Solution structure of a novel K+-channel blocker from the venom of scorpion Tityus cambridgei

Iren Wang1, 2, Shih-Hsiung Wu2, 3, Chinpan Chen1

 1Institute of Biomedical Sciences, Academia Sinica, 2Institute of Biochemical Sciences, National Taiwan University, 3Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan

 

A new K+ channel-blocking peptide, Tc1, was recently identified from the scorpion venom of Tityus cambridgei (FEBS Letters, 486, 2000: 117-120). Tc1, ACGSCRKKCKGSGKCINGRCKCY, is composed of 23 amino acid residues with three disulfide bridges and is the shortest known peptide from scorpion venom that recognizes the Shaker B K+-channels and the voltage dependent K+-channels present in brain. We have synthesized Tc1 by solid-phase synthesis and applied CD, fluorescence, and NMR techniques to obtain the structural characteristics on Tc1. The titration spectra of CD and fluorescence experiments, at different temperatures and concentrations of guanidine-HCl and urea, indicate that Tc1 is a highly stable peptide. The pairings of three disulfide bridges in Tc1, which have not been established previously, were clearly identified by the NOE connectivities of dββ(i, j) of the two cysteine residues in a disulfide bond. The three identified disulfide bridges are Cys2-Cys15, Cys5-Cys20, and Cys9-Cys22. Based on the interproton restraints, stereospecific assignments, 3JNHα coupling constants, and amide-proton exchange rate, we have determined the NMR solution structure of Tc1 using X-PLOR program. The solution structure of Tc1 consists of an N-terminal α-helix from Gly4-Gly11 and a double-stranded antiparallelβ-sheet at residues Gly13-Ile16 and Arg19-Tyr23, with aβturn at residues Asn17-Gly18. Thus, Tc1 is different from the triple-strandedβsheet/cysteine knot motif, which is relatively common in small cysteine-rich toxins. A detailed comparison of the 3D structure of Tc1 and those of other structurally and functionally related scorpion toxins, such as noxiustoxin (NTX) and charybdotoxin, is presented.

 

 

 

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