title: Role of individual disulfide bridges in the conformation and activity of spinoxin (α-KTx6.13), a potassium channel toxin from Heterometrus spinifer scorpion venom.
abstract: Spinoxin (SPX; α-KTx6.13), isolated from venom of the scorpion Heterometrus spinifer, is a K+channel-specific peptide toxin (KTx), which adopts a cysteine-stabilized α/β scaffold that is cross-linked by four disulfide bridges (Cys1-Cys5, Cys2-Cys6, Cys3-Cys7, and Cys4-Cys8). To investigate the role of the individual disulfide bonds in the structure-activity relationship of SPX, we synthesized four SPX analogs in which each pair of cysteine residues was replaced by alanine residues. The analysis of circular dichroism spectra and inhibitory activity against Kv1.3 channels showed that the SPX analogs lacking any of three specific disulfide bonds (Cys1-Cys5, Cys2-Cys6, and Cys3-Cys7) were unable to form the native secondary structure and completely lost inhibitory activities. Thus, we conclude that Cys1-Cys5, Cys2-Cys6, and Cys3-Cys7 are required for the inhibition of the Kv1.3 channel by SPX. In contrast, the analog lacking Cys4-Cys8 retained both native secondary structure and inhibitory activity. Interestingly, one of the isomers of the analog lacking Cys1-Cys5 also showed inhibitory activities, although its inhibition was ∼18-fold weaker than native SPX. This isomer had an atypical disulfide bond pairing (Cys3-Cys4 and Cys7-Cys8) that corresponds to that of maurotoxin (MTX), another α-KTx6 family member. These results indicate that the Cys1-Cys5 and Cys2-Cys6 bonds are important for restricting the toxin from forming an atypical (MTX-type) disulfide bond pairing among the remaining four cysteine residues (Cys3, Cys4, Cys7, and Cys8) in native SPX.
authors.affiliation: Department of Environment Sciences, Fukuoka Women's University, Fukuoka, 813-8529, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Yamaguchi
authors.affiliation: Toxicology and Pharmacology, University of Leuven, Campus Gasthuisberg O&N2, Herestraat 49, P.O. Box 922, Leuven, 3000, Belgium.
authors.forename: Steve
authors.initials: S
authors.lastname: Peigneur
authors.affiliation: Department of Environment Sciences, Fukuoka Women's University, Fukuoka, 813-8529, Japan.
authors.forename: Junyi
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Environment Sciences, Fukuoka Women's University, Fukuoka, 813-8529, Japan.
authors.forename: Shiho
authors.initials: S
authors.lastname: Uemura
authors.affiliation: Faculty of Arts and Science, Kyushu University, Fukuoka, 819-0395, Japan.
authors.forename: Takeru
authors.initials: T
authors.lastname: Nose
authors.affiliation: School of Medicine, Griffith University, Gold Coast, Queensland, 4222, Australia; School of Medical Science, Griffith University, Gold Coast, Queensland, 4222, Australia; Menzies Health Institute Queensland, Gold Coast, Australia.
authors.forename: Selvanayagam
authors.initials: S
authors.lastname: Nirthanan
authors.affiliation: Venom and Toxin Research Program, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 117597, Singapore.
authors.forename: Ponnampalam
authors.initials: P
authors.lastname: Gopalakrishnakone
authors.affiliation: Toxicology and Pharmacology, University of Leuven, Campus Gasthuisberg O&N2, Herestraat 49, P.O. Box 922, Leuven, 3000, Belgium.
authors.forename: Jan
authors.initials: J
authors.lastname: Tytgat
authors.affiliation: Department of Environment Sciences, Fukuoka Women's University, Fukuoka, 813-8529, Japan. Electronic address: sato@fwu.ac.jp.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Sato
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D005260:Female; D015221:Potassium Channels / Q000187:drug effects*; D011487:Protein Conformation; D012604:Scorpion Venoms / Q000737:chemistry*; D012605:Scorpions; D017386:Sequence Homology, Amino Acid; D014981:Xenopus
chemical_list: D004220:Disulfides; D015221:Potassium Channels; D012604:Scorpion Venoms
keywords: Cysteine-stabilized α/β scaffold; Disulfide bond formation; Scorpion toxin; Synthetic analog; Voltage-gated potassium channel