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Neurotoxin Merging: A Strategy Deployed by the Venom of the Spider Cupiennius salei to Potentiate Toxicity on Insects.
Clémençon B
,
Kuhn-Nentwig L
,
Langenegger N
,
Kopp L
,
Peigneur S
,
Tytgat J
,
Nentwig W
,
Lüscher BP
.
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The venom of Cupiennius salei is composed of dozens of neurotoxins, with most of them supposed to act on ion channels. Some insecticidal monomeric neurotoxins contain an α-helical part besides their inhibitor cystine knot (ICK) motif (type 1). Other neurotoxins have, besides the ICK motif, an α-helical part of an open loop, resulting in a heterodimeric structure (type 2). Due to their low toxicity, it is difficult to understand the existence of type 2 peptides. Here, we show with the voltage clamp technique in oocytes of Xenopus laevis that a combined application of structural type 1 and type 2 neurotoxins has a much more pronounced cytolytic effect than each of the toxins alone. In biotests with Drosophila melanogaster, the combined effect of both neurotoxins was enhanced by 2 to 3 log units when compared to the components alone. Electrophysiological measurements of a type 2 peptide at 18 ion channel types, expressed in Xenopus laevis oocytes, showed no effect. Microscale thermophoresis data indicate a monomeric/heterodimeric peptide complex formation, thus a direct interaction between type 1 and type 2 peptides, leading to cell death. In conclusion, peptide mergers between both neurotoxins are the main cause for the high cytolytic activity of Cupienniussalei venom.
Figure 1. Sequence comparison and disulfide bridge arrangement of different neurotoxin structures from Cupiennius salei. Proposed C-terminal α-helical structures are shaded in light green and amino acid residues involved in forming the main part of loop 3 of the ICK structure are shaded in yellow/light brown. The possible docking region of heterodimeric neurotoxins and the proposed corresponding docking region of monomeric neurotoxins are boxed according to their charge. The involved cationic amino acid residues are in red and anionic amino acid residues are colored in blue. CT1-long and CT13-long are indicated by a box. Cysteines are highlighted in gray.
Figure 2. Effect of CsTx-1, CsTx-13, and the combination of both in Xenopus laevis oocytes. The membrane potential of denuded oocytes was adjusted to -40 mV. (A) Exposure of CsTx-1 (blue) at the 0.25 µM concentration results in an inward current amounting to several μA gradually developing. (B) CsTx-13 (red) has no effect on the resting current of oocytes up to the 5 µM concentration. However, (C) CsTx-13 induces an inward current at the 20 µM concentration, comparable to CsTx-1 at the 0.25 µM concentration. (D) The CsTx-1-induced current is amplified 1.8-fold after the application of CsTx-13 at an equal molar concentration (0.25 µM).
Figure 3. Effect of CsTx-9, and in combination with CsTx-13 or CsTx-1 in Xenopus laevis oocytes. (A) CsTx-9 (green) did not induce a current up to the 20 µM concentration. (B) A serial application of CsTx-9 (0.25 µM) followed by an application of CsTx-13 (red) in an equal molar ratio (0.25 µM) resulted in an inward current comparable in size to the current amplitude induced by CsTx-1 and CsTx-13 at an equal molar concentration (Figure 2D). (C) CsTx-9 did not affect the CsTx-1 (blue)-induced current in a serial application after reaching the plateau phase of the CsTx-1-induced current, indicating that no interaction occurred between CsTx-1 and CsTx-9.
Figure 4. Insecticidal activity of the heterodimeric CsTx-13 alone, and in combination with the monomeric neurotoxins CsTx-1 and CsTx-9 on Drosophila flies. For the bioassays, different concentrations of CsTx-1, CsTx-9, and CsTx-13 alone, or in combinations of CsTx-1 and CsTx-9, CsTx-1 and CsTx-13, and CsTx-9 and CsTx-13 (1:1 molar ratio), were injected in 0.1 M ammonium acetate, pH = 6.1 (injected volume 50 nL). Each data point represents 10 injected flies. Calculations of the lethal dose (LD50), where 50% of the injected flies died of intoxication after 24 h, were done with GraphPad PRISM vers. 6.07. A negative control was done with injection of 50 nL of the abovementioned buffer alone into the flies.
Figure 5. Peptide–peptide interaction of CsTx-1/9 with CsTx-13 using MST. (A) Titration using CsTx-13 neurotoxin to CsTx-1 was performed. High-affinity binding of CsTx-13 to CsTx-1 was measured with KD equal to around 430 nM (B) Similar KD was observed for CsTx-9 at 370 nM. (C) No binding interaction occurred between CsTx-1 and CsTx-9. In all cases, data were standardized to bound fractions.
Figure 6. Identification of interacting protein domains in Xenopus laevis oocyte membranes. (A) CT13-long (orange) had no effect on the CsTx-1 (blue)-induced current. (B) CT13-long (orange) was not able to induce a current in combination with CsTx-9 (green). (C) CT1-long (purple)-induced current could not be amplified using CsTx-13 (red). (D) No protein–protein interaction could be observed between CsTx-1 and CT13-long using MST. Data were standardized to the Δ fluorescence shift between bound to unbound fractions of CsTx-1 and CT13-long.
Figure 7. Homology model of CsTx-1, CsTx-9, and CsTx13 based on the NMR structure of purotoxin-2. (A) The predicted 3D structure of CsTx-13 was visualized in a cartoon representation and allows the relative position of the secondary structures within the model to be distinguished. The visualization showed two domains: (i) An α-helical domain at the C-terminal end composed of an α-helix (Y56-G60) responsible for the cytolytic activity of the neurotoxin, and (ii) a globular domain at the N-terminal part exhibiting the ICK motif composed of 3 β-sheets linked with 4 disulfide bonds colored in green (C3-C18), blue (C10-C27), purple (C17-C48), and orange (C29-C48). (B) The surface representation allows evidence of the spatial localization of all positive (red) and negative (blue) amino acid residues within the overall predicted structures and highlights “Y” shape electrostatic interaction domains responsible for neurotoxin merging, which is composed of loop 3 and part of the ICK of CsTx-13, CsTx-1 and CsTx-9, respectively.
Figure 8. Hydrophobicity plot of C-terminal linear and α-helical structures of neurotoxins. (A) The consensus hydrophobicity scale of Eisenberg et al. [18] was used to characterize the hydrophobicity of each amino acid residue. (B) α-helical wheel projection of the C-terminus of CsTx-13 exhibiting distinct hydrophobic and polar regions.
Figure 9. Proposed mode of action of neurotoxin merging. Our model presents neurotoxins as water-soluble molecules with non-structured C-terminal ends. CsTx-13 is able to bind to CsTx1/9 via their N-terminal globular domains, which include loop 3 as well as parts of the ICK motifs. These merged neurotoxins possess spatially closer C-terminal ends, which mimic a hydrophobic environment and may subsequently lead to α-helical formation in the presence of membranes. This conformational change induces an increase of the hydrophobicity pattern of the merged peptides. Insertion of the hydrophobic areas of the α-helices into the membrane induces an alteration of the membrane curvature, leading to a thinning out of the outer leaflet lipids and the formation of lipid/merged neurotoxin micelles, which finally will induce the disruption of the membrane lipid bilayer observed as a cytolytic effect in vitro. A “recycling” process for the peptide merger is hypothesized.
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