved Cys and the C^O atom of the residue after the fourth conserved Cys to the O^C atom immediately preceding the second conserved Cys. (E) The key structural distance, d (defined in D), is conserved within CC and CXC chemokine subfamilies but differs between these two subfamilies. (F–I) Conserved mode of CC chemokine recognition by EVA-P974, chemokine receptor CCR5 (Protein Data Bank [PDB]: 7F1T), chemokine CCL8 (PDB: 7S5A) within a homodimer, and viral CC chemokine inhibitor vCCI (PDB: 4ZLT).

Fig. 3.

Hotspots between the EVA-P974 N terminus and the chemokine N-loop (NL) dominate target preference. (A) Swapping the NL between CCL7 and CCL2 substantially switches their ability to bind to EVA-P974, as shown by SPR. Chimeric chemokines are shown schematically (color-coded) and named to indicate the parental chemokine and the origin of the inserted NL region; for example, CCL7(2NL) consists of the CCL7 sequence with the NL replaced by that of CCL2. (B) EVA-P974:CCL7 complex, highlighting the EVA-P974 hotspots for target chemokine selectivity. (C) Close-up views of EVA-P974 hotspot residues (colored as in B) interacting with CCL7 (green) and CCL17 (pink). (D) N-terminal truncation of EVA-P974 substantially decreases chemokine-binding affinity. (E) Hotspot mutations alter the chemokine-binding profile of EVA-P974. For F31A, each data point is labeled with the one-letter code for the first residue in the NL. (F) Wild-type EVA-P974 binds to the CCL7 Tyr13 side chain in an orientation stabilized by a cation–π interaction with Arg14. (G) Removal of the bulky phenyl ring in the EVA-P974(F31A) variant enables binding to the CCL2 Tyr13 side chain in an alternative orientation not possible in the wild-type evasin. All K_d values were measured using SPR (n = 3). #, no measurable binding at 500-nM chemokine concentration.

Fig. 4.

Interactions of the EVA-P974 C terminus fine-tune chemokine-binding selectivity. (A) Swapping the N terminus (N) and N-loop (NL) between CCL7 and CCL8 substantially switches their ability to bind to EVA-P974, but swapping the NL alone does not, as shown by SPR. Chimeric chemokines are shown schematically (color-coded) and named to indicate the parental chemokine and the origin of the inserted NL or inserted N terminus plus NL (NNL) region; for example, CCL7(8NNL) consists of the CCL7 sequence with both the N-terminal and NL regions replaced by those of CCL8. (B) Detailed interactions between the C terminus of EVA-P974 (R90, blue; K91-N93, gray) and the N termini of CCL7 (left, green) and the chimera CCL7(8NNL) (right, beige). Residues shown in the CCL7(8NNL) but not the CCL7 structure were not resolved in the latter data set. (C) C-terminal truncation of EVA-P974 alters chemokine-binding affinity, with variants 1 through 90 and 1 through 91 exhibiting reduced selectivity among chemokines. All K_d values were measured using SPR (n = 3). #, no measurable binding at 500-nM chemokine concentration.

Fig. 5.

Engineered EVA-P974(F31A) inhibits receptor activation by multiple chemokines. Combined activation of CCR1 and CCR2 in THP-1 monocytes by the chemokine mixture CCL2+CCL5+CCL7 (orange) is fully inhibited by CCR1 inhibitor BX471 plus CCR2 inhibitor INCB3344 (green), partially inhibited by wild-type EVA-P974 (gray), and fully inhibited by EVA-P974(F31A) (magenta). Data points represent the average ± SEM of three independent experiments, each conducted in duplicate. *P < 0.05 and ****P < 0.0001 relative to CCL2+CCL5+CCL7, one-way ANOVA.