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Structural and Functional Elucidation of Peptide Ts11 Shows Evidence of a Novel Subfamily of Scorpion Venom Toxins.
Cremonez CM
,
Maiti M
,
Peigneur S
,
Cassoli JS
,
Dutra AA
,
Waelkens E
,
Lescrinier E
,
Herdewijn P
,
de Lima ME
,
Pimenta AM
,
Arantes EC
,
Tytgat J
.
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To date, several families of peptide toxins specifically interacting with ion channels in scorpion venom have been described. One of these families comprise peptide toxins (called KTxs), known to modulate potassium channels. Thus far, 202 KTxs have been reported, belonging to several subfamilies of KTxs (called α, β, γ, κ, δ, and λ-KTxs). Here we report on a previously described orphan toxin from Tityus serrulatus venom, named Ts11. We carried out an in-depth structure-function analysis combining 3D structure elucidation of Ts11 and electrophysiological characterization of the toxin. The Ts11 structure is highlighted by an Inhibitor Cystine Knot (ICK) type scaffold, completely devoid of the classical secondary structure elements (α-helix and/or β-strand). This has, to the best of our knowledge, never been described before for scorpion toxins and therefore represents a novel, 6th type of structural fold for these scorpion peptides. On the basis of their preferred interaction with voltage-gated K channels, as compared to all the other targets tested, it can be postulated that Ts11 is the first member of a new subfamily, designated as ε-KTx.
Figure 1. Ts11 isolation procedure and molecular mass determination. (A) Reversed-phase fast protein liquid chromatography (RP-FPLC) profile of fraction XIIA from Tityus serrulatus venom on a C8 column; (B) Mass spectra of Ts11 was obtained through MALDI-TOF mass.
Figure 2. NMR solution structure of toxin peptide Ts11. (A) Stereoview of 15 final structures of Ts11 with superimposed backbone heavy atoms (N, CA, C’); (B) Stereo ribbon views of the closest-to-average structure of Ts11. Side chains of Cysteine residues are displayed along with their residue labels. Disulfide bonds are shown in yellow; (C) Amino acid sequence and disulfide connectivity of toxin peptide Ts11 along with a summary of medium and long range NOEs, 3JHNHα (3JαN) couplings, and chemical shift index (CSI) for the Hα protons.
3JHNHα < 6 Hz,
3JHNHα = 6–8 Hz,
3JHNHα > 8 Hz. The filled squares above and below the horizontal line represent CSI values of +1 and −1 respectively.
Figure 3. Electrophysiological study of Ts11 on voltage-gated potassium channels (Kv) expressed in Xenopus oocytes and measured using the two-electrode voltage-clamp. (A) Representative whole cell current traces on 13 different cloned voltage-gated potassium channels in the absence (control) and in the presence (*) of 3 μM native Ts11 (n ≥ 3); (B) Current-Voltage relationship on Kv1.3 in the absence (black circle, control) or in the presence (empty circle) of 5 μM Ts11 (n ≥ 3); (C) Dose-response curve of Ts11 on Kv1.3 (n ≥ 3).
Figure 4. Electrophysiological study of Ts11 on voltage-gated sodium channels (Nav). Representative whole cell current traces in control and in the presence of native Ts11 (1 μM, number of cells ≥ 3) on 5 expressed voltage gated sodium channels, in the absence or presence (*).
Figure 5. Amino acid sequence alignments and identities (%) among Ts11 and representatives of α-β-γ-κ-δ-λ-KTx-subfamilies, λ-KTx/calcine toxins, ϕ-liotoxin-Lw1a and chlorotoxins; and representatives of the five different structural folds adopted for scorpion toxins affecting KvsAlignment performed using ClustalW and MultAlin. The structures were created using Pymol and PDB database.
Figure 6. Comparison of the Ts11 with DDH-fold and ICK-fold toxins disulfide bond patterns. Disulfide patterns were compared with ϕ-Liotoxin-Lw1a (DDH motif), λ-KTxs and λ-KTx/calcine (three disulfide bridges ICK-type toxins, Imperatoxin A and Maurocalcin). Black lines represent the disulfide connectivity unique for Ts11. Red lines represent the DDH motif on ϕ-Liotoxin-Lw1a. Purple lines represent the disulfide connectivity on ICK-type toxins (λ-KTxs and λ-KTx/calcine). Long dashes: disulfide bond shared between Ts11 and ICK-type toxins. Dotted lines: disulfide connectivity shared between DDH motif and the ICK-type toxins. The green arrow indicates the positive charged residue of a possible dyad.
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