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The first chemical synthesis of a short scorpion toxin cross-linked by four disulfide bridges, maurotoxin
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Reports
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zebra-fish embryos, the genotoxicity was detected at doses of OA around 1 PM and also appeared at non-toxic doses. In all cell types, when toxicity occurred, the number of adducts decreased. Some adducts were common to several models and some seemed to be more specific. The first chemical synthesis oj a short scorpion toxin cross-linked by four disuljide bridges, maurotoxin. R. Kharrat,‘,2 K. Mabrouk,’ M. Crest,’ H. Darbon,4 R. Oughideni, ’ M.-F. Martin-Eauclaire,’ G. Jacquet,’ M. El Ayeb,‘, J. Van Rietschoten,’ H. Rochat’ and J.-M. Sabatier’ (‘Laboratoire de Biochimie, CNRS URA 1455, Facultt de Midecine Nord, 13916 Marseille Cedex 20, Fiance; *Laboratoire Venins et Toxines, Institut Pasteur de Tunis, 1002 Tunis, Tunisia; ‘Laboratoire de Neurobiologie, CNRS UPR 9024, 13402 Marseille cedex 20, France; and ‘LCCMB, CNRS URA 1296, 13402 Marseille cedex 20, France). Maurotoxin (MTX) is a novel toxin isolated from the venom of the Tunisian chacto’id scorpion Scorpio maurus. MTX is a basic 34-residue peptide cross-linked by four disulfide bridges, i.e. VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC-NH2 (Kharrat et al., 1997a). Natural MTX competes with radiolabelled apamin and kaliotoxin for binding to rat brain synaptosomes. Thus, it is the first characterized scorpion toxin with four disulfide bridges that acts on K+ channels. Owing to its very low concentration in venom (0.6% of the proteins), MTX was chemically synthesized by the solid-phase technique using optimized Fmoc/tBu strategy. The synthetic peptide (sMTX) was characterized for its physicochemical and pharmacological properties (Kharrat et al., 1997b). sMTX was lethal to mice following i.c.v. injection (LDSO of 80 ng per mouse). The sMTX competed with [‘251]apamin and [‘lSI]kaliotoxin for binding to rat brain synaptosomes with half-maximal effects at concentrations of 5 x 10m9 M and 2 x lo-‘O M, respectively. sMTX was tested on K+ channels and was found to block the KV 1.1, KV 1.2 and KV 1.3 currents with half-maximal blockage (ICS~at 37 nM, 0.8 nm and 150 nM, respectively. Thus, MTX is also the first known scorpion toxin that acts on both Ca’+ activated and voltage-gated K’ channels. The disulfide bridge pairings of sMTX were identified by enzymatic proteolysis. The pairings were unexpectedly between Cysl-Cysu, Cys&ys~, CYSII-CYSIYand Cys31-Cysj4. This disulfide organization is unique among known scorpion toxins, probably because MTX possesses a structural motif, notably with an extra central half-cystine, which differs from the consensus motif reported to be shared by all scorpion toxins (Bontems et al., 1991). The physicochemical and pharmacological properties of sMTX were indistinguishable from those of natural MTX, which suggests that natural MTX adopts the same disulfide pairing pattern. The conformation of sMTX was also investigated using circular dichroism spectroscopy and molecular modelling. In spite of its unusual disulfide pairing pattern, the sMTX conformation appears to be similar to that of other short scorpion toxins. Bontems, F., Roumestand, C., Gilquin, B., Menez, A. and Toma, F. (1991) Science 254, 1521. Kharrat, R., Mansuelle, P., Sampieri, F., Crest, M., Martin-Eauclaire, M.-F. Rochat, H. and El Ayeb, M. (1997a) FEBS Letters, submitted. Kharrat, R., Mabrouk, K., Crest, M., Darbon, H., Oughideni, R., Martin-Eauclaire, M.-F., Jacquet, G., El Ayeb M.. Van Rietschoten, J., Rochat, H. and Sabatier, J.-M. (1997b) European Journal of Biochemistry, submitted. Refined solution structure qf the anti-mammal and anti-insect LqqIII scorpion toxin: comparison with other scorpion toxins and C&/l motive description. C. Landon,’ P. Sodano,’ B. Cornet,’ J,-M. Bonmatin,‘, C. Kopeyan3 H. Rochat,3 F. Vovelle’,’ and M. Ptak’,* (‘Centre de Biophysique Moleculaire; *UniversitC d’Orltans, France; and ‘Laboratoire de Biochimie, Facultt de Medecine nord, Marseille, France). Scorpion toxins form a broad family of proteins with high sequence homologies but with various activities against mammals, insects and/or crustaceans. Neurotoxin III of the scorpion Leiurus quinqucstriatus quinquestriatus (LqqIII) is a small protein (64 residues) containing four disuhide bridges, which presents the characteristics of the long anti-mammal cc-toxins. In addition, LqqIII is particularly toxic to insects (Kopeyan et al., 1993). The study of its 3D structure was undertaken in order to define the particularities which confer on LqqIII this additional activity. We previously determined the global fold of LqqIII (Landon et al., 1996) and now present the refined structure which mainly consists of an a-helix (18-29) connected by two disulfide bridges to a triple-stranded B-sheet (2-6, 33-39 and 43-51). The RMSD between the 15 best structures of LqqIIL and the average structure is 0.1 A for the backbone CG( and reduces to 0.38 A when only secondary structure elements are considered. LqqIII model was superimposed with the structures of AaHII (Housset et al., 1994) and CsE-v3 (Zhao et al., 1992) toxins determined by crystallography. The best global superimposition was obtained with AaHII. The electrostatic and lipophilic potentials were calculated in order to define the charged and hydrophobic surfaces of LqqIII and compare them with known structures of other scorpion toxins interacting with Na+ channels. In a more general context, the interactions between the helix and the sheet were studied in the Cystein Stabilized x-helix /?-sheet (C&/I) motif, first described for the defensin A (Cornet et al., 1995), and common to scorpion neurotoxins, plant defensins and insect defensins. The study of conserved residues or contacts in this region may help in the understanding of this structural motif stability. Cornet, B., Bonmatin, J.-M., Hetru, C., Hoffmann, J. A., Ptak, M. and Vovelle, F. (1995) Structure 3, 435448. Housset, D., Habersetzer-Rochat, C., Astier, J-P. and Fontecilla-Camps. J. C. (1994) \ I Journal of Molecular Biology 238, 88-103. Kopeyan, C., Mansuelle, P., Martin-Eauclaire, M.-F., Rochat, H. and Miranda. F. (1993) Natural Toxins 1, 308-312.
Reports
and Abstracts
zebra-fish embryos, the genotoxicity was detected at doses of OA around 1 PM and also appeared at non-toxic doses. In all cell types, when toxicity occurred, the number of adducts decreased. Some adducts were common to several models and some seemed to be more specific. The first chemical synthesis oj a short scorpion toxin cross-linked by four disuljide bridges, maurotoxin. R. Kharrat,‘,2 K. Mabrouk,’ M. Crest,’ H. Darbon,4 R. Oughideni, ’ M.-F. Martin-Eauclaire,’ G. Jacquet,’ M. El Ayeb,‘, J. Van Rietschoten,’ H. Rochat’ and J.-M. Sabatier’ (‘Laboratoire de Biochimie, CNRS URA 1455, Facultt de Midecine Nord, 13916 Marseille Cedex 20, Fiance; *Laboratoire Venins et Toxines, Institut Pasteur de Tunis, 1002 Tunis, Tunisia; ‘Laboratoire de Neurobiologie, CNRS UPR 9024, 13402 Marseille cedex 20, France; and ‘LCCMB, CNRS URA 1296, 13402 Marseille cedex 20, France). Maurotoxin (MTX) is a novel toxin isolated from the venom of the Tunisian chacto’id scorpion Scorpio maurus. MTX is a basic 34-residue peptide cross-linked by four disulfide bridges, i.e. VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC-NH2 (Kharrat et al., 1997a). Natural MTX competes with radiolabelled apamin and kaliotoxin for binding to rat brain synaptosomes. Thus, it is the first characterized scorpion toxin with four disulfide bridges that acts on K+ channels. Owing to its very low concentration in venom (0.6% of the proteins), MTX was chemically synthesized by the solid-phase technique using optimized Fmoc/tBu strategy. The synthetic peptide (sMTX) was characterized for its physicochemical and pharmacological properties (Kharrat et al., 1997b). sMTX was lethal to mice following i.c.v. injection (LDSO of 80 ng per mouse). The sMTX competed with [‘251]apamin and [‘lSI]kaliotoxin for binding to rat brain synaptosomes with half-maximal effects at concentrations of 5 x 10m9 M and 2 x lo-‘O M, respectively. sMTX was tested on K+ channels and was found to block the KV 1.1, KV 1.2 and KV 1.3 currents with half-maximal blockage (ICS~at 37 nM, 0.8 nm and 150 nM, respectively. Thus, MTX is also the first known scorpion toxin that acts on both Ca’+ activated and voltage-gated K’ channels. The disulfide bridge pairings of sMTX were identified by enzymatic proteolysis. The pairings were unexpectedly between Cysl-Cysu, Cys&ys~, CYSII-CYSIYand Cys31-Cysj4. This disulfide organization is unique among known scorpion toxins, probably because MTX possesses a structural motif, notably with an extra central half-cystine, which differs from the consensus motif reported to be shared by all scorpion toxins (Bontems et al., 1991). The physicochemical and pharmacological properties of sMTX were indistinguishable from those of natural MTX, which suggests that natural MTX adopts the same disulfide pairing pattern. The conformation of sMTX was also investigated using circular dichroism spectroscopy and molecular modelling. In spite of its unusual disulfide pairing pattern, the sMTX conformation appears to be similar to that of other short scorpion toxins. Bontems, F., Roumestand, C., Gilquin, B., Menez, A. and Toma, F. (1991) Science 254, 1521. Kharrat, R., Mansuelle, P., Sampieri, F., Crest, M., Martin-Eauclaire, M.-F. Rochat, H. and El Ayeb, M. (1997a) FEBS Letters, submitted. Kharrat, R., Mabrouk, K., Crest, M., Darbon, H., Oughideni, R., Martin-Eauclaire, M.-F., Jacquet, G., El Ayeb M.. Van Rietschoten, J., Rochat, H. and Sabatier, J.-M. (1997b) European Journal of Biochemistry, submitted. Refined solution structure qf the anti-mammal and anti-insect LqqIII scorpion toxin: comparison with other scorpion toxins and C&/l motive description. C. Landon,’ P. Sodano,’ B. Cornet,’ J,-M. Bonmatin,‘, C. Kopeyan3 H. Rochat,3 F. Vovelle’,’ and M. Ptak’,* (‘Centre de Biophysique Moleculaire; *UniversitC d’Orltans, France; and ‘Laboratoire de Biochimie, Facultt de Medecine nord, Marseille, France). Scorpion toxins form a broad family of proteins with high sequence homologies but with various activities against mammals, insects and/or crustaceans. Neurotoxin III of the scorpion Leiurus quinqucstriatus quinquestriatus (LqqIII) is a small protein (64 residues) containing four disuhide bridges, which presents the characteristics of the long anti-mammal cc-toxins. In addition, LqqIII is particularly toxic to insects (Kopeyan et al., 1993). The study of its 3D structure was undertaken in order to define the particularities which confer on LqqIII this additional activity. We previously determined the global fold of LqqIII (Landon et al., 1996) and now present the refined structure which mainly consists of an a-helix (18-29) connected by two disulfide bridges to a triple-stranded B-sheet (2-6, 33-39 and 43-51). The RMSD between the 15 best structures of LqqIIL and the average structure is 0.1 A for the backbone CG( and reduces to 0.38 A when only secondary structure elements are considered. LqqIII model was superimposed with the structures of AaHII (Housset et al., 1994) and CsE-v3 (Zhao et al., 1992) toxins determined by crystallography. The best global superimposition was obtained with AaHII. The electrostatic and lipophilic potentials were calculated in order to define the charged and hydrophobic surfaces of LqqIII and compare them with known structures of other scorpion toxins interacting with Na+ channels. In a more general context, the interactions between the helix and the sheet were studied in the Cystein Stabilized x-helix /?-sheet (C&/I) motif, first described for the defensin A (Cornet et al., 1995), and common to scorpion neurotoxins, plant defensins and insect defensins. The study of conserved residues or contacts in this region may help in the understanding of this structural motif stability. Cornet, B., Bonmatin, J.-M., Hetru, C., Hoffmann, J. A., Ptak, M. and Vovelle, F. (1995) Structure 3, 435448. Housset, D., Habersetzer-Rochat, C., Astier, J-P. and Fontecilla-Camps. J. C. (1994) \ I Journal of Molecular Biology 238, 88-103. Kopeyan, C., Mansuelle, P., Martin-Eauclaire, M.-F., Rochat, H. and Miranda. F. (1993) Natural Toxins 1, 308-312.