Structure of Membrane-active Toxin from Crab Spider Heriaeus melloteei Suggests Parallel Evolution of Sodium Channel Gating Modifiers in Araneomorphae and Mygalomorphae

Antonina A Berkut,Steve Peigneur,Mikhail Yu Myshkin,Alexander S Paramonov,Ekaterina N Lyukmanova,Alexander S Arseniev,Eugene V Grishin,Jan Tytgat,Zakhar O Shenkarev,Alexander A Vassilevski

doi:10.1074/jbc.m114.595678

Abstract

We present a structural and functional study of a sodium channel activation inhibitor from crab spider venom. Hm-3 is an insecticidal peptide toxin consisting of 35 amino acid residues from the spider Heriaeus melloteei (Thomisidae). We produced Hm-3 recombinantly in Escherichia coli and determined its structure by NMR spectroscopy. Typical for spider toxins, Hm-3 was found to adopt the so-called "inhibitor cystine knot" or "knottin" fold stabilized by three disulfide bonds. Its molecule is amphiphilic with a hydrophobic ridge on the surface enriched in aromatic residues and surrounded by positive charges. Correspondingly, Hm-3 binds to both neutral and negatively charged lipid vesicles. Electrophysiological studies showed that at a concentration of 1 μm Hm-3 effectively inhibited a number of mammalian and insect sodium channels. Importantly, Hm-3 shifted the dependence of channel activation to more positive voltages. Moreover, the inhibition was voltage-dependent, and strong depolarizing prepulses attenuated Hm-3 activity. The toxin is therefore concluded to represent the first sodium channel gating modifier from an araneomorph spider and features a "membrane access" mechanism of action. Its amino acid sequence and position of the hydrophobic cluster are notably different from other known gating modifiers from spider venom, all of which are described from mygalomorph species. We hypothesize parallel evolution of inhibitor cystine knot toxins from Araneomorphae and Mygalomorphae suborders.

Highlights

Several toxins from mygalomorph spiders are known to inhibit sodium channel activation
The target peptide was purified after cleanup of the chimeric protein Trx-Hm-3 by affinity chromatography, its cleavage by cyanogen bromide (CNBr), and separation of the hydrolysate by reversed-phase HPLC
Hm-3 Is a Sodium Channel Gating Modifier—Electrophysiological studies demonstrate that Hm-3 effectively inhibits both mammalian and insect neurotoxins acting on voltage-gated sodium channels (NaVs) (Fig. 4A, left panels)

Summary

Introduction

Several toxins from mygalomorph spiders are known to inhibit sodium channel activation. Results: Hm-3 toxin from the araneomorph spider Heriaeus melloteei inhibits mammalian and insect sodium channel activation and possesses membrane activity. Significance: The first sodium channel activation inhibitor from Araneomorphae points to parallel evolution of H. melloteei and its distant mygalomorph relatives. We present a structural and functional study of a sodium channel activation inhibitor from crab spider venom. Hm-3 is an insecticidal peptide toxin consisting of 35 amino acid residues from the spider Heriaeus melloteei (Thomisidae). The inhibition was voltage-dependent, and strong depolarizing prepulses attenuated Hm-3 activity. Its amino acid sequence and position of the hydrophobic cluster are notably different from other known gating modifiers from spider venom, all of which are described from mygalomorph species. We hypothesize parallel evolution of inhibitor cystine knot toxins from Araneomorphae and Mygalomorphae suborders

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Conclusion