Recombinant Production, NMR Solution Structure, and Membrane Interaction of the Phα1β Toxin, a TRPA1 Modulator from the Brazilian Armed Spider Phoneutria nigriventer

Ph & alpha;1 & beta; (PnTx3-6) is a neurotoxin from the spider Phoneutria nigriventer venom, originally identified as an antagonist of two ion channels involved in nociception: N-type voltage-gated calcium channel (Ca(V)2.2) and TRPA1. In animal models, Ph & alpha;1 & beta; administration reduces both acute and chronic pain. Here, we report the efficient bacterial expression system for the recombinant production of Ph & alpha;1 & beta; and its N-15-labeled analogue. Spatial structure and dynamics of Ph & alpha;1 & beta; were determined via NMR spectroscopy. The N-terminal domain (Ala1-Ala40) contains the inhibitor cystine knot (ICK or knottin) motif, which is common to spider neurotoxins. The C-terminal & alpha;-helix (Asn41-Cys52) stapled to ICK by two disulfides exhibits the & mu;s-ms time-scale fluctuations. The Ph & alpha;1 & beta; structure with the disulfide bond patterns Cys1-5, Cys2-7, Cys3-12, Cys4-10, Cys6-11, Cys8-9 is the first spider knottin with six disulfide bridges in one ICK domain, and is a good reference to other toxins from the ctenitoxin family. Ph & alpha;1 & beta; has a large hydrophobic region on its surface and demonstrates a moderate affinity for partially anionic lipid vesicles at low salt conditions. Surprisingly, 10 & mu;M Ph & alpha;1 & beta; significantly increases the amplitude of diclofenac-evoked currents and does not affect the allyl isothiocyanate (AITC)-evoked currents through the rat TRPA1 channel expressed in Xenopus oocytes. Targeting several unrelated ion channels, membrane binding, and the modulation of TRPA1 channel activity allow for considering Ph & alpha;1 & beta; as a gating modifier toxin, probably interacting with S1-S4 gating domains from a membrane-bound state.

Automated summary

This article reports on the neurotoxin Phα1β (PnTx3-6) from the venom of the spider Phoneutria nigriventer, which acts as an antagonist on two ion channels involved in nociception. The study demonstrates that Phα1β can reduce both acute and chronic pain. NMR spectroscopy was used to determine the spatial structure and dynamics of Phα1β, providing important insights into the structure and function of spider toxins.