An unusual fold for potassium channel blockers: NMR structure of three toxins from the scorpion Opisthacanthus madagascariensis

Abstract

The Om-toxins are short peptides (23-27 amino acids) purified from the venom of the scorpion Opisthacanthus madagascariensis. Their pharmacological targets are thought to be potassium channels. Like Csalpha/beta (cystine-stabilized alpha/beta) toxins, the Om-toxins alter the electrophysiological properties of these channels; however, they do not share any sequence similarity with other scorpion toxins. We herein demonstrate by electrophysiological experiments that Om-toxins decrease the amplitude of the K+ current of the rat channels Kv1.1 and Kv1.2, as well as human Kv1.3. We also determine the solution structure of three of the toxins by use of two-dimensional proton NMR techniques followed by distance geometry and molecular dynamics. The structures of these three peptides display an uncommon fold for ion-channel blockers, Csalpha/alpha (cystine-stabilized alpha-helix-loop-helix), i.e. two alpha-helices connected by a loop and stabilized by two disulphide bridges. We compare the structures obtained and the dipole moments resulting from the electrostatic anisotropy of these peptides with those of the only other toxin known to share the same fold, namely kappa-hefutoxin1.

Figure 1. Purification four peptides from the venom of the scorpion Opisthacanthus madagascariensis

The subfraction containing the Om-toxins was separated by reverse-phase HPLC (TSK gel ODS 120-T column; 0.46 cm×25 cm) with a 40 min linear gradient of 12.5–60% (v/v) acetonitrile/water (1:1, v/v) in 0.1% TFA. The fractions containing the four peptides OmTx1, OmTx2, OmTx3 and OmTx4 were collected, and the peptides were lyophilized and further purified by HPLC on a TSK ODS 120T gel with a linear gradient of 12.5–60% acetonitrile in 0.01 M ammonium acetate, pH 5.8, in 40 min.

Figure 2. Amino acid sequence determination of OmTx1, OmTx2, OmTx3 and OmTx4

The alkylated peptides were digested with trypsin and subjected to LC/MS and LC/MS/MS analysis. The arrows show cleavage sites.

Figure 3. Effects of OmTx1, OmTx2 and OmTx3 on Kv1.1, Kv1.2 and Kv1.3 channels expressed in Xenopus laevis oocytes

Control conditions are compared with the presence of the toxin (500 μM; indicated by *). See the text for details.

Figure 4. Sequence alignment of Om-toxins with related toxins (A), and nOe statistics for OmTx1 (B), OmTx2 (C) and OmTx3 (D)

(A) Sequence alignment of the three Om-toxins with κ-hefutoxin1 isolated from the scorpion Heterometrus fulvipes and BgK toxin from the sea anemone Bunodosoma granulifera. Cysteine residues are highlighted in black, and conserved residues are highlighted in grey. (B) Upper panel: sequence of OmTx1 and sequential assignment. Collected nOes are classified into weak, medium and strong, which are indicated by thin, medium and thick lines respectively. Lower panels: nOe and R.M.S.D. distribution for OmTx1. Intraresidue nOes are designated by black bars, sequential nOes by dark grey bars, medium nOes by light grey bars and long-range nOes by white bars. R.M.S.D. values for backbone and all heavy atoms are indicated by black and grey bars respectively. (C) As in (B), for OmTx2 (C) and OmTx3 (D).

Figure 5. Chemical shift variations in relation to sequence for OmTx2 compared with OmTx1 (□) and for OmTx3 compared with OmTx1 (■)

Figure 6. Three-dimensional structure of Om-toxins and κ-hefutoxin, and their dipole moment

(A) Stereopair views of the best 30 solution structures [backbone (N, Ca, C)] of the molecules OmTx1, OmTx2 and OmTx3. κ-Hefutoxin structure is not available (NA). (B) Molscript representations of OmTx1, OmTx2, OmTx3 and κ-hefutoxin. (C) GRASP representations of OmTx1, OmTx2, OmTx3 and κ-hefutoxin. The dipole moments are indicated by the arrows. The surface and the backbone of the toxins are represented.