J. Biochem. 107, 84-86 (1990)

Preliminary Crystallographic Study of Phospholipase A2 from the Venom of Trimeresurus flavoviridis (Habu Snake)1 YoshihiroShinoda,* AkiraShouji,* Takashi Yamane,' AtsuoSuzuki,* Tamaichi Ashida,* 2 Hiroshi Kihara,** and Motonori Ohno*** 'Department of Applied Chemistry, Faculty of Engineering, Nagoya University, Chikusa-ku, Nagoya, Aichi 464; "Department of Physiology, Faculty of Medicine, Kagoshima University, Kagoshima, Kagoshima 890; and '"Department of Chemistry, Faculty of Science, Kyushu University, Higashi-ku, Fukuoka, Fukuoka 812 Received for publication, July 25, 1989

Crystallization and a preliminary crystallographic study of Trimeresurus flavoviridis (habu snake) phospholipase A2 (PLA2) were carried out. Although crystals were obtained from various solutions, crystals suitable for X-ray analysis could be obtained from polyethylene glycol solutions only when a repeated seeding technique was applied starting from twinned crystals. The crystal is monoclinic with space group P2,, with a = 44.1, b = 55.7, c = 48.8A, and £ = 92.4'. An asymmetric unit contains a dimer consisting of two identical subunits made of 122 amino acids. The crystal reflects X-rays beyond 2.5 A. A Pt derivative gave a good isomorphous crystal.

Phospholipase A2 (PLA2) [EC 3.1.1.4] hydrolyzes the /?-eater linkage of phosphatidylcholine in the presence of Ca2+ to produce lysophosphatidylcholine and fatty acids. It occurs in snake and bee venom as a major component or is secreted from the mammalian pancreas. The amino acid sequences of this enzyme from different origins are highly homologous, though there are significant differences in the details of the catalytic function. Pancreatic PLA2 is monomeric, while snake venom PLA2 is active as a dimer. The crystal structure of bovine pancreatic PLA2 and that of porcine pancreatic PLA2 have already been studied (1-4), while for snake venom PLA2 the structure of only the Ca-free form from Crotalus atrox is known (5). Phospholipase A2 from the venom of Trimeresurus flavoviridis (habu snake) was isolated by Ishihara et al. (6). It is composed of two identical subunits of a single polypeptide chain of 122 amino acids (7). In order to elucidate calcium modulated changes of its tertiary and quaternary structure and to obtain further information on the catalytic mechanism, an X-ray crystallographic study of the present PLA2 was undertaken. EXPERIMENTAL PROCEDURES Crystallization--Thin rhombic crystals were easily grown from a water solution. They belonged to space group P2,, with a=35.2,6=55.5, c = 66.0 A, and£ = 90\ Vm (8) was 2.34 A3/Da if two dimers were included in the unit cell. Polyethylene glycol (PEG) solutions gave crystals with the hanging drop vapor diffusion method; protein at 7.5 mg/ml was dissolved in l m M acetate buffer (pH 4.5-5.5). The reservoir solution was the same buffer containing 5% PEG (At- 6,000) and 2 mM CaCl2. One droplet was composed of ' This work was supported in part by a Grant-in-Aid for Scientific Research (No. 63580044) from the Ministry of Education, Science and Culture of Japan and by a Grant from Asahi Glass Foundation. * To whom correspondence should be addressed. Abbreviations: PLA,, phospholipase A,: PEG, polyethylene glycol.

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5 /xl reservoir solution and an equal amount of the protein solution. In one or two days crystals appeared. The crystals were essentially the same as those from water solutions. They were, however, heavily twinned and not suitable for the X-ray structure determination. Ammonium sulfate solutions also gave crystals with a maximum dimension of 0.5 mm by the hanging drop vapor diffusion method; the crystal was cubic with space group 14,32 (or I4332) with a = 210 A, and an asymmetric unit contained six subunits with ym = 2.34A 3 /Da. Thus crystals were rather easily obtained, but not all of them were suitable for precise X-ray structure determination. After several trials, however, good crystals could be obtained with a repeated seeding technique starting from twinned crystals. The protein and reservoir solutions were prepared with the same buffer solution as that of the PEG solutions mentioned above, the pH being set to the optimum value of 4.9. To the reservoir solution, 1.5% (w/v) 1,4-dioxane was added. A 10//I mixed droplet was seeded with crushed crystals, those obtained from the PEG solutions. Tiny crystals appeared in 4-7 days. They were washed several times with a 1 mM acetate buffer with 2.5% (w/v) PEG. Then the crystallization was continued under the same conditions. A few large crystals appeared as oblique blocks with maximum dimensions of 0.4 X 0.3 X 0.2 mm within a week after seeding (Fig. 1). Precession photographs (Fig. 2) showed that these crystals were not twinned and that they belong to the monoclinic system, space group P2,, with a = 44.1, 6=55.7, c=48.8A, 0 = 92.4*, and the unit cell volume was 1.198 x 10s A3. The Vm value was 2.18 A3/Da if an asymmetric unit contained a dimer, the solvent content in the crystal being 42% by volume. Heavy-Atom Derivatives—Heavy atom replaced crystals were prepared by soaking in 5 mM acetate buffer (pH 4.9) containing 8% (w/v) PEG6000. K2PtCl, solution (2 mM) yielded promising replaced crystals. The unit cell dimensions were: o=44.1, 6=55.8, c=48.9 A, and /9 = 92.2\ J. Biochem.

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Preliminary X-Ray Study of Snake Venom PhosphoUpase A2

RESULTS AND DISCUSSION

Fig. 1. A single crystal of Trimeresurus flavoviridis PLA2 from a PEG solution.

Crystallization—The protein crystallizes easily, but it mostly gives twinned crystals. They could not be separated mechanically. Trials of crystallization with various protein concentrations and/or addition of 1,4-dioxane reduced twinning, but did not eliminate it. A trial of getting true intensities by an analytical method using the intensity data also failed to give satisfactory results. The crystals from water are fragile and not suitable for preparing heavy atom derivatives. The crystals from ammonium sulfate solutions contain too many independent subunits in an asymmetric unit to make a high resolution structure analysis feasible. But finally the repeated seeding technique produced a good single crystal. It is to be noted that essentially different crystals grew from the seed crystals. The crystals thus obtained have the same symmetry and almost the same b axis length as those of the twinned crystals used as the seed, but definitely different a and c axis lengths and unit cell volume. The repeated seeding technique appears to be a powerful one in protein crystallization experiments. Preliminary X-Ray Study—The mean isomorphous difference (Sl-th-^pl/S^p) w a s 0.13, and the mean Bijvoet difference was 0.049, for 796 independent reflections. The isomorphous and anomalous difference Patterson maps synthesized at 6.5 A resolution are shown in Fig. 3, a major heavy atom site being clearly shown in the maps. The refinement of the heavy atom parameters showed that the isomorphism of this derivative is not good for phase angle determination beyond 6.5 A resolution. The mean figure of merit is currently 0.66 for 447 reflections within 15 to 6.5 A resolution range. Further study by the molecular replace-

Fig. 2. Precession photograph (if = 15') of the (Ml) zone of a Trimeresurus flavoviridis PLA, crystal.

X-Ray Experiment—Preliminary X-ray experiments were done with a Precession camera. The intensity data of the native and K^PtCU -replaced crystals were collected on a Rigaku four-circle diffractometer (AFC-5) mounted on a rotating-anode X-ray generator (50 kV, 54 mA, fine focus), using graphite monochromated CuKa radiation with A = 1.5418 A. The ordinate analysis method (9) was applied, and the absorption correction was made based on the azimuthal reflection intensity. The deterioration of crystals was monitored and corrected by standard reflections measured every 100 reflections. For the native crystal a total of 4,372 reflections up to 3.1 A resolution was collected with one crystal, of which the number of observed reflections with|F 0 |>3cr(F) was 3,730. The intensities of the derivative crystal were collected up to 5 A resolution including Bijvoet pairs. Vol. 107, No. 1, 1990

Fig. 3. Harker sections at u = l/2 of the (a) isomorphous and (b) anomalous difference Patterson functions calculated at 6.5 A resolution. The cross represents the self-vector from the major heavy atom binding site. Contours are drawn in arbitrary units at equal intervals.

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ment method and the search for the second derivative are being carried out REFERENCES 1. Dijkstra, B.W., Kalk, K.H., Hoi, W.G.J., & Drenth, J. (1981) J. Mol. Biol. 147, 97-123 2. Dijkstra, B.W., Renetseder, R, Kalk, K.H., Hoi, W.G.J., & Drenth, J. (1983) J. Mol BioL 168, 163-179 3. Dijkstra, B.W., van Nes, G.J.H., Kalk, K.H., Brandenburg, N.P., Hoi, W.G.J., & Drenth, J. (1982) Acta CrystaUogr. B38, 793-

799 - Renetaeder, R., Dijkstra, B.W., Hniringa, K., Kalk, K.H., & Drenth, J. (1988) J. MoL BioL 200, 181-188 5. Brunie, S., Bolin, J., Gewirth, D., & Sigler, P.B. (1985) J. Biol. Chan. 260, 9742-9749 6. Iahimura, K., Kihara, H., & Ohno, M. (1980) J. Biochem. 88, 443-451 7. Tanaka, S., Mohri, N., Kihara, H., & Ohno, M. (1986) J. Biochem. 99, 281-289 8. Matthews, B.W. (1968) J. MoL BioL 33, 491-497 9. Tickel, I.J. (1975) Acta CrystaUogr. B31 329-331

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J. Biochem.

Preliminary crystallographic study of phospholipase A2 from the venom of Trimeresurus flavoviridis (habu snake).

Crystallization and a preliminary crystallographic study of Trimeresurus flavoviridis (habu snake) phospholipase A2 (PLA2) were carried out. Although ...
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