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Preliminary X-ray diffraction studies on a scorpion neurotoxin: Toxin II of Androctonus australis Hector
J. Mol. Biol. (1978) 126, 289-291
LETTER
Preliminary
TO THE EDITOR
X-ray Diffraction Studies on a Scorpion Neurotoxin Toxin II of Androctonus australis Hector
:
The most toxic scorpion neurotoxin (toxin II), isolated from the North African scorpion Androctonus australis Hector, has been crystallized. The space group is C2 with one molecule of protein in the asymmetric unit. The cell parameters are are very stable a = 51.9 A, b = 33.3 A, c = 40.4 A, p = 113”. The crystals under radiation.
Interest in the neurotoxins has increased over the past deca,de, since they appear to be very useful tools in the study of neurochemical mechanisms. Particularly, the toxic polypeptides isolated from snake and scorpion venoms are a good example of such tools. Much work has been done on Elapidae or Hydrophidae snake toxins (neurotoxins and cardio- or cytot’oxins) including X-ray structure analysis (Low predictions. Scorpion et al., 1976; Tsernoglou & Petsko, 1976) and conformational neurotoxins show some molecular analogies with snake neurotoxins but t.heir biological activity appears quite different : snake neurotoxins induce genera’lly a flaccid paralysis in mouse, which is due to their irreversible binding to the aoetylcholine receptor at the postsynaptic neuromuscular junction level; scorpion neurotoxins provoke a spastic paralysis and act at t,he Na+ channel level, inducing the depolarisation of excitable membranes (Romey et al., 1975; Couraud et al., 1976: Catterall, 1977a,b; Bernard et al., 1977). Scorpion neurotoxins are ma,de of a single peptide cha#in of a,bout 60 amino acid residues cross-linked by four disulfide bridges. Comparison of part,ial or complete sequences of the neurotoxins already studied shows that they displa,y a high degree of homology (Roohat et aE., 1970; Zlotkin et al., 1978). Toxin II of A. australis H. is of special interest, since its toxicity on mice is the highest when compared to other scorpion neurotoxins (Roohat et al., 1970 ; Zlotkin et al., 1978). Its complete amino acid sequence is known (Roohat et al., 1972), as well as the position of the disulfide bridges (Kopeyan et al., 1974). Some physico-chemical properties have been studied (Chicheportiche & Lazdunski, 1970). Moreover, structure-function relationship studies have already been investigated by chemical modifications of some t,rifunotional amino a,oid residues (Habersetzer-Roohat & Sampieri, 1976). In this paper, preliminary crystallographic data concerning this toxin are reported. h’eurotoxin II was purified from the venom of the scorpion A. australis H. as described elsewhere (Roohat et al., 1967; Miranda et aZ., 1970). Its molecular weight is 7250 (64 amino acid residues). The LD,, value is 9 pg per kg of mouse (Roohat et al., 1970; Zlotkin et al., 1978). Disulfide bridges link half-cystine residues number 12 and 63, 16 and 36: 22 and 46, and 26 and 48 (Kopeyan et al., 1974). Crystallization
Toxin II was dissolved in low3 M-sodium azide and centrifugated. Tris.HCl buffer was added to the supernatant to give a final ooncentJration of 2% (w/w) protein in 289 0022-2836/78/340289-03
$02.00/O
0 1978 Academic
Press Inc. (London)
Ltd.
290
F. SAMPIERI
ET
AL.
O-01 rvr-Tris (pH 7.7). Then the pH of the solution was slowly raised by diffusion of ammonia from an ammonium bicarbonate solution. Crystals suitable for X-ray diffraction studies (0.6 mm x 0.15 mm i< 0.3 mm) were grown in a few weeks at room temperature. They were mounted and sealed in thin-walled Lindeman glass capillaries in the usual way. X-ray precession photographs reveal that crystals of neurotoxin II of A. australis H. are monoclinic (space group C2). Crystal data as refined through diffractomet’er measurements are : a = 51.9 8, b = 33.3 8, c = 40.4 8; p = 113”: tl = y = 90”, V = 64,154 A3. Assuming
the presence of one molecule of protein in the asymmetric unit, one finds of 2.21 A” per dalton, which is within the usual range found for protein crystals (Matthews, 1968). The crystals diffract down to 2.8 A and are very stable under radiation. Screening of possible heavy-atom derivatives through the usual soaking techniques is underway in our laborat’ories.
a specific volume
The authors are greatly indebted to Professor Miranda for his friendly support and constant encouragement. They thank Mrs M. F. Martin for purification of neurotoxin. They also thank Dr M. Pierrot and Professor Kern for their kind interest in this work. This work was supported by the Institut National de la Sante et la Reoherche Medicale (U172), the Centre National de la Recherche Scientifique (ERA070617), the Direction des Recherches, Etudes et Techniques (contract no. 77/247), the Direction G&n&ale de la Recherche Scientifique et Technique (contract no. 770494) and the Fondation pour la Recherche Medicale Franqaise. Department of Biochemistry, Faculte de Medecine Secteur Nord, Boulevard Pierre Dramard 13326 Marseille Cedex 3, France
FRANFOIS SAMPIERI CATHERINE HABERSETZER-ROCHAT
Centre de Recherche Croissance Cristalline, Scientiflque de Saint Cedex 4, France
sur les Mkcanismes de la C.N.R.S., Centre Jerirme, 13397 Marseille
JEAN-PIERRE ASTIER MICHELFREY RICHARD HASER
Received
1978
14 August
REFERENCES Bernard, P., Couraud, F. & Lissitzky, S. (1977). Biochem. Biophys. Res. Common. 77, 782-788. Catterall, W. A. (1977a). J. Biol. Chem. 252, 8660-8668. Catterall, W. A. (19773). J. Biol. Chem. 252, 8669-8676. Chicheportiche, R. & Lazdunski, M. (1970). EAT. J. Biochem. 14, 5499555. Couraud, F., Rochat, H. & Lissitzky, S. (1976). Biochim. Biophys. Acta, 433, 90-100. Habersetzer-Rochat, C. & Sampieri, F. (1976). Biochemisby, 15, 2254-2261. Kopeyan, C., Martinez, G., Lissitzky, S., Miranda, F. & Rochat, H. (1974). Eur. J. Biochem. 47, 4833489. Low, B. W., Preston, H. S., Sato, A., Rosen, L. S., Searl, J. E., Rudko, A. D. & Richardson, J. S. (1976). Proc. Nat. Acad. sci., U.S.A. 73, 2991-2994. Matthews, B. W. (1968). J. Mol. Biol. 33, 491-497. Miranda, F., Kopeyan, C.: Rochat, H., Rochat, C. & Lissitzky, S. (1970). Eur. J. Biochem. 16, 514-523. Rochat, C., Rochat, H., Miranda, F. & Lissitzky, S. (1967). Biochemistry, 6, 578-585.
LETTER
TO THE
EDITOR
291
F., Lissitzky, S. & Edman, P. (1970). Rochat, H., Rochat, C., Kopeyan, C., Miranda, FEBS Letters, 10, 349-351. Rochat, H., Rochat, C., Sampieri, F., Miranda, F. & Lissitzky, S. (1972). Eur. J. Biochem. 28, 381-388. Romey, G., Chicheportiche, R., Lazdunski, M., Rochat, H., Miranda, F. & Lissitzky, S. (1975). Biochem. Bio@ys. Res. Commu~. 64, 115-121. Tsernoglou, D. & Petsko, G. A. (1976). FEBS Letters, 68, l-4. Zlotkin, E., Miranda, F. & Rochat, H. (1978). In Handbook of Experimental Pharmacology (Bettini, S., ed.). vol. 48, pp. 317-369, Springer Verlag, Berlin.
LETTER
Preliminary
TO THE EDITOR
X-ray Diffraction Studies on a Scorpion Neurotoxin Toxin II of Androctonus australis Hector
:
The most toxic scorpion neurotoxin (toxin II), isolated from the North African scorpion Androctonus australis Hector, has been crystallized. The space group is C2 with one molecule of protein in the asymmetric unit. The cell parameters are are very stable a = 51.9 A, b = 33.3 A, c = 40.4 A, p = 113”. The crystals under radiation.
Interest in the neurotoxins has increased over the past deca,de, since they appear to be very useful tools in the study of neurochemical mechanisms. Particularly, the toxic polypeptides isolated from snake and scorpion venoms are a good example of such tools. Much work has been done on Elapidae or Hydrophidae snake toxins (neurotoxins and cardio- or cytot’oxins) including X-ray structure analysis (Low predictions. Scorpion et al., 1976; Tsernoglou & Petsko, 1976) and conformational neurotoxins show some molecular analogies with snake neurotoxins but t.heir biological activity appears quite different : snake neurotoxins induce genera’lly a flaccid paralysis in mouse, which is due to their irreversible binding to the aoetylcholine receptor at the postsynaptic neuromuscular junction level; scorpion neurotoxins provoke a spastic paralysis and act at t,he Na+ channel level, inducing the depolarisation of excitable membranes (Romey et al., 1975; Couraud et al., 1976: Catterall, 1977a,b; Bernard et al., 1977). Scorpion neurotoxins are ma,de of a single peptide cha#in of a,bout 60 amino acid residues cross-linked by four disulfide bridges. Comparison of part,ial or complete sequences of the neurotoxins already studied shows that they displa,y a high degree of homology (Roohat et aE., 1970; Zlotkin et al., 1978). Toxin II of A. australis H. is of special interest, since its toxicity on mice is the highest when compared to other scorpion neurotoxins (Roohat et al., 1970 ; Zlotkin et al., 1978). Its complete amino acid sequence is known (Roohat et al., 1972), as well as the position of the disulfide bridges (Kopeyan et al., 1974). Some physico-chemical properties have been studied (Chicheportiche & Lazdunski, 1970). Moreover, structure-function relationship studies have already been investigated by chemical modifications of some t,rifunotional amino a,oid residues (Habersetzer-Roohat & Sampieri, 1976). In this paper, preliminary crystallographic data concerning this toxin are reported. h’eurotoxin II was purified from the venom of the scorpion A. australis H. as described elsewhere (Roohat et al., 1967; Miranda et aZ., 1970). Its molecular weight is 7250 (64 amino acid residues). The LD,, value is 9 pg per kg of mouse (Roohat et al., 1970; Zlotkin et al., 1978). Disulfide bridges link half-cystine residues number 12 and 63, 16 and 36: 22 and 46, and 26 and 48 (Kopeyan et al., 1974). Crystallization
Toxin II was dissolved in low3 M-sodium azide and centrifugated. Tris.HCl buffer was added to the supernatant to give a final ooncentJration of 2% (w/w) protein in 289 0022-2836/78/340289-03
$02.00/O
0 1978 Academic
Press Inc. (London)
Ltd.
290
F. SAMPIERI
ET
AL.
O-01 rvr-Tris (pH 7.7). Then the pH of the solution was slowly raised by diffusion of ammonia from an ammonium bicarbonate solution. Crystals suitable for X-ray diffraction studies (0.6 mm x 0.15 mm i< 0.3 mm) were grown in a few weeks at room temperature. They were mounted and sealed in thin-walled Lindeman glass capillaries in the usual way. X-ray precession photographs reveal that crystals of neurotoxin II of A. australis H. are monoclinic (space group C2). Crystal data as refined through diffractomet’er measurements are : a = 51.9 8, b = 33.3 8, c = 40.4 8; p = 113”: tl = y = 90”, V = 64,154 A3. Assuming
the presence of one molecule of protein in the asymmetric unit, one finds of 2.21 A” per dalton, which is within the usual range found for protein crystals (Matthews, 1968). The crystals diffract down to 2.8 A and are very stable under radiation. Screening of possible heavy-atom derivatives through the usual soaking techniques is underway in our laborat’ories.
a specific volume
The authors are greatly indebted to Professor Miranda for his friendly support and constant encouragement. They thank Mrs M. F. Martin for purification of neurotoxin. They also thank Dr M. Pierrot and Professor Kern for their kind interest in this work. This work was supported by the Institut National de la Sante et la Reoherche Medicale (U172), the Centre National de la Recherche Scientifique (ERA070617), the Direction des Recherches, Etudes et Techniques (contract no. 77/247), the Direction G&n&ale de la Recherche Scientifique et Technique (contract no. 770494) and the Fondation pour la Recherche Medicale Franqaise. Department of Biochemistry, Faculte de Medecine Secteur Nord, Boulevard Pierre Dramard 13326 Marseille Cedex 3, France
FRANFOIS SAMPIERI CATHERINE HABERSETZER-ROCHAT
Centre de Recherche Croissance Cristalline, Scientiflque de Saint Cedex 4, France
sur les Mkcanismes de la C.N.R.S., Centre Jerirme, 13397 Marseille
JEAN-PIERRE ASTIER MICHELFREY RICHARD HASER
Received
1978
14 August
REFERENCES Bernard, P., Couraud, F. & Lissitzky, S. (1977). Biochem. Biophys. Res. Common. 77, 782-788. Catterall, W. A. (1977a). J. Biol. Chem. 252, 8660-8668. Catterall, W. A. (19773). J. Biol. Chem. 252, 8669-8676. Chicheportiche, R. & Lazdunski, M. (1970). EAT. J. Biochem. 14, 5499555. Couraud, F., Rochat, H. & Lissitzky, S. (1976). Biochim. Biophys. Acta, 433, 90-100. Habersetzer-Rochat, C. & Sampieri, F. (1976). Biochemisby, 15, 2254-2261. Kopeyan, C., Martinez, G., Lissitzky, S., Miranda, F. & Rochat, H. (1974). Eur. J. Biochem. 47, 4833489. Low, B. W., Preston, H. S., Sato, A., Rosen, L. S., Searl, J. E., Rudko, A. D. & Richardson, J. S. (1976). Proc. Nat. Acad. sci., U.S.A. 73, 2991-2994. Matthews, B. W. (1968). J. Mol. Biol. 33, 491-497. Miranda, F., Kopeyan, C.: Rochat, H., Rochat, C. & Lissitzky, S. (1970). Eur. J. Biochem. 16, 514-523. Rochat, C., Rochat, H., Miranda, F. & Lissitzky, S. (1967). Biochemistry, 6, 578-585.
LETTER
TO THE
EDITOR
291
F., Lissitzky, S. & Edman, P. (1970). Rochat, H., Rochat, C., Kopeyan, C., Miranda, FEBS Letters, 10, 349-351. Rochat, H., Rochat, C., Sampieri, F., Miranda, F. & Lissitzky, S. (1972). Eur. J. Biochem. 28, 381-388. Romey, G., Chicheportiche, R., Lazdunski, M., Rochat, H., Miranda, F. & Lissitzky, S. (1975). Biochem. Bio@ys. Res. Commu~. 64, 115-121. Tsernoglou, D. & Petsko, G. A. (1976). FEBS Letters, 68, l-4. Zlotkin, E., Miranda, F. & Rochat, H. (1978). In Handbook of Experimental Pharmacology (Bettini, S., ed.). vol. 48, pp. 317-369, Springer Verlag, Berlin.