Role of Disulfide Bonds in Folding and Activity of Leiurotoxin I:  Just Two Disulfides Suffice

Abstract

The aim of this study is to investigate the contribution of each disulfide bond in the folding and function of leiurotoxin I, a short scorpion toxin that blocks small conductance K(+) channels. The structure of leiurotoxin I contains a motif conserved in all scorpion toxins, formed by a helix and a double-stranded beta-sheet and stabilized by three disulfide bridges. We synthesized three analogues, each presenting two alpha-aminobutyric acid (Abu) moieties replacing two bridged cysteine residues: LeTx1 ([Abu 3,21] Leiurotoxin I), LeTx2 ([Abu 8,26] Leiurotoxin I), and LeTx3 ([Abu 12,28] Leiurotoxin I). All three analogues fold into a major product containing two native disulfide bonds, while LeTx3 forms an additional isomer, containing non-native disulfides. In denaturing conditions, analogues LeTx2 and LeTx3 yield non-native isomers, while LeTx1 only forms the isomer with native disulfides. All isomers with native disulfides contain nativelike alpha-helical conformations and bind to synaptosomal membranes with affinities within a log of that shown by the native toxin. By contrast, the non-native LeTx3A analogue exhibits a disordered conformation and a decreased biological potency. Our results indicate that the "CxxxC, CxC" cysteine spacing, conserved in all scorpion toxins and preserved in LeTx1, may play an active role in folding, and that only two native disulfide bonds in leiurotoxin I are sufficient to preserve a nativelike and active conformation. Thus, in the scorpion toxin scaffold, modifications of conserved and interior cysteine residues may permit modulation of function, without significantly affecting folding efficiency and structure.

Role of N-Glycosylation Sites and CXC Motifs in Trafficking of Medicago truncatula Nod Factor Perception Protein to Plasma Membrane

“…CXC motifs are found in several protein families, including some plant proteins (e.g. prolamins and lipid transfer proteins containing an 8-Cys motif (48)), some scorpion toxins and related natural peptides (49), and the animal CXC-type chemokines (50). In some of these proteins, the CXC motif has been shown to be involved in S-S bridges.…”

“…In some of these proteins, the CXC motif has been shown to be involved in S-S bridges. In most of these cases, each Cys residue of the CXC motif forms an S-S bridge with another Cys residue in the protein, and in only a few very rare examples does the CXC motif form an intramotif S-S bridge leading to a thermodynamically disfavored 11-member ring structure (49,51).…”

Structure-Activity Relationships of Hainantoxin-IV and Structure Determination of Active and Inactive Sodium Channel Blockers

“…The linear peptides were oxidized with glutathione and purified using the method recently described by Zhuqi (15). The molecular weights of the reduced peptides or oxidized peptides were checked by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry on a Voyager-DE TM STR Biospectrometry TM work station.…”

Btdb - Database on Structural and Functional Information of Biotoxins

“…The biotoxins were also analyzed by the number of disulphide bridges present in the predicted models. The more disulphide bridges present, the stability of the biotoxin structure (Zhu et al, 2002;Possani et al, 1999) improves and exerts increased inhibitor activity as in the case of voltage gated ion channels (Boubaker et al, 2004;Kharrat et al, 1997 ) In the snake dataset, nearly 94% of structure has RMSD value below 4Å (Fig. 2a).…”