Crystallization and preliminary X-ray diffraction analysis of a novel Arg49 phospholipase A₂ homologue from Zhaoermia mangshanensis venom

A single crystal of zhaoermiatoxin with maximum dimensions of 0.2 × 0.2 × 0.5 mm was used for X-ray diffraction data collection to a resolution of 2.05 Å using synchrotron radiation and the diffraction pattern was indexed in the hexagonal space group P6₄, with unit-cell parameters a = 72.9, b = 72.9, c = 93.9 Å.

Abstract

Zhaoermiatoxin, an Arg49 phospholipase A₂ homologue from Zhaoermia mangshanensis (formerly Trimeresurus mangshanensis, Ermia mangshanensis) venom is a novel member of the PLA₂-homologue family that possesses an arginine residue at position 49, probably arising from a secondary Lys49→Arg substitution that does not alter the catalytic inactivity towards phospholipids. Like other Lys49 PLA₂ homologues, zhaoermiatoxin induces oedema and strong myonecrosis without detectable PLA₂ catalytic activity. A single crystal with maximum dimensions of 0.2 × 0.2 × 0.5 mm was used for X-ray diffraction data collection to a resolution of 2.05 Å using synchrotron radiation and the diffraction pattern was indexed in the hexagonal space group P6₄, with unit-cell parameters a = 72.9, b = 72.9, c = 93.9 Å.

1. Introduction

Lys49 phospholipase A₂ homologues (Lys49 PLA₂s) belong to a subfamily of snake-venom group II PLA₂s in which Asp49 is replaced by a lysine residue (reviewed by Ownby et al., 1999 ▶; Murakami & Arni, 2003 ▶). These proteins are considered to be catalytically inactive owing to their inability to bind the cofactor (a calcium ion) and thus stabilize the tetrahedral intermediate observed in the calcium-dependent catalytic reaction promoted by Asp49 PLA₂s (van den Bergh et al., 1989 ▶). Despite their catalytic inactivity, Lys49 PLA₂s are very potent in the induction of myonecrosis, a major and clinically highly challenging event in envenomation by most viperid snakes, based on one or more mechanisms that continue to be enigmatic to toxinologists. Consequently, a number of different strategies, such as structural analysis (Arni & Ward, 1996 ▶; Ownby et al., 1999 ▶; Murakami & Arni, 2003 ▶), chemical modification (Andrião-Escarso et al., 2000 ▶; Soares et al., 2001 ▶), sequence-comparison analyses (Selistre de Araujo et al., 1996 ▶; Ward et al., 1998 ▶), charge-distribution analysis (Kini & Iwanaga, 1986 ▶; Kini & Evans, 1989 ▶), hydrophobicity profiles (Kini & Iwanaga, 1986 ▶), synthetic peptide studies (Lomonte et al., 1994 ▶; Nuñez et al., 2001 ▶) and site-directed mutagenesis (Ward et al., 2002 ▶), have attempted to elucidate the structural determinants for the myotoxicity expressed by Lys49 PLA₂s. The three-dimensional structures of a large number of Lys49 PLA₂s in both native and complexed states have recently been elucidated (Arni et al., 1995 ▶; Lee et al., 2001 ▶; Ambrosio et al., 2005 ▶; Murakami et al., 2005 ▶, 2006 ▶, 2007 ▶) and have contributed to addressing the role of Lys122 in the myotoxicity of Lys49 PLA₂s (Ambrosio et al., 2005 ▶; Watanabe et al., 2005 ▶).

Compounds such as suramin, heparin, heparin-like glycosaminoglycans, related polyanions and polyethylene glycol derivatives have been shown to effectively inhibit the activity of myotoxic Lys49 PLA₂s both in vitro and in vivo (Murakami et al., 2005 ▶, 2007 ▶). Recently, a new role has been proposed for Lys49 PLA₂s in the inhibition of the vascular endothelial growth factor and its receptor system, which plays a central role in angiogenic processes (Yamazaki et al., 2005 ▶).

In this work, we report the crystallization and preliminary X-ray diffraction analysis of zhaoermiatoxin, isolated from the venom of the Chinese pit viper Zhaoermia mangshanensis, which contains a Lys49→Arg substitution and represents a novel member of the catalytically inactive PLA₂-homologue subfamily (Mebs et al., 2006 ▶). Zhaoermiatoxin causes vasodilatation and oedema and also acute myonecrosis as part of a synergistic system which contributes to the overall oedematous and myotoxic response caused by the venom.

2. Materials and methods

2.1. Protein purification

Zhaoermiatoxin was purified from the crude venom of an adult male Z. mangshanensis following a two-step purification procedure which consisted of gel filtration on Sephadex G-100 followed by cation-exchange chromatography on a Resource S column as described previously (Mebs et al., 2006 ▶). Prior to the crystallization trials, the zhaoermiatoxin sample was further purified by high-performance liquid chromatography (HPLC) on a C18 reversed-phase (RP) column. Standard SDS–PAGE performed according to Laemmli (1970 ▶) indicated the presence of zhaoermiatoxin as a single band with an apparent molecular weight of approximately 14 000 Da; the theoretical value obtained from the sequence (121 amino-acid residues, 14 of which are cysteines) was calculated to be 13 971.3 Da (Mebs et al., 2006 ▶). Protein concentrations were measured by the Bradford method (Bradford, 1976 ▶).

2.2. Dynamic light scattering

Dynamic light-scattering experiments were carried out using a DynaPro 810 (Protein Solutions) equipped with a temperature stabilizer. A 1.0 mg ml⁻¹ protein solution was prepared in 0.1 M HEPES pH 7.5. Standard curves of bovine serum albumin were used for calibration and the experiments were conducted at 291 K.

2.3. Crystallization

The sample was dissolved to a concentration of 30 mg ml⁻¹ in 20 mM HEPES buffer pH 7.5. Crystallization was performed by the hanging-drop vapour-diffusion method using 24-well tissue-culture plates. Initial trials were carried out by the sparse-matrix method with some modifications using Crystal Screen HR2-110 and HR2-112 kits (Hampton Research). Typically, 1 µl protein solution was mixed with an equal volume of screening solution and equilibrated over 0.6 ml of the latter in the reservoir. Large single crystals were obtained when a 1.5 µl protein droplet was mixed with an equal volume of reservoir solution consisting of 0.2 M ammonium sulfate, 0.1 M sodium cacodylate trihydrate pH 6.5 and 30%(w/v) polyethylene glycol 8000. The crystals were obtained after equilibrating the above solution for two months at room temperature.

2.4. X-ray diffraction data collection and analysis

X-ray diffraction data were collected at the DB03-MX1 beamline at the Laboratório Nacional de Luz Síncrotron (LNLS, Campinas, Brazil; Polikarpov et al., 1998 ▶). The crystal was transferred to a cryoprotectant solution containing 10% glycerol and flash-frozen in a gaseous nitrogen stream at 100 K. The wavelength of the radiation source was set to 1.427 Å and a MAR CCD detector was used to record the X-ray diffraction data. The crystal-to-detector distance was set to 95 mm. The data were indexed, integrated, reduced and scaled using the DENZO and SCALEPACK programs from the HKL program suite (Otwinowski & Minor, 1997 ▶).

3. Results and discussion

Structural homogeneity in solution was demonstrated by the dynamic light-scattering experiments, which presented a monomodal distribution. Single crystals with dimensions of 0.2 × 0.2 × 0.5 mm (Fig. 1 ▶) were used for the X-ray diffraction experiments and the data were collected to 2.05 Å resolution (Fig. 2 ▶) under cryogenic conditions (100 K). The diffraction data were indexed in space group P6, with unit-cell parameters a = 72.9, b = 72.9, c = 93.9 Å. An examination of the systematic absences indicated that the crystals belonged to either space group P6₂ or its enantiomorph P6₄.

Figure 1.

Photomicrograph of a zhaoermiatoxin crystal.

Figure 2.

X-ray diffraction pattern of zhaoermiatoxin crystals with a different contrast level at low and high resolution.

Processing of the 305 151 measured reflections to 2.05 Å resolution led to 17 842 unique reflections with an R _merge of 7.1% (33.3% in the last shell, 2.12–2.05 Å resolution) and a completeness of 99.9% (99.86% in the last shell). Taking into consideration the molecular weight of 13 972 Da (Mebs et al., 2006 ▶), one dimer is present in the asymmetric unit, with a corresponding Matthews coefficient of 2.44 ų Da⁻¹ and a solvent content of 49.25% (Matthews, 1968 ▶). Data-processing statistics are presented in Table 1 ▶.

Table 1. Data-collection and processing statistics.

Values in parentheses are for the highest resolution shell.

Data collection
Source	Synchrotron radiation
Temperature (K)	100
Detector	MAR CCD
Cryoprotectant	10% glycerol
Data-processing software	DENZO/SCALEPACK
Data collection and processing
Wavelength (Å)	1.427
Space group	P64
Unit-cell parameters (Å)	a = 72.9, b = 72.9, c = 93.9
Matthews coefficient (Å3 Da−1)	2.44
Solvent content (%)	49.25
No. of molecules in the asymmetric unit	2
No. of unique reflections	17842
No. of observed reflections	305151
Redundancy	12.5 (9.5)
Resolution range (Å)	25.0–2.05 (2.12–2.05)
Completeness (%)	99.9 (99.8)
Rmerge† (%)	7.1 (33.3)
〈I/σ(I)〉	33.4 (7)
Wilson B factor (Å2)	39.1

^† R _merge = , where I_i(hkl) is the ith intensity measurement of reflection hkl, including symmetry-related reflections, and 〈I(hkl)〉 is its average.

The atomic coordinates of the Lys49 PLA₂ from Bothrops asper venom, which displays a sequence identity of 70%, were used to generate a search model (Murakami et al., 2005 ▶; PDB code 1y4l) and molecular-replacement calculations were carried out using the program AMoRe in the resolution range 25.0–4.0 Å (Navaza, 1994 ▶). A clear solution was obtained for two molecules in the asymmetric unit in space group P6₄. REFMAC5 (Murshudov et al., 1997 ▶) was used for rigid-body refinement of this solution in the resolution range 25.0–3.0 Å (excluding 5% of reflections for R _free calculations), resulting in a correlation coefficient of 60.3% and an R factor of 40.1% (R _free = 48.3%). Structure refinement and analysis are currently in progress.