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

Abstract

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 α-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 α/β scaffold of scorpion toxins. It is compared to the structure of the other α-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.