Differential roles for disulfide bonds in the structural integrity and biological activity of kappa-Bungarotoxin, a neuronal nicotinic acetylcholine receptor antagonist.
kappa-Bungarotoxin, a kappa-neurotoxin derived from the venom of the banded Krait, Bungarus multicinctus, is a homodimeric protein composed of subunits of 66 amino acid residues containing five disulfide bonds. kappa-Bungarotoxin is a potent, selective, and slowly reversible antagonist of alpha3 beta2 neuronal nicotinic acetylcholine receptors. kappa-Bungarotoxin is structurally related to the alpha-neurotoxins, such as alpha-bungarotoxin derived from the same snake, which are monomeric in solution and which effectively antagonize muscle type receptors (alpha1 beta1 gamma delta) and the homopentameric neuronal type receptors (alpha7, alpha8, and alpha9). Like the kappa-neurotoxins, the long alpha-neurotoxins contain the same five conserved disulfide bonds, while the short alpha-neurotoxins only contain four of the five. Systematic removal of single disulfide bonds in kappa-bungarotoxin by site-specific mutagenesis reveals a differential role for each of the disulfide bonds. Removal of either of the two disulfides connecting elements of the carboxy terminal loop of this toxin (Cys 46-Cys 58 and Cys 59-Cys 64) interferes with the ability of the toxin to fold. In contrast, removal of each of the other three disulfides does not interfere with the general folding of the toxin and yields molecules with biological activity. In fact, when either C3-C21 or C14-C42 are removed individually, no loss in biological activity is seen. However, removing both produces a polypeptide chain which fails to fold properly. Removal of the C27-C31 disulfide only reduces the activity of the toxin 46.6-fold. This disulfide may play a role in specific interaction of the toxin with specific neuronal receptors.
Grant
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Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA. ggrant@pharmdec.wusl.edu
Luetje
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Summers
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Xu
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D000409:Alanine / Q000235:genetics; D000818:Animals; D002038:Bungarotoxins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D002942:Circular Dichroism; D003545:Cysteine / Q000235:genetics; D004220:Disulfides / Q000737:chemistry*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D018733:Nicotinic Antagonists / Q000494:pharmacology*; D009865:Oocytes / Q000378:metabolism; D017510:Protein Folding; D017433:Protein Structure, Secondary; D014982:Xenopus laevis
D002038:Bungarotoxins; D004220:Disulfides; D018733:Nicotinic Antagonists; D003545:Cysteine; D000409:Alanine