Engineering broad-spectrum inhibitors of inflammatory chemokines from subclass A3 tick evasins

Abstract

Chemokines are key regulators of leukocyte trafficking and attractive targets for anti-inflammatory therapy. Evasins are chemokine-binding proteins from tick saliva, whose application as anti-inflammatory therapeutics will require manipulation of their chemokine target selectivity. Here we describe subclass A3 evasins, which are unique to the tick genus Amblyomma and distinguished from "classical" class A1 evasins by an additional disulfide bond near the chemokine recognition interface. The A3 evasin EVA-AAM1001 (EVA-A) bound to CC chemokines and inhibited their receptor activation. Unlike A1 evasins, EVA-A was not highly dependent on N- and C-terminal regions to differentiate chemokine targets. Structures of chemokine-bound EVA-A revealed a deep hydrophobic pocket, unique to A3 evasins, that interacts with the residue immediately following the CC motif of the chemokine. Mutations to this pocket altered the chemokine selectivity of EVA-A. Thus, class A3 evasins provide a suitable platform for engineering proteins with applications in research, diagnosis or anti-inflammatory therapy.

Fig. 1. Discovery and characterisation of class A3 evasins.

a Sequence logo based on alignments of class A evasins (class A1: n = 149, class A2: n = 65 and class A3: n = 17). Conserved cysteine and disulfide bond connectivity are indicated for class A1 evasins (orange). The additional cysteine and disulfide bond of class A3 are indicated in red. The secondary structure of EVA-P974 (class A1) is shown above the alignment. b A midpoint-rooted, neighbour-joining tree represented as a cladogram, based on MUSCLE alignments of the class A evasins. Background colours show subclasses; A1 (light yellow), A2 (green) and A3 (purple). The genus of each node is indicated by coloured segments in the outer ring and shown in the legend. c Representative binding sensorgrams of human chemokines (5 injections at consecutive concentrations of 31.25, 62.5, 125, 250 and 500 nM) measured by SPR using single-cycle kinetics. d Binding affinities (K_d) of EVA-A for CC chemokines, measured by SPR. Data are presented as mean ± SEM from three independent experiments. $, K_d < 0.1 nM; #, no measurable binding at 500 nM chemokine concentration. e Concentration response curves showing the inhibition of chemokines CCL3, CCL4 and CCL8, but not CCL2, by EVA-A. FlpInCHO cells stably expressing CCR5 (for CCL3, CCL4 and CCL8) or CCR2 (for CCL2) and transfected with the cAMP biosensor CAMYEL, were treated with coelenterazine h (5 μM, 10 min), followed by forskolin (10 μM, 10 min) to induce cAMP production, followed by CCL3 (60 nM), CCL4 (80 nM), CCL8 (100 nM) or CCL2 (100 nM), either alone or pre-incubated with the indicated concentrations of EVA-A. cAMP was detected 10 min after chemokine addition. Data are represented as a percentage of the inhibition of cAMP production observed upon chemokine treatment in the absence of EVA-A, and presented as mean ± SEM from three or four independent experiments.

Fig. 2. Structure of EVA- A bound to the human CC chemokines.

a Overall structure of EVA-A (grey; conserved disulfides, orange; additional disulfide, red) in complex with CCL7 (sky blue; CC motif, yellow) showing all the major features as labelled. b Overlay of EVA-A complexes with CCL7 (blue), CCL11 (green), CCL16 (cyan) and CCL17 (magenta). c Cartoon representation of overlaid structures of EVA-A (grey) bound to CCL17 (magenta) and EVA-P (pale green) bound to CCL17 (deep teal) (PDB ID:7S4N) showing the different orientation of CCL17 by 35°. d Conserved mode of CC chemokine recognition by EVA-A and EVA-P. e Truncations of the N- and C-termini of EVA-A have subtle effects on the chemokine binding affinities of EVA-A. Data are presented as mean ± SEM from three independent experiments. $, K_d < 0.1 nM; #, no measurable binding at 500 nM chemokine concentration; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, versus wild type EVA-A (one-way ANOVA with Šídák correction for multiple comparisons for each chemokine; or a two-tailed t test if only a single comparison was possible).

Fig. 3. The fifth disulfide defines a critical binding pocket for chemokine target selectivity (CC + 1 residue binding).

a Cartoon representation of EVA-A CCL7, with expanded view showing the EVA-A residues (grey, additional disulfide: red) of the hydrophobic pocket holding the CC + 1 residue (magenta) of CCL17. b Surface representations of the EVA-A binding pocket (grey) fitting the CC + 1 residue of four different chemokines; CCL7 (sky blue), CCL11 (green), CCL16 (cyan) and CCL17 (magenta). c Chemokine affinities (K_d) of EVA-A (grey) and EVA-A(C₈) (sand). Data are presented as mean ± SEM from three independent experiments. $, K_d < 0.1 nM; #, no measurable binding at 500 nM chemokine concentration; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, versus wild type EVA-A (two-tailed t test with Holm- Šídák correction for multiple comparisons). d Overlay of the binding pocket residues of EVA-A (grey) bound to the CC + 1 residue of CCL17 (magenta) and EVA-A(C₈) (sand) bound to the CC + 1 residue of CCL17 (purple). e Representative SPR sensorgrams for binding of EVA-A to wild type chemokines (CCL7, sky blue; CCL11, green; and CCL16, cyan) and CC + 1 residue-mutated versions of each chemokine (red) measured using single-cycle kinetics (5 chemokine injections at consecutive concentrations of 31.25, 63.5, 125, 250 and 500 nM). f EVA-A affinities (K_d) for wild type and CC + 1 residue-mutated chemokines (coloured as in e). Data are presented as mean ± SEM from three independent experiments. $, K_d < 0.1 nM; ****p < 0.0001, versus wild type chemokine (two-tailed t test). g Overlay of the binding pocket residues of EVA-A (grey) bound to CCL7 (sky blue) and EVA-A (forest green) bound to CCL7(Y13A) (violet).

Fig. 4. Engineering of the EVA-A hydrophobic pocket.

a CC chemokine affinities (K_d) of EVA-A (grey), EVA-A(Y44A) (green) and EVA-A(L39P) (blue). Upper panel, chemokines with aromatic CC + 1 residues; lower panel, chemokines with aliphatic CC + 1 residues. Data are presented as mean ± SEM from three independent experiments. $, K_d < 0.1 nM; #, no measurable binding at 500 nM chemokine concentration; *p < 0.05, ****p < 0.0001, versus wild type EVA-A (one-way ANOVA with Šídák correction for multiple comparisons; or a two-tailed t test if only a single comparison was possible). b Concentration-response curves showing the inhibition of CCL2 (100 nM) (top Panel) and CCL7 (100 nM) (bottom panel) by EVA-A, EVA-A(Y44A) and EVA-A(L39P). FlpInCHO cells stably expressing CCR2 transfected with the cAMP biosensor CAMYEL, were treated with coelenterazine h (5 μM, 10 min), followed by CCL2 (100 nM) or CCL7 (100 nM), either alone or pre-incubated with the indicated concentrations of EVA-A, followed by forskolin (10 μM, 10 min) to induce cAMP production. cAMP was detected 10 min after chemokine addition. Data are represented as a percentage of the inhibition of cAMP production observed upon chemokine treatment in the absence of EVA-A, and presented as mean ± SEM from three independent experiments. c Interactions of chemokine CC + 1 residues (sticks with mesh) with hydrophobic pocket side chains (sticks) of EVA-A, EVA-A(Y44A) and EVA-A(L39P). Top (left to right): EVA-A (grey) bound to CCL7 (sky blue) and EVA-A(Y44A) (green) bound to CCL7 (sky blue). Bottom (left to right): EVA-A(L39P) (deep blue) bound to CCL7 (sky blue) and EVA-A(Y44A) (green) bound to CCL2 (olive). d EVA-A(Y44A) (top) and EVA-A(L39P) (bottom) affinities (K_d) for wild type and CC + 1 residue-mutated chemokines (coloured as in Fig. 3e, f). Data are presented as mean ± SEM from three independent experiments. $, K_d < 0.1 nM; **p < 0.01, ***p < 0.001, ****p < 0.0001, versus wild type chemokine (two-tailed t test).

Fig. 5. Class A3 evasins achieve broad chemokine binding through flexible structure.

a, b Plots comparing the root-mean-square fluctuation (RMSF) values during MD simulations for EVA-A (grey) and EVA-P (green teal). The secondary structure of EVA-A in the crystal structure is shown at the top. a Free evasins only. b Bound state with chemokine. c RMSF of CCL17 bound to EVA-A (grey) and EVA-P (teal) with MD trajectories aligned at the evasins. Data are presented as mean ± SEM from three independent simulations. d The EVA-A:CCL17 complex forms high occupancy intermolecular hydrogen bonds at both ends of CC the motif. e The EVA-P:CCL17 complex forms a single high occupancy intermolecular hydrogen bond in the centre of the interface.