Structural characterization of anti-CCL5 activity of the tick salivary protein evasin-4

Ticks, as blood-sucking parasites, have developed a complex strategy to evade and suppress host immune responses during feeding. The crucial part of this strategy is expression of a broad family of salivary proteins, called Evasins, to neutralize chemokines responsible for cell trafficking and recruitment. However, structural information about Evasins is still scarce, and little is known about the structural determinants of their binding mechanism to chemokines. Here, we studied the structurally uncharacterized Evasin-4, which neutralizes a broad range of CC-motif chemokines, including the chemokine CC-motif ligand 5 (CCL5) involved in atherogenesis. Crystal structures of Evasin-4 and E66S CCL5, an obligatory dimeric variant of CCL5, were determined to a resolution of 1.3–1.8 Å. The Evasin-4 crystal structure revealed an L-shaped architecture formed by an N- and C-terminal subdomain consisting of eight β-strands and an α-helix that adopts a substantially different position compared with closely related Evasin-1. Further investigation into E66S CCL5–Evasin-4 complex formation with NMR spectroscopy showed that residues of the N terminus are involved in binding to CCL5. The peptide derived from the N-terminal region of Evasin-4 possessed nanomolar affinity to CCL5 and inhibited CCL5 activity in monocyte migration assays. This suggests that Evasin-4 derivatives could be used as a starting point for the development of anti-inflammatory drugs.

In order to obtain uniformly 13 C, 15 N-labeled proteins, bacteria containing met-Evasin-4 pET30a or E66S MKKKWPR-CCL5 CCL5 pET24a vector were grown in LB media as described above. After OD600 reached 0.6-0.8, cells were harvested by centrifugation at 4,000 rpm at 4°C and transferred to M9 media. Prior to induction with IPTG, cells were incubated in media for 1 h to deplete internal carbon and nitrogen sources. At induction, media was supplemented with 1 g/L of 13 C-glucose and 15 NH4Cl. To achieve a higher level of stable isotope enrichment, the concentration of 13 C-glucose was measured using a domestic glucosemeter (Medisana). Typically, after 3-4 h about 80% of glucose was consumed and cells were harvested by centrifugation at 4,000 rpm for 20 min at 4°C. For 2 D, 13 C, 15 N-labeled met-Evasin-4, M9 media containing 70% D2O was used.
In the case of met-Evasin-4 pET30a pellets, the soluble fraction was dialyzed overnight against 0.5% acetic acid using a 3.5 kDa Spectra/Por RC membrane (Replige) and subsequently lyophilized. The lyophilized material was dissolved in a minimal volume of 6 M Gdn-HCl, 50 Mm Tris, pH 8, and diluted with 0.1 M Tris, 0.5 M NaCl, 1 mM EDTA, 2 mM GSH, 1 mM GSSG, 10% acetonitrile, pH 8 to a final concentration of 1 mg/ml and stirred overnight at 4°C.
The cleared lysate of His6-SUMO-Evasin-4 pET23a pellets was supplemented with 20 mM imidazole, applied to Ni-NTA Agarose (Quagen) and stirred for 1 h at RT. The suspension was transferred to an empty PD10 column and filtered under mild vacuum. Then, Ni-NTA Agarose was washed with one column volume of lysis buffer. Bound proteins were eluted by one column volume of 20 mM phosphate, 0.5 M NaCl, 0.5 M imidazole, pH 7.4. Eluted fractions were dialyzed against PBS buffer, pH 7.4. In order to remove the His6-SUMO tag, 5 U/ml of SUMO protease 1 (Sigma-Aldrich) and 0.6 mg of GSH were added and the resulting solution was gently stirred at 30℃. After cleavage completion, the solution was diluted 4 times by 0.1 M Tris, 0.5 M NaCl, 1 mM EDTA, 2 mM GSH, 1 mM GSSG, 10% acetonitrile, pH 8 and stirred overnight at 4°C. The folded proteins were purified by HPLC using a 22 mm x 250 mm Vydac C18 column, analyzed by LC-MS and lyophilized.

E66S CCL5 folding and purification.
Pellets were resuspended in 50 mM Tris, 2 mM MgSO4, pH 8 and lysed with 1x Bugbuster (Novagen) and 0.1 U/ml benzonaze ® nuclease (Sigma-Aldrich). The insoluble fraction was removed by centrifugation at 10,000 rpm for 20 min at 4°C, washed with 50 mM, 0.5% Tween-20, Tris, pH 8 and twice with 50 mM Tris, pH 8. Then, the insoluble fraction was dissolved in 0.1 M Tris, 200 mM DTT, pH 8, 6 M Gdn-HCl and dialyzed overnight against 0.5% acetic acid using 3.5 kDa Spectra/Por RC membrane (Repligen) and lyophilized. Lyophilized material was then dissolved in a minimal volume of 6 M Gdn-HCl, 50 mM Tris, pH 8, then diluted with 6 M Gdn-HCl, 50 Mm Tris, 10 mM cysteine, 1mM cystine, pH 8 to a final concentration of 1 mg/ml and stirred overnight at 4°C. The folded protein was purified by HPLC using a 22 mm x 250 mm Vydac C18 column, analyzed by LC-MS and S3 lyophilized. In order to remove MKKKWPR-tag, the purified protein was dissolved in 100 mM ammonium bicarbonate buffer pH 8.5 and treated with trypsin (Sigma-Aldrich). After cleavage completion, E66S CCL5 was purified by HPLC and lyophilized.
The obligatory CCL5 monomer which contains a methylated amide nitrogen of T7 ([NMe-7 T] CCL5) (1) was synthesized using Boc-based solid-phase peptide synthesis (SPPS) and native chemical ligation (NCL). In short, the N-terminal NH2-Ser 1 -Lys 33 -MPAL-COOH and C-terminal NH2-Cys 34 -Ser 68 -CONH2 segment were synthesized on Boc-Leu-PAM and MBHA resin, respectively. After chain assembly, the peptides were deprotected and cleaved from the resin by treatment with anhydrous hydrogen fluoride (HF) for 1 h at 0°C, using 4% (v/v) p-cresol as a scavenger. Crude peptides were lyophilized and purified by HPLC as described above. To allow NCL, unprotected peptides (10 mg/ml) were dissolved in 6 M Gdn-HCl, 100 mM Tris buffer, pH 8, containing 1% (v/v) benzylmercaptan and thiophenol and incubated at 37℃. Reaction progress was analyzed by analytical HPLC and ESI/MS or with UPLC-MS (Fig. S7). Ligated material was purified by HPLC, lyophilized and subjected to oxidative folding. Oxidative folding of the proteins was performed at 4℃ in 1 M Gdn-HCl, 100 mM Tris buffer, pH 8.0, containing 1 mM cystine/10 mM cysteine as a redox couple. After folding completion, [NMe-7 T] CCL5 was purified by HPLC and lyophilized (Fig. S8).
For labeling with a fluorescent probe, Boc-Lys(Fmoc)-OH was coupled to Ev4 E14-N31 and Ev4 E14-A23 peptidyl resin. The fluorenylmethoxycarbonyl (Fmoc) protecting group was removed by treatment by 20% piperidine in DMF. Subsequently, excess of 5-(and-6)-Carboxyfluorescein, Succinimidyl Ester (5(6)-Fam, SE) in DMF was added to the peptidyl resin in the presence of DIPEA and incubated overnight at 37°C and constant shaking. Then the peptidyl resin was deprotected by TFA and peptides were cleaved from resin by HF treatment. Labeling yielded two isomers with the ratio close to 1:1. Isomers were purified, pooled together and used as a mixture. Table S1. Gene sequences used for expression.

catatggaagtaccccaaatgacttcaagttcagctccagatctagaagaagaagatgat H M E V P Q M T S S S A P D L E E E D D tacaccgcgtatgcgccgctgacctgctacttcaccaacagcaccctgggtctgctggct Y T A Y A P L T C Y F T N S T L G L L A ccgccgaactgcagcgtgctgtgcaacagcaccaccacctggttcaacgagaccagcccg P P N C S V L C N S T T T W F N E T S P aacaacgcgagctgcctgctgaccgttgactttctgacccaggatgcgatcctgcaggaa N N A S C L L T V D F L T Q D A I L Q E aaccaaccgtacaactgcagcgttggtcactgcgacaatggcacctgcgcgggtccgccg N Q P Y N C S V G H C D N G T C A G P P cgtcatgcgcaatgctggtaagaattc R H A Q C W -E F
His6-SUMO-