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. 2004 Jan 1;377(Pt 1):37–49. doi: 10.1042/BJ20030977

Cobatoxin 1 from Centruroides noxius scorpion venom: chemical synthesis, three-dimensional structure in solution, pharmacology and docking on K+ channels.

Besma Jouirou 1, Amor Mosbah 1, Violeta Visan 1, Stephan Grissmer 1, Sarrah M'Barek 1, Ziad Fajloun 1, Jurphaas Van Rietschoten 1, Christiane Devaux 1, Hervé Rochat 1, Guy Lippens 1, Mohamed El Ayeb 1, Michel De Waard 1, Kamel Mabrouk 1, Jean-Marc Sabatier 1
PMCID: PMC1223841  PMID: 14498829

Abstract

CoTX1 (cobatoxin 1) is a 32-residue toxin with three disulphide bridges that has been isolated from the venom of the Mexican scorpion Centruroides noxius Hoffmann. Here we report the chemical synthesis, disulphide bridge organization, 3-D (three-dimensional) solution structure determination, pharmacology on K+ channel subtypes (voltage-gated and Ca2+-activated) and docking-simulation experiments. An enzyme-based cleavage of the synthetic folded/oxidized CoTX1 indicated half-cystine pairs between Cys3-Cys22, Cys8-Cys27 and Cys12-Cys29. The 3-D structure of CoTX1 (solved by 1H-NMR) showed that it folds according to the common alpha/beta scaffold of scorpion toxins. In vivo, CoTX1 was lethal after intracerebroventricular injection to mice (LD50 value of 0.5 microg/mouse). In vitro, CoTX1 tested on cells expressing various voltage-gated or Ca2+-activated (IKCa1) K+ channels showed potent inhibition of currents from rat K(v)1.2 ( K(d) value of 27 nM). CoTX1 also weakly competed with 125I-labelled apamin for binding to SKCa channels (small-conductance Ca2+-activated K+ channels) on rat brain synaptosomes (IC50 value of 7.2 microM). The 3-D structure of CoTX1 was used in docking experiments which suggests a key role of Arg6 or Lys10, Arg14, Arg18, Lys21 (dyad), Ile23, Asn24, Lys28 and Tyr30 (dyad) residues of CoTX1 in its interaction with the rat K(v)1.2 channel. In addition, a [Pro7,Gln9]-CoTX1 analogue (ACoTX1) was synthesized. The two residue replacements were selected aiming to restore the RPCQ motif in order to increase peptide affinity towards SKCa channels, and to alter the CoTX1 dipole moment such that it is expected to decrease peptide activity on K(v) channels. Unexpectedly, ACoTX1 exhibited an activity similar to that of CoTX1 towards SKCa channels, while it was markedly more potent on IKCa1 and several voltage-gated K+ channels.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Blanc E., Sabatier J. M., Kharrat R., Meunier S., el Ayeb M., Van Rietschoten J., Darbon H. Solution structure of maurotoxin, a scorpion toxin from Scorpio maurus, with high affinity for voltage-gated potassium channels. Proteins. 1997 Nov;29(3):321–333. [PubMed] [Google Scholar]
  2. Bontems F., Roumestand C., Gilquin B., Ménez A., Toma F. Refined structure of charybdotoxin: common motifs in scorpion toxins and insect defensins. Science. 1991 Dec 6;254(5037):1521–1523. doi: 10.1126/science.1720574. [DOI] [PubMed] [Google Scholar]
  3. Brünger A. T., Adams P. D., Clore G. M., DeLano W. L., Gros P., Grosse-Kunstleve R. W., Jiang J. S., Kuszewski J., Nilges M., Pannu N. S. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):905–921. doi: 10.1107/s0907444998003254. [DOI] [PubMed] [Google Scholar]
  4. Carbone E., Wanke E., Prestipino G., Possani L. D., Maelicke A. Selective blockage of voltage-dependent K+ channels by a novel scorpion toxin. Nature. 1982 Mar 4;296(5852):90–91. doi: 10.1038/296090a0. [DOI] [PubMed] [Google Scholar]
  5. Chicchi G. G., Gimenez-Gallego G., Ber E., Garcia M. L., Winquist R., Cascieri M. A. Purification and characterization of a unique, potent inhibitor of apamin binding from Leiurus quinquestriatus hebraeus venom. J Biol Chem. 1988 Jul 25;263(21):10192–10197. [PubMed] [Google Scholar]
  6. Christie M. J. Molecular and functional diversity of K+ channels. Clin Exp Pharmacol Physiol. 1995 Dec;22(12):944–951. doi: 10.1111/j.1440-1681.1995.tb02331.x. [DOI] [PubMed] [Google Scholar]
  7. Crest M., Jacquet G., Gola M., Zerrouk H., Benslimane A., Rochat H., Mansuelle P., Martin-Eauclaire M. F. Kaliotoxin, a novel peptidyl inhibitor of neuronal BK-type Ca(2+)-activated K+ channels characterized from Androctonus mauretanicus mauretanicus venom. J Biol Chem. 1992 Jan 25;267(3):1640–1647. [PubMed] [Google Scholar]
  8. Doyle D. A., Morais Cabral J., Pfuetzner R. A., Kuo A., Gulbis J. M., Cohen S. L., Chait B. T., MacKinnon R. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science. 1998 Apr 3;280(5360):69–77. doi: 10.1126/science.280.5360.69. [DOI] [PubMed] [Google Scholar]
  9. Fajloun Z., Carlier E., Lecomte C., Geib S., Di Luccio E., Bichet D., Mabrouk K., Rochat H., De Waard M., Sabatier J. M. Chemical synthesis and characterization of Pi1, a scorpion toxin from Pandinus imperator active on K+ channels. Eur J Biochem. 2000 Aug;267(16):5149–5155. doi: 10.1046/j.1432-1327.2000.01577.x. [DOI] [PubMed] [Google Scholar]
  10. GRAY E. G., WHITTAKER V. P. The isolation of nerve endings from brain: an electron-microscopic study of cell fragments derived by homogenization and centrifugation. J Anat. 1962 Jan;96:79–88. [PMC free article] [PubMed] [Google Scholar]
  11. Gimenez-Gallego G., Navia M. A., Reuben J. P., Katz G. M., Kaczorowski G. J., Garcia M. L. Purification, sequence, and model structure of charybdotoxin, a potent selective inhibitor of calcium-activated potassium channels. Proc Natl Acad Sci U S A. 1988 May;85(10):3329–3333. doi: 10.1073/pnas.85.10.3329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Grissmer S., Nguyen A. N., Aiyar J., Hanson D. C., Mather R. J., Gutman G. A., Karmilowicz M. J., Auperin D. D., Chandy K. G. Pharmacological characterization of five cloned voltage-gated K+ channels, types Kv1.1, 1.2, 1.3, 1.5, and 3.1, stably expressed in mammalian cell lines. Mol Pharmacol. 1994 Jun;45(6):1227–1234. [PubMed] [Google Scholar]
  13. Gómez-Lagunas F., Olamendi-Portugal T., Zamudio F. Z., Possani L. D. Two novel toxins from the venom of the scorpion Pandinus imperator show that the N-terminal amino acid sequence is important for their affinities towards Shaker B K+ channels. J Membr Biol. 1996 Jul;152(1):49–56. doi: 10.1007/s002329900084. [DOI] [PubMed] [Google Scholar]
  14. Güntert P., Braun W., Wüthrich K. Efficient computation of three-dimensional protein structures in solution from nuclear magnetic resonance data using the program DIANA and the supporting programs CALIBA, HABAS and GLOMSA. J Mol Biol. 1991 Feb 5;217(3):517–530. doi: 10.1016/0022-2836(91)90754-t. [DOI] [PubMed] [Google Scholar]
  15. Güntert P., Wüthrich K. Improved efficiency of protein structure calculations from NMR data using the program DIANA with redundant dihedral angle constraints. J Biomol NMR. 1991 Nov;1(4):447–456. doi: 10.1007/BF02192866. [DOI] [PubMed] [Google Scholar]
  16. Hamill O. P., Marty A., Neher E., Sakmann B., Sigworth F. J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981 Aug;391(2):85–100. doi: 10.1007/BF00656997. [DOI] [PubMed] [Google Scholar]
  17. Ishii T. M., Silvia C., Hirschberg B., Bond C. T., Adelman J. P., Maylie J. A human intermediate conductance calcium-activated potassium channel. Proc Natl Acad Sci U S A. 1997 Oct 14;94(21):11651–11656. doi: 10.1073/pnas.94.21.11651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jiang Youxing, Lee Alice, Chen Jiayun, Ruta Vanessa, Cadene Martine, Chait Brian T., MacKinnon Roderick. X-ray structure of a voltage-dependent K+ channel. Nature. 2003 May 1;423(6935):33–41. doi: 10.1038/nature01580. [DOI] [PubMed] [Google Scholar]
  19. Johnson W. C., Jr Circular dichroism and its empirical application to biopolymers. Methods Biochem Anal. 1985;31:61–163. doi: 10.1002/9780470110522.ch2. [DOI] [PubMed] [Google Scholar]
  20. Lecomte C., Ferrat G., Fajloun Z., Van Rietschoten J., Rochat H., Martin-Eauclaire M. F., Darbon H., Sabatier J. M. Chemical synthesis and structure-activity relationships of Ts kappa, a novel scorpion toxin acting on apamin-sensitive SK channel. J Pept Res. 1999 Nov;54(5):369–376. doi: 10.1034/j.1399-3011.1999.00107.x. [DOI] [PubMed] [Google Scholar]
  21. Logsdon N. J., Kang J., Togo J. A., Christian E. P., Aiyar J. A novel gene, hKCa4, encodes the calcium-activated potassium channel in human T lymphocytes. J Biol Chem. 1997 Dec 26;272(52):32723–32726. doi: 10.1074/jbc.272.52.32723. [DOI] [PubMed] [Google Scholar]
  22. Marion D., Wüthrich K. Application of phase sensitive two-dimensional correlated spectroscopy (COSY) for measurements of 1H-1H spin-spin coupling constants in proteins. Biochem Biophys Res Commun. 1983 Jun 29;113(3):967–974. doi: 10.1016/0006-291x(83)91093-8. [DOI] [PubMed] [Google Scholar]
  23. Merrifield B. Solid phase synthesis. Science. 1986 Apr 18;232(4748):341–347. doi: 10.1126/science.3961484. [DOI] [PubMed] [Google Scholar]
  24. Miller C. The charybdotoxin family of K+ channel-blocking peptides. Neuron. 1995 Jul;15(1):5–10. doi: 10.1016/0896-6273(95)90057-8. [DOI] [PubMed] [Google Scholar]
  25. Mosbah A., Belaïch A., Bornet O., Belaïch J. P., Henrissat B., Darbon H. Solution structure of the module X2 1 of unknown function of the cellulosomal scaffolding protein CipC of Clostridium cellulolyticum. J Mol Biol. 2000 Nov 24;304(2):201–217. doi: 10.1006/jmbi.2000.4192. [DOI] [PubMed] [Google Scholar]
  26. Mosbah A., Kharrat R., Fajloun Z., Renisio J. G., Blanc E., Sabatier J. M., El Ayeb M., Darbon H. A new fold in the scorpion toxin family, associated with an activity on a ryanodine-sensitive calcium channel. Proteins. 2000 Aug 15;40(3):436–442. doi: 10.1002/1097-0134(20000815)40:3<436::aid-prot90>3.0.co;2-9. [DOI] [PubMed] [Google Scholar]
  27. Nicholls A., Sharp K. A., Honig B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins. 1991;11(4):281–296. doi: 10.1002/prot.340110407. [DOI] [PubMed] [Google Scholar]
  28. Nolde D. E., Sobol A. G., Pluzhnikov K. A., Grishin E. V., Arseniev A. S. Three-dimensional structure of ectatomin from Ectatomma tuberculatum ant venom. J Biomol NMR. 1995 Jan;5(1):1–13. doi: 10.1007/BF00227465. [DOI] [PubMed] [Google Scholar]
  29. Olamendi-Portugal T., Gómez-Lagunas F., Gurrola G. B., Possani L. D. A novel structural class of K+-channel blocking toxin from the scorpion Pandinus imperator. Biochem J. 1996 May 1;315(Pt 3):977–981. doi: 10.1042/bj3150977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Palma P. N., Krippahl L., Wampler J. E., Moura J. J. BiGGER: a new (soft) docking algorithm for predicting protein interactions. Proteins. 2000 Jun 1;39(4):372–384. [PubMed] [Google Scholar]
  31. Piotto M., Saudek V., Sklenár V. Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J Biomol NMR. 1992 Nov;2(6):661–665. doi: 10.1007/BF02192855. [DOI] [PubMed] [Google Scholar]
  32. Rauer H., Grissmer S. Evidence for an internal phenylalkylamine action on the voltage-gated potassium channel Kv1.3. Mol Pharmacol. 1996 Dec;50(6):1625–1634. [PubMed] [Google Scholar]
  33. Rogowski R. S., Collins J. H., O'Neill T. J., Gustafson T. A., Werkman T. R., Rogawski M. A., Tenenholz T. C., Weber D. J., Blaustein M. P. Three new toxins from the scorpion Pandinus imperator selectively block certain voltage-gated K+ channels. Mol Pharmacol. 1996 Nov;50(5):1167–1177. [PubMed] [Google Scholar]
  34. Rogowski R. S., Krueger B. K., Collins J. H., Blaustein M. P. Tityustoxin K alpha blocks voltage-gated noninactivating K+ channels and unblocks inactivating K+ channels blocked by alpha-dendrotoxin in synaptosomes. Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1475–1479. doi: 10.1073/pnas.91.4.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sabatier J. M., Zerrouk H., Darbon H., Mabrouk K., Benslimane A., Rochat H., Martin-Eauclaire M. F., Van Rietschoten J. P05, a new leiurotoxin I-like scorpion toxin: synthesis and structure-activity relationships of the alpha-amidated analog, a ligand of Ca(2+)-activated K+ channels with increased affinity. Biochemistry. 1993 Mar 23;32(11):2763–2770. doi: 10.1021/bi00062a005. [DOI] [PubMed] [Google Scholar]
  36. Selisko B., Garcia C., Becerril B., Gómez-Lagunas F., Garay C., Possani L. D. Cobatoxins 1 and 2 from Centruroides noxius Hoffmann constitute a subfamily of potassium-channel-blocking scorpion toxins. Eur J Biochem. 1998 Jun 15;254(3):468–479. doi: 10.1046/j.1432-1327.1998.2540468.x. [DOI] [PubMed] [Google Scholar]
  37. Vincent J. P., Schweitz H., Lazdunski M. Structure-function relationships and site of action of apamin, a neurotoxic polypeptide of bee venom with an action on the central nervous system. Biochemistry. 1975 Jun 3;14(11):2521–2525. doi: 10.1021/bi00682a035. [DOI] [PubMed] [Google Scholar]
  38. Zerrouk H., Laraba-Djebari F., Fremont V., Meki A., Darbon H., Mansuelle P., Oughideni R., van Rietschoten J., Rochat H., Martin-Eauclaire M. F. Characterization of PO1, a new peptide ligand of the apamin-sensitive Ca2+ activated K+ channel. Int J Pept Protein Res. 1996 Dec;48(6):514–521. doi: 10.1111/j.1399-3011.1996.tb00870.x. [DOI] [PubMed] [Google Scholar]

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