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

Phα1β (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, Phα1β administration reduces both acute and chronic pain. Her...

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Autores principales: Lyukmanova, Ekaterina N., Mironov, Pavel A., Kulbatskii, Dmitrii S., Shulepko, Mikhail A., Paramonov, Alexander S., Chernaya, Elizaveta M., Logashina, Yulia A., Andreev, Yaroslav A., Kirpichnikov, Mikhail P., Shenkarev, Zakhar O.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10305275/
https://www.ncbi.nlm.nih.gov/pubmed/37368679
http://dx.doi.org/10.3390/toxins15060378
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author Lyukmanova, Ekaterina N.
Mironov, Pavel A.
Kulbatskii, Dmitrii S.
Shulepko, Mikhail A.
Paramonov, Alexander S.
Chernaya, Elizaveta M.
Logashina, Yulia A.
Andreev, Yaroslav A.
Kirpichnikov, Mikhail P.
Shenkarev, Zakhar O.
author_facet Lyukmanova, Ekaterina N.
Mironov, Pavel A.
Kulbatskii, Dmitrii S.
Shulepko, Mikhail A.
Paramonov, Alexander S.
Chernaya, Elizaveta M.
Logashina, Yulia A.
Andreev, Yaroslav A.
Kirpichnikov, Mikhail P.
Shenkarev, Zakhar O.
author_sort Lyukmanova, Ekaterina N.
collection PubMed
description Phα1β (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, Phα1β administration reduces both acute and chronic pain. Here, we report the efficient bacterial expression system for the recombinant production of Phα1β and its (15)N-labeled analogue. Spatial structure and dynamics of Phα1β 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 α-helix (Asn41–Cys52) stapled to ICK by two disulfides exhibits the µs–ms time-scale fluctuations. The Phα1β 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. Phα1β has a large hydrophobic region on its surface and demonstrates a moderate affinity for partially anionic lipid vesicles at low salt conditions. Surprisingly, 10 µM Phα1β 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 Phα1β as a gating modifier toxin, probably interacting with S1–S4 gating domains from a membrane-bound state.
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spelling pubmed-103052752023-06-29 Recombinant Production, NMR Solution Structure, and Membrane Interaction of the Phα1β Toxin, a TRPA1 Modulator from the Brazilian Armed Spider Phoneutria nigriventer Lyukmanova, Ekaterina N. Mironov, Pavel A. Kulbatskii, Dmitrii S. Shulepko, Mikhail A. Paramonov, Alexander S. Chernaya, Elizaveta M. Logashina, Yulia A. Andreev, Yaroslav A. Kirpichnikov, Mikhail P. Shenkarev, Zakhar O. Toxins (Basel) Article Phα1β (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, Phα1β administration reduces both acute and chronic pain. Here, we report the efficient bacterial expression system for the recombinant production of Phα1β and its (15)N-labeled analogue. Spatial structure and dynamics of Phα1β 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 α-helix (Asn41–Cys52) stapled to ICK by two disulfides exhibits the µs–ms time-scale fluctuations. The Phα1β 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. Phα1β has a large hydrophobic region on its surface and demonstrates a moderate affinity for partially anionic lipid vesicles at low salt conditions. Surprisingly, 10 µM Phα1β 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 Phα1β as a gating modifier toxin, probably interacting with S1–S4 gating domains from a membrane-bound state. MDPI 2023-06-03 /pmc/articles/PMC10305275/ /pubmed/37368679 http://dx.doi.org/10.3390/toxins15060378 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Lyukmanova, Ekaterina N.
Mironov, Pavel A.
Kulbatskii, Dmitrii S.
Shulepko, Mikhail A.
Paramonov, Alexander S.
Chernaya, Elizaveta M.
Logashina, Yulia A.
Andreev, Yaroslav A.
Kirpichnikov, Mikhail P.
Shenkarev, Zakhar O.
Recombinant Production, NMR Solution Structure, and Membrane Interaction of the Phα1β Toxin, a TRPA1 Modulator from the Brazilian Armed Spider Phoneutria nigriventer
title Recombinant Production, NMR Solution Structure, and Membrane Interaction of the Phα1β Toxin, a TRPA1 Modulator from the Brazilian Armed Spider Phoneutria nigriventer
title_full Recombinant Production, NMR Solution Structure, and Membrane Interaction of the Phα1β Toxin, a TRPA1 Modulator from the Brazilian Armed Spider Phoneutria nigriventer
title_fullStr Recombinant Production, NMR Solution Structure, and Membrane Interaction of the Phα1β Toxin, a TRPA1 Modulator from the Brazilian Armed Spider Phoneutria nigriventer
title_full_unstemmed Recombinant Production, NMR Solution Structure, and Membrane Interaction of the Phα1β Toxin, a TRPA1 Modulator from the Brazilian Armed Spider Phoneutria nigriventer
title_short Recombinant Production, NMR Solution Structure, and Membrane Interaction of the Phα1β Toxin, a TRPA1 Modulator from the Brazilian Armed Spider Phoneutria nigriventer
title_sort recombinant production, nmr solution structure, and membrane interaction of the phα1β toxin, a trpa1 modulator from the brazilian armed spider phoneutria nigriventer
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10305275/
https://www.ncbi.nlm.nih.gov/pubmed/37368679
http://dx.doi.org/10.3390/toxins15060378
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