Role of individual disulfide bridges in the conformation and activity of spinoxin (α-KTx6.13), a potassium channel toxin from Heterometrus spinifer scorpion venom

Yoko Yamaguchi, Steve Peigneur, Junyi Liu, Shiho Uemura, Takeru Nose, Selvanayagam Nirthanan, Ponnampalam Gopalakrishnakone, Jan Tytgat, Kazuki Sato

Research output: Contribution to journalArticle

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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.

Original languageEnglish
Pages (from-to)31-38
Number of pages8
JournalToxicon
Volume122
DOIs
Publication statusPublished - Nov 1 2016

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Scorpion Venoms
Potassium Channels
Disulfides
Conformations
Cysteine
Isomers
Structure-Activity Relationship
Circular Dichroism
Scaffolds
Alanine
Peptides

All Science Journal Classification (ASJC) codes

  • Toxicology

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Role of individual disulfide bridges in the conformation and activity of spinoxin (α-KTx6.13), a potassium channel toxin from Heterometrus spinifer scorpion venom. / Yamaguchi, Yoko; Peigneur, Steve; Liu, Junyi; Uemura, Shiho; Nose, Takeru; Nirthanan, Selvanayagam; Gopalakrishnakone, Ponnampalam; Tytgat, Jan; Sato, Kazuki.

In: Toxicon, Vol. 122, 01.11.2016, p. 31-38.

Research output: Contribution to journalArticle

Yamaguchi, Yoko ; Peigneur, Steve ; Liu, Junyi ; Uemura, Shiho ; Nose, Takeru ; Nirthanan, Selvanayagam ; Gopalakrishnakone, Ponnampalam ; Tytgat, Jan ; Sato, Kazuki. / Role of individual disulfide bridges in the conformation and activity of spinoxin (α-KTx6.13), a potassium channel toxin from Heterometrus spinifer scorpion venom. In: Toxicon. 2016 ; Vol. 122. pp. 31-38.
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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.",
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T1 - Role of individual disulfide bridges in the conformation and activity of spinoxin (α-KTx6.13), a potassium channel toxin from Heterometrus spinifer scorpion venom

AU - Yamaguchi, Yoko

AU - Peigneur, Steve

AU - Liu, Junyi

AU - Uemura, Shiho

AU - Nose, Takeru

AU - Nirthanan, Selvanayagam

AU - Gopalakrishnakone, Ponnampalam

AU - Tytgat, Jan

AU - Sato, Kazuki

PY - 2016/11/1

Y1 - 2016/11/1

N2 - 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.

AB - 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.

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