Crystallization and preliminary X-ray crystallographic analysis of agkicetin-C from Deinagkistrodon acutus venom

Abstract

The crystallization and preliminary crystallographic analysis of agkicetin-C, a well known platelet glycoprotein Ib (GPIb) antagonist from the venom of Deinagkistrodon acutus found in Anhui Province, China is reported. Crystals of agkicetin-C suitable for structure determination were obtained from 1.8 M ammonium sulfate, 40 mM MES pH 6.5 with 2%(v/v) PEG 400. Interestingly, low buffer concentrations of MES seem to be necessary for crystal growth. The crystals of agkicetin-C belong to space group C2, with unit-cell parameters a = 177.5, b = 97.7, c = 106.8 A, beta = 118.5 degrees, and diffract to 2.4 A resolution. Solution of the phase problem by the molecular-replacement method shows that there are four agkicetin-C molecules in the asymmetric unit, with a VM value of 3.4 A3 Da(-1), which corresponds to a high solvent content of approximately 64%. Self-rotation function calculations show a single well defined non-crystallographic twofold axis with features that may represent additional elements of non-crystallographic symmetry.

Figure 1

Purification of agkicetin-C. (a) 1 g crude venom from D. acutus was dissolved in 20 ml buffer A (20 mM Tris–HCl pH 8.0) and centrifuged at 12 000 rev min⁻¹ for 15 min. The supernatant was applied onto a DEAE-Sepharose Fast Flow column (1.6 × 40 cm) pre-equilibrated with buffer A. The column was washed at a flow rate of 120 ml h⁻¹ initially with buffer A until peaks 1 and 2 were eluted and then with an 800 ml linear NaCl gradient (0–200 mM in buffer A). Finally, the column was exhaustively eluted with a high concentration of NaCl (500 mM in buffer A). Peak 4 containing agkicetin-C was pooled. (b) Peak 4 mentioned above was condensed and dialyzed against buffer B (50 mM sodium citrate pH 5.0) overnight at 277 K and then applied onto a CM-Sepharose Fast Flow column (1.6 × 40 cm) pre-equilibrated with buffer B. A similar strategy to the linear NaCl gradient used above was applied with buffer B and peak 2 was pooled. (c) Peak 2 mentioned above was condensed and dialyzed against buffer A overnight at 277 K and then applied onto a Mono Q HR 5/5 column (3.6 × 100 mm) pre-equilibrated with buffer A. The column was washed at a flow rate of 60 ml h⁻¹ initially with 5 ml buffer A and then with a linear NaCl gradient (0–200 mM in buffer A). Finally, the column was eluted with a high concentration of NaCl (500 mM in buffer A) as mentioned above. The main fraction collected proved to be agkicetin-C.

Figure 2

Preparation of reduced and S-pyridylethylated agkicetin-C. 3 mg agkicetin-C was dissolved in 0.5 ml 0.5 M Tris–HCl buffer pH 8.0 containing 6 M guanidine hydrochloride and 2 mM EDTA. 7 mg dithiothreitol was added to the protein solution, mixed and then incubated for 3 h at 323 K. After the addition of 4-vinylpyridine with a 3:1 molar ratio of 4-vinylpyridine:dithiothreitol, the mixture was further incubated for another 3 h at 323 K and then dialyzed against distilled water and 0.1% TFA solution. Finally, the mixture was applied onto a Delta-Pak C4 column (3.9 × 150 mm) and sequentially eluted at room temperature with two linear gradients of acetonitrile solution (0–20% and 20–50% containing 0.1% TFA).

Figure 3

Crystals of agkicetin-C from 1.8 M ammonium sulfate, 2% PEG 400, 40 mM MES pH 6.5.