Scorpion Neurotoxin BeM9 Derivative Uncovers Unique Interaction Mode with Nav1.5 Sodium Channel Isoform

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Abstract

Scorpion α-neurotoxins are classical ligands of voltage-gated sodium channels that inhibit their inactivation. The strength of this effect depends on the organism and channel isoform, and the precise mechanisms explaining the differences in activity are still unknown. Previously, we have shown that scorpion α-toxins are characterized by a modular structure. They consist of a conserved and structurally stable core module and a variable and mobile specificity module, which determines the selectivity for different channels. We noted a higher mobility of the specificity module in toxins active against mammals compared to insect-active toxins. We then hypothesized that the enhanced mobility in mammal toxins was provided by two conserved glycine residues that enclose the N-terminal loop of the specificity module. To test this assumption, we obtained a derivative of the neurotoxin BeM9 from the venom of the scorpion Mesobuthus eupeus with two replacements of amino acid residues in the corresponding positions with glycine (A4G and Y17G). Unexpectedly, it turned out that BeM9GG lost its activity against Nav1.5 channel isoform, characteristic of mammalian cardiac muscle. A comparison of two known structures of voltage-gated sodium channel complexes with scorpion toxins made it possible to explain the observed effect. We hypothesize an essential role of the membrane in the interaction of toxins with the Nav1.5 isoform.

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About the authors

M. A. Chernykh

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: avas@ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

M. A. Duzheva

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences; D.I. Mendeleev Russian University of Chemical Technology

Email: avas@ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997; Miusskaya pl. 9, Moscow, 125047

N. A. Kuldyushev

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: avas@ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

S. Peigneur

KU Leuven, ON II

Email: avas@ibch.ru
Belgium, Herestraat 49, box 922, 3000, Leuven, Belgium

A. A. Berkut

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: avas@ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

J. Tytgat

KU Leuven, ON II

Email: avas@ibch.ru
Belgium, Herestraat 49, box 922, 3000, Leuven

A. A. Vassilevski

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Author for correspondence.
Email: avas@ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

A. O. Chugunov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: avas@ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

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Supplementary files

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2. Fig. 1. Structural features of α-NaTx. (a) Comparison of amino acid sequences of representatives of different α-NaTx groups. Pink shows the α-helix, blue – the β-strands, shades of green – different regions of the specificity module: light – RT-loop, darker – β₂–β₃-loop, dark – C-terminal region. The “hinge” glycines (residues 4 and 17 according to the Aah2 numbering) are highlighted in magenta; (b) – spatial organization of α-NaTx using the Aah2 mammalotoxin as an example. Color coding is similar to that in panel (a). Disulfide bridges are shown in yellow; (c) – general view of the interaction of α-NaTx with sodium channels using the Lqh3–hNaᵥ1.5 complex as an example [7]. Naᵥ is shown as a colored surface with individually colored homologous repeats D I–IV (PD is paler, VCD is bright). Lqh3 is shown in burgundy.

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3. Fig. 2. Differences in the structure of Naᵥ complexes with α-NaTx. (a, b) – Binding site of α-NaTx with Naᵥ in the membrane. VSD IV channel is highlighted in blue, PD I is highlighted in green (glycan is visible), toxins are shown in pink and crimson; (c) – comparison of amino acid sequence fragments S2 and S3 of VSD IV. The so-called “support” residues that determine the nature of the attachment of the β₂–β₃-loop of α-NaTx are highlighted in yellow; (d, d) – attachment of the β₂–β₃-loop of α-NaTx to the “support” residues S2–S3 of VSD IV. Color designations are similar to panels (a, b), the “support” residues are shown in yellow.

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4. Fig. 3. Effect of the Y17G mutation on BeM9 binding to Naᵥ. Channel IV VSD is shown in blue, channel I VSD is shown in green, BeM9 toxin is shown in pink, and the Y17 residue directed toward the membrane in BeM9 and replaced by glycine in BeM9GG is shown in dark red. (a) – Model of the BeM9–hNaᵥ1.5 complex obtained by spatial superposition with Lqh3 from the 7K18 complex. It is seen that the Y17 residue in BeM9 is buried in the membrane; (b) – model of the BeM9–mNaᵥ1.6 complex (for construction of the model, see the “Experimental section”).

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