Guijarro J Iñaki, M'Barek Sarrah, Gómez-Lagunas Froylan, Garnier Damien, Rochat Hervé, Sabatier Jean-Marc, Possani Lourival, Delepierre Muriel
Unité de RMN des Biomolécules (CNRS URA 2185), Dépt. de Biologie Structurale et Chimie, Institut Pasteur, 75724 Paris Cedex 15, France.
Protein Sci. 2003 Sep;12(9):1844-54. doi: 10.1110/ps.03186703.
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 alpha-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 (1)H 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 (6)C-(27)C, (12)C-(32)C, (16)C-(34)C, and (22)C-(37)C. 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 alpha/beta scaffold of scorpion toxins. It is compared to the structure of the other alpha-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.
Pi4是一种在帝王蝎毒液中含量极低的短毒素。它是一种有效的钾离子通道阻滞剂。与它所属的α-KTX6亚家族的其他成员一样，它通过四个二硫键交联。合成类似物（sPi4）和天然毒素（nPi4）分别通过固相合成或从蝎毒中获得。对nPi4和sPi4的二维氢核磁共振谱分析表明，这两种肽具有相同的结构。此外，sPi4对Shaker B钾离子通道阻滞的电生理记录（解离常数K(D)=8.5 nM）表明，sPi4具有与先前描述的nPi4相同的阻滞活性（K(D)=8.0 nM）。二硫键已通过核磁共振和结构计算独立确定，以及通过对nPi4进行蛋白水解得到的肽段进行埃德曼降解/质谱鉴定。两种方法均表明半胱氨酸的配对为（6）C-（27）C、（12）C-（32）C、（16）C-（34）C和（22）C-（37）C。毒素的结构已通过使用来自sPi4核磁共振数据的705个约束条件确定。该结构定义明确，显示出蝎毒素特有的α/β支架。它与其他α-KTX6亚家族成员的结构进行了比较，特别是与马罗毒素的结构进行了比较，尽管马罗毒素与Pi4的序列同一性很高（76%），但其二硫键模式不同。Pi4的结构以及大量可用的合成肽，将有助于详细分析Pi4与钾离子通道的相互作用。
