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Int J Mol Sci
2024 Sep 28;2519:. doi: 10.3390/ijms251910472.
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Structure-Function Relationship of a Novel MTX-like Peptide (MTX1) Isolated and Characterized from the Venom of the Scorpion Maurus palmatus.
ElFessi R
,
Khamessi O
,
De Waard M
,
Srairi-Abid N
,
Ghedira K
,
Marrouchi R
,
Kharrat R
.
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Maurotoxin (MTX) is a 34-residue peptide from Scorpio maurus venom. It is reticulated by four disulfide bridges with a unique arrangement compared to other scorpion toxins that target potassium (K+) channels. Structure-activity relationship studies have not been well performed for this toxin family. The screening of Scorpio maurus venom was performed by different steps of fractionation, followed by the ELISA test, using MTX antibodies, to isolate an MTX-like peptide. In vitro, in vivo and computational studies were performed to study the structure-activity relationship of the new isolated peptide. We isolated a new peptide designated MTX1, structurally related to MTX. It demonstrated toxicity on mice eight times more effectively than MTX. MTX1 blocks the Kv1.2 and Kv1.3 channels, expressed in Xenopus oocytes, with IC50 values of 0.26 and 180 nM, respectively. Moreover, MTX1 competitively interacts with both 125I-apamin (IC50 = 1.7 nM) and 125I-charybdotoxin (IC50 = 5 nM) for binding to rat brain synaptosomes. Despite its high sequence similarity (85%) to MTX, MTX1 exhibits a higher binding affinity towards the Kv1.2 and SKCa channels. Computational analysis highlights the significance of specific residues in the β-sheet region, particularly the R27, in enhancing the binding affinity of MTX1 towards the Kv1.2 and SKCa channels.
Figure 1. Purification of MTX1 from Scorpio maurus palmatus scorpion venom. (A) Chromatography of Sephadex G-50 column. Fractions I–VII were collected. (B) Chromatography of fraction IV by using C18 RP-HPLC. (C) Chromatography of pics P1 and P2 eluted at 14.31 min and 14.62 min on C18-RP-HPLC. MTX1 and MTX were collected at 25.19 min and 41.3 min, respectively.
Figure 2. (A) The sequence of MTX1 aligned with Kv scorpion toxins. The MTX1 amino acid sequence was revealed by automatic Edman degradation. The alignment of MTX1 with members of the alpha-KTx subfamily is shown. Cysteine residues are highlighted in green. The numbers next to each toxin name represent the percentage identity (ID) of MTX1. (B) A phylogenetic tree depicting the relationship between MTX, MTX1 and the alpha-KTx toxin family is shown. Phylogenetic analyses were conducted using the JTT matrix-based method, considering the sequence homology of all the toxins. This analysis encompassed 34 to 38 amino acid sequences. The topology of the tree was confirmed using 1000 bootstrap replicates. Bootstraps around 70 are highlighted in red, whereas those around 100 are shown in green.
Figure 3. Cross-antigenicity characterization of MTX1, MTX and CHTX. Binding of anti-MTX serum to plates coated with 5 µg/mL of MTX (●), MTX1 (■) or CHTX (▲).
Figure 4. Competitive binding on rat brain synaptosomes of MTX1 and potassium channel toxins. (A) Inhibition of 125I-CHTX binding to rat brain synaptosomes by unlabeled CHTX (▪), MTX1 (▲) and MTX (●). Instances of non-specific binding, defined as less than 20% of total binding of 125I-CHTX, were subtracted from the ratio calculation. (B) Inhibition of 125I-apamin binding to rat brain synaptosomes by unlabeled Apamin (⧫), MTX (▲) and MTX1 (■) and CHTX (●). Instances of non-specific binding, defined as less than 10% of total binding of 125I-apamin, were subtracted from the ratio calculation. BO represents radiolabeled toxins binding in the absence of a ligand, and B represents binding in the presence of the indicated concentrations of competitors. The values shown on the curves represent the average of three experimental values. The errors less than or equal to 0.05.
Figure 5. The blocking efficacy of MTX1 on the Kv1.2 and Kv1.3 channels. (A,D) Oocytes expressing the Kv1.2 or Kv1.3 channel were recorded using a two-electrode voltage clamp. K+ currents were obtained by depolarization from a holding potential of −80 mV to +70 mV under different concentrations of MTX1 toxin, illustrating Kv1 channel block. (B,E). Dose-response effects of MTX1 on Kv1.2 and Kv1.3 channel currents with IC50 values of 0.26 nM and 180 nM, respectively, with a Hill coefficient of 1.1 ± 0.1. (C,F) Comparison of K+ control current (○) and inhibition by MTX1 at 2.5 µM for Kv1.2 and 2.5 nM for Kv1.3 (●). Data points represent the mean ± SEM. The tail current likely consists of a combination of inward K+ current and outward Cl− current. The addition of niflumic acid aimed to inhibit chloride currents, yet its efficacy remains incomplete. In the case of the inward potassium current, it is plausible that, at a holding potential of −80 mV, we surpass the ion’s reversal potential, possibly accounting for the inward current during repolarization and the toxin’s partial impact. Reduction in tail current mirrored the ionic current’s amplitude, indicating the toxin’s specific action on the channel.
Figure 6. A cartoon representation of the 3D structures of the MTX1 and MTX. The hotspots at positions 23 and 32 are represented as sticks, the disulfide bridges are represented in spheres and colored olive and the different amino acids between MTX1 and MTX are represented by white colored sticks.
Figure 7. Interaction modes of MTX1 with SK2, Kv1.2 and KV1.3 as predicted by a protein–protein docking study. Residues defining the toxin–channel interactions are highlighted in black for the ligand (MTX1) and in red for the targeted channel. Elementary interactions are shown in zoom.
