Synthesis and characterization of leiurotoxin I analogs lacking one disulfide bridge: evidence that disulfide pairing 3-21 is not required for full toxin activity.

Leiurotoxin I (Lei-NH2), a toxin isolated from the venom of the scorpion Leiurus quinquestriatus hebraeus, is a blocker of the apamin-sensitive Ca(2+)-activated K+ channels. It is a 31-residue polypeptide cross-linked by three disulfide bridges which are presumably between Cys3-Cys21, Cys8-Cys26, and Cys12-Cys28. To investigate the role of these disulfides, analogs of Lei-NH2 lacking one disulfide bridge (i.e., [Abu3,21]Lei-NH2, [Abu8,26]Lei-NH2, and [Abu12,28]Lei-NH2) were chemically synthesized by selective replacement of each pair of half-cystines forming a bridge by two alpha-aminobutyrate (Abu) residues. The two disulfide pairings of the main folded form of the synthetic analogs were established by enzymatic proteolysis. They were as expected between Cys8-Cys26 and Cys12-Cys28 for [Abu3,21]Lei-NH2 but were unexpectedly between Cys3-Cys12 and Cys21-Cys28 for [Abu8,26]Lei-NH2 and between Cys3-Cys8 and Cys21-Cys26 for [Abu12,28]Lei-NH2. The synthetic peptides were tested in vitro for their capacity to compete with the binding of [125I]apamin to rat brain synaptosomes and in vivo for their neurotoxicity in mice. In both assays, [Abu3,21]Lei-NH2 exhibited full Lei-NH2-like activity whereas [Abu8,26]Lei-NH2 and [Abu12,28]-Lei-NH2 possessed only residual activities (< 2% native toxin activity). This suggests that disulfide bridge Cys3-Cys21 is not essential per se for high toxin activity. Circular dichroism (CD) spectroscopy of the three analogs showed that only [Abu3,21]Lei-NH2 exhibited a CD spectrum similar to that of Lei-NH2, suggesting they both adopt closely related conformations, in agreement with the pharmacological data. Structural models of the analogs were constructed on the basis of the disulfide pairing assignment and compared with that of Lei-NH2.