Dataset Information

Scorpion toxin inhibits the voltage-gated proton channel using a Zn²⁺ -like long-range conformational coupling mechanism.

ABSTRACT:

Background and purpose

Blocking the voltage-gated proton channel H_V 1 is a promising strategy for the treatment of diseases like ischaemia stroke and cancer. However, few H_V 1 channel antagonists have been reported. Here, we have identified a novel H_V 1 channel antagonist from scorpion venom and have elucidated its action mechanism.

Experimental approach

H_V 1 and NaV channels were heterologously expressed in mammalian cell lines and their currents recorded using whole-cell patch clamp. Site-directed mutagenesis was used to generate mutants. Toxins were recombinantly produced in Escherichia coli. AGAP/W38F-H_V 1 interaction was modelled by molecular dynamics simulations.

Key results

The scorpion toxin AGAP (anti-tumour analgesic peptide) potently inhibited H_V 1 currents. One AGAP mutant has reduced Na_V channel activity but intact H_V 1 activity (AGAP/W38F). AGAP/W38F inhibited H_V 1 channel activation by trapping its S4 voltage sensor in a deactivated state and inhibited H_V 1 currents with less pH dependence than Zn²⁺ . Mutation analysis showed that the binding pockets of AGAP/W38F and Zn²⁺ in H_V 1 channel partly overlapped (common sites are His140 and His193). The E153A mutation at the intracellular Coulombic network (ICN) in H_V 1 channel markedly reduced AGAP/W38F inhibition, as observed for Zn²⁺ . Experimental data and MD simulations suggested that AGAP/W38F inhibited H_V 1 channel using a Zn²⁺ -like long-range conformational coupling mechanism.

Conclusion and implications

Our results suggest that the Zn²⁺ binding pocket in H_V 1 channel might be a hotspot for modulators and valuable for designing H_V 1 channel ligands. Moreover, AGAP/W38F is a useful molecular probe to study H_V 1 channel and a lead compound for drug development.

SUBMITTER: Tang D

PROVIDER: S-EPMC7174885 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The recognition of the scorpion toxin maurotoxin (MTX) by the voltage-gated potassium (Kv1) channels, Kv1.1, Kv1.2, and Kv1.3, has been studied by means of Brownian dynamics (BD) simulations. All of the 35 available structures of MTX in the Protein Data Bank (http://www.rcsb.org/pdb) determined by nuclear magnetic resonance were considered during the simulations, which indicated that the conformation of MTX significantly affected both the recognition and the binding between MTX and the Kv1 channels. Comparing the top five highest-frequency structures of MTX binding to the Kv1 channels, we found that the Kv1.2 channel, with the highest docking frequencies and the lowest electrostatic interaction energies, was the most favorable for MTX binding, whereas Kv1.1 was intermediate, and Kv1.3 was the least favorable one. Among the 35 structures of MTX, the 10th structure docked into the binding site of the Kv1.2 channel with the highest probability and the most favorable electrostatic interactions. From the MTX-Kv1.2 binding model, we identified the critical residues for the recognition of these two proteins through triplet contact analyses. MTX locates around the extracellular mouth of the Kv1 channels, making contacts with its beta-sheets. Lys23, a conserved amino acid in the scorpion toxins, protrudes into the pore of the Kv1.2 channel and forms two hydrogen bonds with the conserved residues Gly401(D) and Tyr400(C) and one hydrophobic contact with Gly401(C) of the Kv1.2 channel. The critical triplet contacts for recognition between MTX and the Kv1.2 channel are Lys23(MTX)-Asp402(C)(Kv1), Lys27(MTX)-Asp378(D)(Kv1), and Lys30(MTX)-Asp402(A)(Kv1). In addition, six hydrogen-bonding interactions are formed between residues Lys23, Lys27, Lys30, and Tyr32 of MTX and residues Gly401, Tyr400, Asp402, Asp378, and Thr406 of Kv1.2. Many of them are formed by side chains of residues of MTX and backbone atoms of the Kv1.2 channel. Five hydrophobic contacts exist between residues Pro20, Lys23, Lys30 and Tyr32 of MTX and residues Asp402, Val404, Gly401, and Arg377 of the Kv1.2 channel. The simulation results are in agreement with the previous molecular biology experiments and explain the binding phenomena between MTX and Kv1 channels at the molecular level. The consistency between the results of the BD simulations and the experimental data indicated that our three-dimensional model of the MTX-Kv1.2 channel complex is reasonable and can be used in additional biological studies, such as rational design of novel therapeutic agents blocking the voltage-gated channels and in mutagenesis studies in both the toxins and the Kv1 channels. In particular, both the BD simulations and the molecular mechanics refinements indicate that residue Asp378 of the Kv1.2 channel is critical for its recognition and binding functionality toward MTX. This phenomenon has not been appreciated in the previous mutagenesis experiments, indicating this might be a new clue for additional functional study of Kv1 channels.

Project description:Pi1 is a 35-residue scorpion toxin cross-linked by four disulphide bridges that acts potently on both small-conductance Ca2+-activated (SK) and voltage-gated (Kv) K+ channel subtypes. Two approaches were used to investigate the relative contribution of the Pi1 functional dyad (Tyr-33 and Lys-24) to the toxin action: (i) the chemical synthesis of a [A24,A33]-Pi1 analogue, lacking the functional dyad, and (ii) the production of a Pi1 analogue that is phosphorylated on Tyr-33 (P-Pi1). According to molecular modelling, this phosphorylation is expected to selectively impact the two amino acid residues belonging to the functional dyad without altering the nature and three-dimensional positioning of other residues. P-Pi1 was directly produced by peptide synthesis to rule out any possibility of trace contamination by the unphosphorylated product. Both Pi1 analogues were compared with synthetic Pi1 for bioactivity. In vivo, [A24,A33]-Pi1 and P-Pi1 are lethal by intracerebroventricular injection in mice (LD50 values of 100 and 40 microg/mouse, respectively). In vitro, [A24,A33]-Pi1 and P-Pi1 compete with 125I-apamin for binding to SK channels of rat brain synaptosomes (IC50 values of 30 and 10 nM, respectively) and block rat voltage-gated Kv1.2 channels expressed in Xenopus laevis oocytes (IC50 values of 22 microM and 75 nM, respectively), whereas they are inactive on Kv1.1 or Kv1.3 channels at micromolar concentrations. Therefore, although both analogues are less active than Pi1 both in vivo and in vitro, the integrity of the Pi1 functional dyad does not appear to be a prerequisite for the recognition and binding of the toxin to the Kv1.2 channels, thereby highlighting the crucial role of other toxin residues with regard to Pi1 action on these channels. The computed simulations detailing the docking of Pi1 peptides on to the Kv1.2 channels support an unexpected key role of specific basic amino acid residues, which form a basic ring (Arg-5, Arg-12, Arg-28 and Lys-31 residues), in toxin binding.

Dataset Information

Scorpion toxin inhibits the voltage-gated proton channel using a Zn²⁺ -like long-range conformational coupling mechanism.

Background and purpose

Experimental approach

Key results

Conclusion and implications

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Dataset Information

Scorpion toxin inhibits the voltage-gated proton channel using a Zn2+ -like long-range conformational coupling mechanism.

Background and purpose

Experimental approach

Key results

Conclusion and implications

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Scorpion toxin inhibits the voltage-gated proton channel using a Zn²⁺ -like long-range conformational coupling mechanism.