Role of disulfide bonds in folding and activity of leiurotoxin I: just two disulfides suffice.
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.
Zhu
Qi
Q
College of Life Sciences, Peking University, Beijing, 100871, P. R. China.
Liang
Songping
S
Martin
Loïc
L
Gasparini
Sylvaine
S
Ménez
André
A
Vita
Claudio
C
D000595:Amino Acid Sequence; D000818:Animals; D001030:Apamin / Q000378:metabolism; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003553:Cystine; D004220:Disulfides / Q000737:chemistry*; D005978:Glutathione / Q000737:chemistry; D019803:Glutathione Disulfide / Q000737:chemistry; D036341:Intercellular Signaling Peptides and Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010456:Peptides, Cyclic / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D012604:Scorpion Venoms / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D004220:Disulfides; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D010456:Peptides, Cyclic; D012604:Scorpion Venoms; C077585:leiurutoxin III; C056574:leiurotoxin I; D001030:Apamin; D003553:Cystine; D005978:Glutathione; D019803:Glutathione Disulfide