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Solution structure of Pi4, a short four-disulfide-bridged scorpion toxin specific of potassium channels

¹ Unité de RMN des Biomolécules (CNRS URA 2185), Dépt. de Biologie Structurale et Chimie, Institut Pasteur, 75724 Paris Cedex 15, France
² Laboratoire de Biochimie CNRS UMR 6560, and Laboratoire International Associé d’Ingéniérie Biomoléculaire, Faculté de Médecine Nord, 13916 Marseille Cedex 20, France
³ Department of Physiology, School of Medicine, National Autonomous University of Mexico, D.F. 04510 Mexico
⁴ Department of Molecular Medicine and Bioprocesses, Institute of Biotechnology, National Autonomous University of Mexico, Cuernavaca 62210 Mexico

Reprint requests to: Muriel Delepierre, Unité de RMN des Biomolécules (CNRS URA 2185), Dépt. de Biologie Structurale et Chimie, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France; e-mail: murield{at}pasteur.fr; fax: 33-145688929.

Pi4 is a short toxin found at very low abundance in the venom of Pandinus imperator scorpions. It is a potent blocker of K⁺ channels. Like the other members of the -KTX6 subfamily to which it belongs, it is cross-linked by four disulfide bonds. The synthetic analog (sPi4) and the natural toxin (nPi4) have been obtained by solid-phase synthesis or from scorpion venom, respectively. Analysis of two-dimensional ¹H NMR spectra of nPi4 and sPi4 indicates that both peptides have the same structure. Moreover, electrophysiological recordings of the blocking of Shaker B K⁺ channels by sPi4 (K_D = 8.5 nM) indicate that sPi4 has the same blocking activity of nPi4 (K_D = 8.0 nM), previously described. The disulfide bonds have been independently determined by NMR and structure calculations, and by Edman-degradation/mass-spectrometry identification of peptides obtained by proteolysis of nPi4. Both approaches indicate that the pairing of the half-cystines is ⁶C–²⁷C, ¹²C–³²C, ¹⁶C–³⁴C, and ²²C–³⁷C. The structure of the toxin has been determined by using 705 constraints derived from NMR data on sPi4. The structure, which is well defined, shows the characteristic /ß scaffold of scorpion toxins. It is compared to the structure of the other -KTX6 subfamily members and, in particular, to the structure of maurotoxin, which shows a different pattern of disulfide bridges despite its high degree of sequence identity (76%) with Pi4. The structure of Pi4 and the high amounts of synthetic peptide available, will enable the detailed analysis of the interaction of Pi4 with K⁺ channels.

Keywords: Cysteine-stabilized ß motif; disulfide bridges; NMR; Pandinus imperator; potassium channel; scorpion toxin

Abbreviations: a.m.u., atomic mass unit • COSY, correlation spectroscopy • CSI, chemical shift index • DQF-COSY, double-quantum • filtered COSY • HPLC, high-performance liquid chromatography • MTX, maurotoxin • NOE, nuclear Overhauser effect • NOESY, nuclear Overhauser effect spectroscopy • nPi4, Pi4 purified from scorpion venom • Pi4, Pandinus imperator toxin 4 • RMSD, root mean square deviation • sPi4, synthetic Pi4 • TFA, trifluoroacetic acid • TOCSY, total correlation spectroscopy

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