- Email: [email protected]
Crystallization and preliminary X-ray diffraction analysis of a myotoxic phospholipase A2 homologue from Bothrops neuwiedi pauloensis venom
Biochimica et Biophysica Acta 1432 (1999) 393^395 www.elsevier.com/locate/bba
Rapid report
Crystallization and preliminary X-ray di¡raction analysis of a myotoxic phospholipase A2 homologue from Bothrops neuwiedi pauloensis venom Marcos R.M. Fontes a; *, Andreimar M. Soares b , Veridiana M. Rodrigues b , Andre¨ C. Fernandes a , Reinaldo J. Da Silva c , Jose¨ R. Giglio b a
Departamento de F|¨sica e Biof|¨sica, Instituto de Biocieªncias, UNESP, C.P. 510, CEP 18618-000, Botucatu-SP, Brazil b Departamento de Bioqu|¨mica, Faculdade de Medicina de Ribeira¬o Preto, USP, Ribeira¬o Preto-SP, Brazil c Centro de Estudo de Venenos e Animais Pec° onhentos, UNESP, Botucatu-SP, Brazil Received 12 May 1999; accepted 21 May 1999
Abstract î Crystals of a myotoxic phospholipase A2 from Bothrops neuwiedi pauloensis have been obtained. They diffracted at 2.5 A resolution using a synchrotron radiation source and belong to space group P31 21. Preliminary analysis shows that there are two molecules in the asymmetric unit. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Crystallization ; Lys-49 myotoxin; Phospholipase A2 ; Snake venom
In Latin America, snakes of the genus Bothrops are responsible for most of the ophidian accidents [1^3]. In addition to systemic alterations, these poisonings are characterized by prominent local tissue damage due to myonecrosis, hemorrhage and edema [4]. Acute muscle damage induced by these venoms is mainly due to one or two basic myotoxic phospholipases A2 (PLA2 ) [5]. Phospholipases A2 (EC 3.1.1.4) hydrolyze the sn-2 acyl group of phospholipids and release fatty acids and lysophospholipids [6]. However, in addition to their catalytic role, PLA2 s cause diverse pharmacological e¡ects, including myonecrosis, which may be due to a direct action of myotoxic phospholipases A2 upon the plasma membranes, muscle cells or, indiAbbreviations: PLA2 , phospholipase A2 ; BnSP-7, myotoxin-II from Bothrops neuwiedi pauloensis * Corresponding author. Fax: +55 (14) 8213744; E-mail: [email protected]
rectly, to vascular degeneration and ischemia caused by hemorrhage. It is believed that mycotoxins do act on the sarcoplasma membrane, thus inducing a disorganization of the phospholipids, the loss of intracellular components and an in£ux of calcium ions [7^ 9]. PLA2 s with skeletal muscle-damaging activity are widely distributed among venomous snakes and are usually classi¢ed into two categories: enzymatically active (Asp-49) and enzymatically inactive (Lys-49) PLA2 s [10]. In the last years, the isolation, characterization and crystallization of several myotoxins [11^18], as well as some structural studies [19^22], have been reported. We have recently studied Bothrops neuwiedi venoms from di¡erent regions and identi¢ed two basic myotoxins solely in B. neuwiedi pauloensis, a subspecies from Sa¬o Paulo State, Botucatu city region [23] and we report now the crystallization experiments
0167-4838 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 9 9 ) 0 0 1 2 0 - X
BBAPRO 30442 5-7-99
394
M.R.M. Fontes et al. / Biochimica et Biophysica Acta 1432 (1999) 393^395
and preliminary X-ray di¡raction data of myotoxinII (BnSP-7) from this venom. The venom was collected individually from several snakes and BnSP-7 was puri¢ed as previously described [23]. The lyophilized sample of BnSP-7 was dissolved in ultra-pure water at a concentration of 12 mg/ml. The sparse matrix method [24] was used to perform initial screening of the crystallization conditions. Small hexagonal crystals of BnSP-7 were obtained by the conventional hanging drop vapor di¡usion method [25] in which the protein solution was equilibrated against a reservoir containing 0.1 M sodium cacodylate pH 6.5, 0.2 M ammonium sulfate and 30% (w/v) polyethylene glycol 8000, after 2 days. In order to improve the crystal quality, we have made some modi¢cations in the original reservoir solution. The better crystals were obtained with 0.1 M sodium cacodylate pH 6.7, 0.2 M ammonium sulfate and 27% (w/v) polyethylene glycol 6000 and they measured approx. 0.15U0.25U0.25 mm after 7^10 days (Fig. 1). X-Ray di¡raction data of a single BnSP-7 crystal î (at 7³C) were collected at a wavelength of 1.38 A using a Synchrotron Radiation Source (Laborato¨rio Nacional de Luz Sincrotron, LNLS, Campinas, Brazil) and a 30 cm MAR imaging plate detector (MAR Research). The crystal detector distance was 200 mm and it was used at an oscillation range of 1³, resulting in 40 images collected. The data were processed î resolution using the DENZO program and to 2.5 A scaled by SCALEPACK program [26].
Table 1 X-Ray di¡raction data collection for BnSP-7 from B. neuwiedi pauloensis Space group P31 21 î Cell dimensions a = b = 58.2, c = 132.0 A Number of unique re£ections 10 146 (I s 2c(I)) î Maximum resolution 2.5 A î Completeness at 2.5 A 90.2% 5.4% Rasym î 3 /Da 2.31 A VM = V/Mw Molecules in the asymmetric 2 unit Calculated density 1.19 g/cm3 Solvent 48% P P P a Rsym = h i MI hi 3GI h fM= h GI h f where Ih is the observed intensity and GIh f is the mean intensity of re£ection h over all measurements of Ih .
The data collection statistics for BnSP-7 crystals are shown in Table 1. A total of 15 425 re£ections were measured from which 10 146 were independent with I s 2c(I). The merging of all equivalent re£ections resulting in a data set is about 90.2% complete î with Rmerge = 5.4%, as shown in Table 1. The at 2.5 A crystals belong to the trigonal system, space group P31 21 and have average unit cell dimensions î. a = b = 58.2, c = 132.0 A î 3 compatThe volume of the unit cell is 388.103 A ible with a dimer in the asymmetric unit with a VM î 3 /Da. Assuming (volume per Dalton) value of 2.31 A 3 a value of 0.74 cm /g for the protein partial speci¢c volume, the calculated solvent content in the crystal is 48% and the calculated crystal density is 1.19 g/ cm3 . The crystal structure is currently being determined using molecular replacement techniques implemented in the program AMoRe [27] and the coordinates of Lys-PLA2 from Bothrops asper [20]. Acknowledgements
Fig. 1. Crystal of BnSP-7, maximum dimensions 0.5U0.5U 0.25 mm.
This work was supported by FAPESP, FUNDUNESP, CNPq and National Synchrotron Light Laboratory (LNLS) (Brazil). We thank Mr. J.R. Branda¬o Neto (LNLS) for his help during the X-ray data collection.
BBAPRO 30442 5-7-99
M.R.M. Fontes et al. / Biochimica et Biophysica Acta 1432 (1999) 393^395
References [1] G. Rosenfeld, in: W. Bucherl, E. Buckley, V. Deulofeu (Eds.), Venomous Animals and Their Venoms II, Academic Press, New York, 1971, pp. 345^384. [2] M.L. Ferreira, A.M. Moura-da-Silva, F.O.S. Franc°a, J.L. Cardoso, I. Mota, Toxicon 30 (1992) 1603^1608. [3] L.A. Ribeiro, V.A.F. Pires de Campos, M.J. Albuquerque, N.Y. Takoaka, Rev. Assoc. Med. Bras. 39 (1993) 4^7. [4] P. Rosenberg, in: W. Shier, D. Mebs (Eds.), Handbook of Toxinology, Marcel Dekker, New York, 1990, pp. 67^277. [5] J.M. Gutie¨rrez, B. Lomonte, Toxicon 33 (1995) 1405^1424. [6] L.L.M. van Deenen, G.H. de Haas, Advances in Lipid Research, vol. 2, Academic Press, New York, 1964, p. 167. [7] J.M. Gutie¨rrez, F. Chaves, J.A. Gene¨, B. Lomonte, Z. Camacho, K. Schosinsky, Toxicon 27 (1989) 735^746. [8] J.M. Gutie¨rrez, J. Nunez, C. D|¨az, A.C.O. Cintra, M.I. Homsi-Brandeburgo, J.R. Giglio, Exp. Mol. Pathol. 55 (1991) 217^229. [9] C. D|¨az, J.M. Gut|¨errez, B. Lomonte, Arch. Biochem. Biophys. 298 (1992) 135^142. [10] J.M. Maraganore, G. Merutka, W. Cho, W. Welches, F.J. Kezdy, R.L. Heinrikson, J. Biol. Chem. 259 (1984) 13839^ 13843. [11] M.I. Homsi-Brandeburgo, L.S. Queiroz, H. Santo-Neto, L. Rodrigues-Simioni, J.R. Giglio, Toxicon 26 (1988) 615^627. [12] I.I. Kaiser, J.M. Gutie¨rrez, D. Plummer, S.D. Aird, G.V. Odell, Arch. Biochem. Biophys. 278 (1990) 319^325. [13] B. Lomonte, J.M. Gutie¨rrez, M.F. Furtado, R. Otero, J.P. Rosso, O. Vargas, E. Carmona, M.E. Rovira, Toxicon 28 (1990) 1137^1146. [14] A.C.O. Cintra, S. Marangoni, B. Oliveira, J.R. Giglio, J. Protein Chem. 12 (1993) 57^64.
395
[15] L.C. Mancuso, M.M. Correa, C.A. Vieira, O.A.B. Cunha, J.-J. Lachat, H.S. Selistre de Arau¨jo, C.L. Ownby, J.R. Giglio, Toxicon 33 (1995) 615^626. [16] A.M. Soares, L.H. Anzaloni-Pedrosa, M.R.M. Fontes, R.J. da Silva, J.R. Giglio, J. Venomous Anim. Toxins 4 (1998) 137^142. [17] A.M. Soares, V.M. Rodrigues, M.I. Homsi-Brandeburgo, M.H. Toyama, F.R. Lombardi, R.K. Arni, J.R. Giglio, Toxicon 36 (1998) 503^516. [18] W.F. de Azevedo Jr., M.R.M. Fontes, R.J. Ward, F.R. Lombardi, A.C.O. Cintra, J.R. Giglio, R.K. Arni, Protein Pept. Lett. 5 (1998) 181^184. [19] D.R. Holland, L.L. Clancy, S.W. Muchmore, T.J. Rydel, H.M. Einspahr, B.C. Finzel, R.L. Heinrickson, K.D. Watenpaugh, J. Biol. Chem. 266 (1990) 17649^17656. [20] R.K. Arni, R.J. Ward, J.M. Gutie¨rrez, A. Tulinsky, Acta Cryst. D51 (1995) 311^317. [21] W.F. de Azevedo Jr., R.J. Ward, F.R. Lombardi, J.R. Giglio, A.M. Soares, M.R.M. Fontes, R.K. Arni, Protein Pept. Lett. 4 (1997) 329^334. [22] M.T. da Silva-Giotto, R.C. Garratt, G. Oliva, Y.P. Mascarenhas, J.R. Giglio, A.C.O. Cintra, W.F. de Azevedo Jr., R.K. Arni, R.J. Ward, Proteins Struct. Funct. Genet. 30 (1998) 442^454. [23] V.M. Rodrigues, A.M. Soares, A.C. Mancin, M.R.M. Fontes, M.I. Homsi-Brandeburgo, J.R. Giglio, Comp. Biochem. Physiol. 121A (1998) 215^222. [24] J. Jancarik, S.H. Kim, J. Appl. Cryst. 24 (1991) 409^411. [25] A. MacPherson, Preparation and Analysis of Protein Crystals, Wiley, New York, 1982. [26] Z. Otwinowski, W. Minor, Methods Enzymol. 276 (1997) 307^326. [27] J. Navaza, Acta Cryst. A50 (1994) 157^163.
BBAPRO 30442 5-7-99
Rapid report
Crystallization and preliminary X-ray di¡raction analysis of a myotoxic phospholipase A2 homologue from Bothrops neuwiedi pauloensis venom Marcos R.M. Fontes a; *, Andreimar M. Soares b , Veridiana M. Rodrigues b , Andre¨ C. Fernandes a , Reinaldo J. Da Silva c , Jose¨ R. Giglio b a
Departamento de F|¨sica e Biof|¨sica, Instituto de Biocieªncias, UNESP, C.P. 510, CEP 18618-000, Botucatu-SP, Brazil b Departamento de Bioqu|¨mica, Faculdade de Medicina de Ribeira¬o Preto, USP, Ribeira¬o Preto-SP, Brazil c Centro de Estudo de Venenos e Animais Pec° onhentos, UNESP, Botucatu-SP, Brazil Received 12 May 1999; accepted 21 May 1999
Abstract î Crystals of a myotoxic phospholipase A2 from Bothrops neuwiedi pauloensis have been obtained. They diffracted at 2.5 A resolution using a synchrotron radiation source and belong to space group P31 21. Preliminary analysis shows that there are two molecules in the asymmetric unit. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Crystallization ; Lys-49 myotoxin; Phospholipase A2 ; Snake venom
In Latin America, snakes of the genus Bothrops are responsible for most of the ophidian accidents [1^3]. In addition to systemic alterations, these poisonings are characterized by prominent local tissue damage due to myonecrosis, hemorrhage and edema [4]. Acute muscle damage induced by these venoms is mainly due to one or two basic myotoxic phospholipases A2 (PLA2 ) [5]. Phospholipases A2 (EC 3.1.1.4) hydrolyze the sn-2 acyl group of phospholipids and release fatty acids and lysophospholipids [6]. However, in addition to their catalytic role, PLA2 s cause diverse pharmacological e¡ects, including myonecrosis, which may be due to a direct action of myotoxic phospholipases A2 upon the plasma membranes, muscle cells or, indiAbbreviations: PLA2 , phospholipase A2 ; BnSP-7, myotoxin-II from Bothrops neuwiedi pauloensis * Corresponding author. Fax: +55 (14) 8213744; E-mail: [email protected]
rectly, to vascular degeneration and ischemia caused by hemorrhage. It is believed that mycotoxins do act on the sarcoplasma membrane, thus inducing a disorganization of the phospholipids, the loss of intracellular components and an in£ux of calcium ions [7^ 9]. PLA2 s with skeletal muscle-damaging activity are widely distributed among venomous snakes and are usually classi¢ed into two categories: enzymatically active (Asp-49) and enzymatically inactive (Lys-49) PLA2 s [10]. In the last years, the isolation, characterization and crystallization of several myotoxins [11^18], as well as some structural studies [19^22], have been reported. We have recently studied Bothrops neuwiedi venoms from di¡erent regions and identi¢ed two basic myotoxins solely in B. neuwiedi pauloensis, a subspecies from Sa¬o Paulo State, Botucatu city region [23] and we report now the crystallization experiments
0167-4838 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 9 9 ) 0 0 1 2 0 - X
BBAPRO 30442 5-7-99
394
M.R.M. Fontes et al. / Biochimica et Biophysica Acta 1432 (1999) 393^395
and preliminary X-ray di¡raction data of myotoxinII (BnSP-7) from this venom. The venom was collected individually from several snakes and BnSP-7 was puri¢ed as previously described [23]. The lyophilized sample of BnSP-7 was dissolved in ultra-pure water at a concentration of 12 mg/ml. The sparse matrix method [24] was used to perform initial screening of the crystallization conditions. Small hexagonal crystals of BnSP-7 were obtained by the conventional hanging drop vapor di¡usion method [25] in which the protein solution was equilibrated against a reservoir containing 0.1 M sodium cacodylate pH 6.5, 0.2 M ammonium sulfate and 30% (w/v) polyethylene glycol 8000, after 2 days. In order to improve the crystal quality, we have made some modi¢cations in the original reservoir solution. The better crystals were obtained with 0.1 M sodium cacodylate pH 6.7, 0.2 M ammonium sulfate and 27% (w/v) polyethylene glycol 6000 and they measured approx. 0.15U0.25U0.25 mm after 7^10 days (Fig. 1). X-Ray di¡raction data of a single BnSP-7 crystal î (at 7³C) were collected at a wavelength of 1.38 A using a Synchrotron Radiation Source (Laborato¨rio Nacional de Luz Sincrotron, LNLS, Campinas, Brazil) and a 30 cm MAR imaging plate detector (MAR Research). The crystal detector distance was 200 mm and it was used at an oscillation range of 1³, resulting in 40 images collected. The data were processed î resolution using the DENZO program and to 2.5 A scaled by SCALEPACK program [26].
Table 1 X-Ray di¡raction data collection for BnSP-7 from B. neuwiedi pauloensis Space group P31 21 î Cell dimensions a = b = 58.2, c = 132.0 A Number of unique re£ections 10 146 (I s 2c(I)) î Maximum resolution 2.5 A î Completeness at 2.5 A 90.2% 5.4% Rasym î 3 /Da 2.31 A VM = V/Mw Molecules in the asymmetric 2 unit Calculated density 1.19 g/cm3 Solvent 48% P P P a Rsym = h i MI hi 3GI h fM= h GI h f where Ih is the observed intensity and GIh f is the mean intensity of re£ection h over all measurements of Ih .
The data collection statistics for BnSP-7 crystals are shown in Table 1. A total of 15 425 re£ections were measured from which 10 146 were independent with I s 2c(I). The merging of all equivalent re£ections resulting in a data set is about 90.2% complete î with Rmerge = 5.4%, as shown in Table 1. The at 2.5 A crystals belong to the trigonal system, space group P31 21 and have average unit cell dimensions î. a = b = 58.2, c = 132.0 A î 3 compatThe volume of the unit cell is 388.103 A ible with a dimer in the asymmetric unit with a VM î 3 /Da. Assuming (volume per Dalton) value of 2.31 A 3 a value of 0.74 cm /g for the protein partial speci¢c volume, the calculated solvent content in the crystal is 48% and the calculated crystal density is 1.19 g/ cm3 . The crystal structure is currently being determined using molecular replacement techniques implemented in the program AMoRe [27] and the coordinates of Lys-PLA2 from Bothrops asper [20]. Acknowledgements
Fig. 1. Crystal of BnSP-7, maximum dimensions 0.5U0.5U 0.25 mm.
This work was supported by FAPESP, FUNDUNESP, CNPq and National Synchrotron Light Laboratory (LNLS) (Brazil). We thank Mr. J.R. Branda¬o Neto (LNLS) for his help during the X-ray data collection.
BBAPRO 30442 5-7-99
M.R.M. Fontes et al. / Biochimica et Biophysica Acta 1432 (1999) 393^395
References [1] G. Rosenfeld, in: W. Bucherl, E. Buckley, V. Deulofeu (Eds.), Venomous Animals and Their Venoms II, Academic Press, New York, 1971, pp. 345^384. [2] M.L. Ferreira, A.M. Moura-da-Silva, F.O.S. Franc°a, J.L. Cardoso, I. Mota, Toxicon 30 (1992) 1603^1608. [3] L.A. Ribeiro, V.A.F. Pires de Campos, M.J. Albuquerque, N.Y. Takoaka, Rev. Assoc. Med. Bras. 39 (1993) 4^7. [4] P. Rosenberg, in: W. Shier, D. Mebs (Eds.), Handbook of Toxinology, Marcel Dekker, New York, 1990, pp. 67^277. [5] J.M. Gutie¨rrez, B. Lomonte, Toxicon 33 (1995) 1405^1424. [6] L.L.M. van Deenen, G.H. de Haas, Advances in Lipid Research, vol. 2, Academic Press, New York, 1964, p. 167. [7] J.M. Gutie¨rrez, F. Chaves, J.A. Gene¨, B. Lomonte, Z. Camacho, K. Schosinsky, Toxicon 27 (1989) 735^746. [8] J.M. Gutie¨rrez, J. Nunez, C. D|¨az, A.C.O. Cintra, M.I. Homsi-Brandeburgo, J.R. Giglio, Exp. Mol. Pathol. 55 (1991) 217^229. [9] C. D|¨az, J.M. Gut|¨errez, B. Lomonte, Arch. Biochem. Biophys. 298 (1992) 135^142. [10] J.M. Maraganore, G. Merutka, W. Cho, W. Welches, F.J. Kezdy, R.L. Heinrikson, J. Biol. Chem. 259 (1984) 13839^ 13843. [11] M.I. Homsi-Brandeburgo, L.S. Queiroz, H. Santo-Neto, L. Rodrigues-Simioni, J.R. Giglio, Toxicon 26 (1988) 615^627. [12] I.I. Kaiser, J.M. Gutie¨rrez, D. Plummer, S.D. Aird, G.V. Odell, Arch. Biochem. Biophys. 278 (1990) 319^325. [13] B. Lomonte, J.M. Gutie¨rrez, M.F. Furtado, R. Otero, J.P. Rosso, O. Vargas, E. Carmona, M.E. Rovira, Toxicon 28 (1990) 1137^1146. [14] A.C.O. Cintra, S. Marangoni, B. Oliveira, J.R. Giglio, J. Protein Chem. 12 (1993) 57^64.
395
[15] L.C. Mancuso, M.M. Correa, C.A. Vieira, O.A.B. Cunha, J.-J. Lachat, H.S. Selistre de Arau¨jo, C.L. Ownby, J.R. Giglio, Toxicon 33 (1995) 615^626. [16] A.M. Soares, L.H. Anzaloni-Pedrosa, M.R.M. Fontes, R.J. da Silva, J.R. Giglio, J. Venomous Anim. Toxins 4 (1998) 137^142. [17] A.M. Soares, V.M. Rodrigues, M.I. Homsi-Brandeburgo, M.H. Toyama, F.R. Lombardi, R.K. Arni, J.R. Giglio, Toxicon 36 (1998) 503^516. [18] W.F. de Azevedo Jr., M.R.M. Fontes, R.J. Ward, F.R. Lombardi, A.C.O. Cintra, J.R. Giglio, R.K. Arni, Protein Pept. Lett. 5 (1998) 181^184. [19] D.R. Holland, L.L. Clancy, S.W. Muchmore, T.J. Rydel, H.M. Einspahr, B.C. Finzel, R.L. Heinrickson, K.D. Watenpaugh, J. Biol. Chem. 266 (1990) 17649^17656. [20] R.K. Arni, R.J. Ward, J.M. Gutie¨rrez, A. Tulinsky, Acta Cryst. D51 (1995) 311^317. [21] W.F. de Azevedo Jr., R.J. Ward, F.R. Lombardi, J.R. Giglio, A.M. Soares, M.R.M. Fontes, R.K. Arni, Protein Pept. Lett. 4 (1997) 329^334. [22] M.T. da Silva-Giotto, R.C. Garratt, G. Oliva, Y.P. Mascarenhas, J.R. Giglio, A.C.O. Cintra, W.F. de Azevedo Jr., R.K. Arni, R.J. Ward, Proteins Struct. Funct. Genet. 30 (1998) 442^454. [23] V.M. Rodrigues, A.M. Soares, A.C. Mancin, M.R.M. Fontes, M.I. Homsi-Brandeburgo, J.R. Giglio, Comp. Biochem. Physiol. 121A (1998) 215^222. [24] J. Jancarik, S.H. Kim, J. Appl. Cryst. 24 (1991) 409^411. [25] A. MacPherson, Preparation and Analysis of Protein Crystals, Wiley, New York, 1982. [26] Z. Otwinowski, W. Minor, Methods Enzymol. 276 (1997) 307^326. [27] J. Navaza, Acta Cryst. A50 (1994) 157^163.
BBAPRO 30442 5-7-99