The molecular mechanism of snake short-chain α-neurotoxin binding to muscle-type nicotinic acetylcholine receptors

Mieke Nys; Eleftherios Zarkadas; Marijke Brams; Aujan Mehregan; Kumiko Kambara; Jeroen Kool; Nicholas R. Casewell; Daniel Bertrand; John E. Baenziger; Hugues Nury; Chris Ulens

doi:10.1038/s41467-022-32174-7

The molecular mechanism of snake short-chain α-neurotoxin binding to muscle-type nicotinic acetylcholine receptors

Mieke Nys^*, Eleftherios Zarkadas, Marijke Brams, Aujan Mehregan, Kumiko Kambara, Jeroen Kool, Nicholas R. Casewell, Daniel Bertrand, John E. Baenziger, Hugues Nury, Chris Ulens

^*Corresponding author for this work

Research output: Contribution to Journal › Article › Academic › peer-review

Abstract

Bites by elapid snakes (e.g. cobras) can result in life-threatening paralysis caused by venom neurotoxins blocking neuromuscular nicotinic acetylcholine receptors. Here, we determine the cryo-EM structure of the muscle-type Torpedo receptor in complex with ScNtx, a recombinant short-chain α-neurotoxin. ScNtx is pinched between loop C on the principal subunit and a unique hairpin in loop F on the complementary subunit, thereby blocking access to the neurotransmitter binding site. ScNtx adopts a binding mode that is tilted toward the complementary subunit, forming a wider network of interactions than those seen in the long-chain α-Bungarotoxin complex. Certain mutations in ScNtx at the toxin-receptor interface eliminate inhibition of neuronal α7 nAChRs, but not of human muscle-type receptors. These observations explain why ScNtx binds more tightly to muscle-type receptors than neuronal receptors. Together, these data offer a framework for understanding subtype-specific actions of short-chain α-neurotoxins and inspire strategies for design of new snake antivenoms.

Original language	English
Article number	4543
Pages (from-to)	1-12
Number of pages	12
Journal	Nature Communications
Volume	13
DOIs	https://doi.org/10.1038/s41467-022-32174-7
Publication status	Published - 4 Aug 2022

Bibliographical note

Funding Information:
This work was supported by grants KU Leuven C32/16/035 (CU) and the Wellcome Trust 221710/Z/20/Z (C.U., J.K. and N.R.C.). MN is a recipient of a FWO postdoctoral fellowship 12X2722N (M.N.). The microscale thermophoresis data were obtained at the KU Leuven Molecular Biophysics Platform of the SWITCH laboratory (Prof. Joost Schymkowitz and Prof. Frederic Rousseau) with support from Flanders Institute for Biotechnology (VIB, grant no. C0401); KU Leuven; and The Research Foundation - Flanders (FWO, equipment grant AKUL/15/34 - G0H1716N). This work used the EM facilities at the Grenoble Instruct-ERIC Center (ISBG; UAR 3518 CNRS CEA-UGA-EMBL) with support from the French Infrastructure for Integrated Structural Biology (FRISBI; ANR-10-INSB-05-02) and GRAL, a project of the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR- 17-EURE-0003) within the Grenoble Partnership for Structural Biology. We thank Guy Schoehn and the IBS Electron Microscope facility, supported by the Auvergne Rhône-Alpes Region, the Fonds Feder, the Fondation pour la Recherche Médicale and GIS-IBiSA. This work was funded by the ERC Starting grant 637733 Pentabrain (HN). We thank Guillermo de la Rosa for his advice on the expression of an earlier version of the ScNtx expression plasmid.

Funding Information:
This work was supported by the National Institutes of Health grant R37HD083217 (RMS) and NYU Wasserman and DURF fellowships (NN).

Publisher Copyright:
© 2022, The Author(s).

Funding

This work was supported by grants KU Leuven C32/16/035 (CU) and the Wellcome Trust 221710/Z/20/Z (C.U., J.K. and N.R.C.). MN is a recipient of a FWO postdoctoral fellowship 12X2722N (M.N.). The microscale thermophoresis data were obtained at the KU Leuven Molecular Biophysics Platform of the SWITCH laboratory (Prof. Joost Schymkowitz and Prof. Frederic Rousseau) with support from Flanders Institute for Biotechnology (VIB, grant no. C0401); KU Leuven; and The Research Foundation - Flanders (FWO, equipment grant AKUL/15/34 - G0H1716N). This work used the EM facilities at the Grenoble Instruct-ERIC Center (ISBG; UAR 3518 CNRS CEA-UGA-EMBL) with support from the French Infrastructure for Integrated Structural Biology (FRISBI; ANR-10-INSB-05-02) and GRAL, a project of the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR- 17-EURE-0003) within the Grenoble Partnership for Structural Biology. We thank Guy Schoehn and the IBS Electron Microscope facility, supported by the Auvergne Rhône-Alpes Region, the Fonds Feder, the Fondation pour la Recherche Médicale and GIS-IBiSA. This work was funded by the ERC Starting grant 637733 Pentabrain (HN). We thank Guillermo de la Rosa for his advice on the expression of an earlier version of the ScNtx expression plasmid. This work was supported by the National Institutes of Health grant R37HD083217 (RMS) and NYU Wasserman and DURF fellowships (NN).

Funders	Funder number
DURF
Fonds Feder
GIS-IBiSA
KU Leuven C32/16/035
NYU Wasserman
National Institutes of Health	R37HD083217
National Institutes of Health
Wellcome Trust	221710/Z/20/Z
Wellcome Trust
European Resuscitation Council	637733
European Resuscitation Council
Fondation pour la Recherche Médicale
Fonds Wetenschappelijk Onderzoek	UAR 3518 CNRS CEA-UGA-EMBL, 12X2722N, AKUL/15/34 - G0H1716N
Fonds Wetenschappelijk Onderzoek
KU Leuven
Vlaams Instituut voor Biotechnologie	C0401
Vlaams Instituut voor Biotechnologie
Région Auvergne-Rhône-Alpes
French Infrastructure for Integrated Structural Biology	ANR- 17-EURE-0003, ANR-10-INSB-05-02
French Infrastructure for Integrated Structural Biology

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@article{68154aeaafa749ef8f409352dd1c2702,

title = "The molecular mechanism of snake short-chain α-neurotoxin binding to muscle-type nicotinic acetylcholine receptors",

abstract = "Bites by elapid snakes (e.g. cobras) can result in life-threatening paralysis caused by venom neurotoxins blocking neuromuscular nicotinic acetylcholine receptors. Here, we determine the cryo-EM structure of the muscle-type Torpedo receptor in complex with ScNtx, a recombinant short-chain α-neurotoxin. ScNtx is pinched between loop C on the principal subunit and a unique hairpin in loop F on the complementary subunit, thereby blocking access to the neurotransmitter binding site. ScNtx adopts a binding mode that is tilted toward the complementary subunit, forming a wider network of interactions than those seen in the long-chain α-Bungarotoxin complex. Certain mutations in ScNtx at the toxin-receptor interface eliminate inhibition of neuronal α7 nAChRs, but not of human muscle-type receptors. These observations explain why ScNtx binds more tightly to muscle-type receptors than neuronal receptors. Together, these data offer a framework for understanding subtype-specific actions of short-chain α-neurotoxins and inspire strategies for design of new snake antivenoms.",

author = "Mieke Nys and Eleftherios Zarkadas and Marijke Brams and Aujan Mehregan and Kumiko Kambara and Jeroen Kool and Casewell, {Nicholas R.} and Daniel Bertrand and Baenziger, {John E.} and Hugues Nury and Chris Ulens",

note = "Funding Information: This work was supported by grants KU Leuven C32/16/035 (CU) and the Wellcome Trust 221710/Z/20/Z (C.U., J.K. and N.R.C.). MN is a recipient of a FWO postdoctoral fellowship 12X2722N (M.N.). The microscale thermophoresis data were obtained at the KU Leuven Molecular Biophysics Platform of the SWITCH laboratory (Prof. Joost Schymkowitz and Prof. Frederic Rousseau) with support from Flanders Institute for Biotechnology (VIB, grant no. C0401); KU Leuven; and The Research Foundation - Flanders (FWO, equipment grant AKUL/15/34 - G0H1716N). This work used the EM facilities at the Grenoble Instruct-ERIC Center (ISBG; UAR 3518 CNRS CEA-UGA-EMBL) with support from the French Infrastructure for Integrated Structural Biology (FRISBI; ANR-10-INSB-05-02) and GRAL, a project of the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR- 17-EURE-0003) within the Grenoble Partnership for Structural Biology. We thank Guy Schoehn and the IBS Electron Microscope facility, supported by the Auvergne Rh{\^o}ne-Alpes Region, the Fonds Feder, the Fondation pour la Recherche M{\'e}dicale and GIS-IBiSA. This work was funded by the ERC Starting grant 637733 Pentabrain (HN). We thank Guillermo de la Rosa for his advice on the expression of an earlier version of the ScNtx expression plasmid. Funding Information: This work was supported by the National Institutes of Health grant R37HD083217 (RMS) and NYU Wasserman and DURF fellowships (NN). Publisher Copyright: {\textcopyright} 2022, The Author(s).",

year = "2022",

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doi = "10.1038/s41467-022-32174-7",

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journal = "Nature Communications",

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T1 - The molecular mechanism of snake short-chain α-neurotoxin binding to muscle-type nicotinic acetylcholine receptors

AU - Nys, Mieke

AU - Zarkadas, Eleftherios

AU - Brams, Marijke

AU - Mehregan, Aujan

AU - Kambara, Kumiko

AU - Kool, Jeroen

AU - Casewell, Nicholas R.

AU - Bertrand, Daniel

AU - Baenziger, John E.

AU - Nury, Hugues

AU - Ulens, Chris

N1 - Funding Information: This work was supported by grants KU Leuven C32/16/035 (CU) and the Wellcome Trust 221710/Z/20/Z (C.U., J.K. and N.R.C.). MN is a recipient of a FWO postdoctoral fellowship 12X2722N (M.N.). The microscale thermophoresis data were obtained at the KU Leuven Molecular Biophysics Platform of the SWITCH laboratory (Prof. Joost Schymkowitz and Prof. Frederic Rousseau) with support from Flanders Institute for Biotechnology (VIB, grant no. C0401); KU Leuven; and The Research Foundation - Flanders (FWO, equipment grant AKUL/15/34 - G0H1716N). This work used the EM facilities at the Grenoble Instruct-ERIC Center (ISBG; UAR 3518 CNRS CEA-UGA-EMBL) with support from the French Infrastructure for Integrated Structural Biology (FRISBI; ANR-10-INSB-05-02) and GRAL, a project of the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR- 17-EURE-0003) within the Grenoble Partnership for Structural Biology. We thank Guy Schoehn and the IBS Electron Microscope facility, supported by the Auvergne Rhône-Alpes Region, the Fonds Feder, the Fondation pour la Recherche Médicale and GIS-IBiSA. This work was funded by the ERC Starting grant 637733 Pentabrain (HN). We thank Guillermo de la Rosa for his advice on the expression of an earlier version of the ScNtx expression plasmid. Funding Information: This work was supported by the National Institutes of Health grant R37HD083217 (RMS) and NYU Wasserman and DURF fellowships (NN). Publisher Copyright: © 2022, The Author(s).

PY - 2022/8/4

Y1 - 2022/8/4

N2 - Bites by elapid snakes (e.g. cobras) can result in life-threatening paralysis caused by venom neurotoxins blocking neuromuscular nicotinic acetylcholine receptors. Here, we determine the cryo-EM structure of the muscle-type Torpedo receptor in complex with ScNtx, a recombinant short-chain α-neurotoxin. ScNtx is pinched between loop C on the principal subunit and a unique hairpin in loop F on the complementary subunit, thereby blocking access to the neurotransmitter binding site. ScNtx adopts a binding mode that is tilted toward the complementary subunit, forming a wider network of interactions than those seen in the long-chain α-Bungarotoxin complex. Certain mutations in ScNtx at the toxin-receptor interface eliminate inhibition of neuronal α7 nAChRs, but not of human muscle-type receptors. These observations explain why ScNtx binds more tightly to muscle-type receptors than neuronal receptors. Together, these data offer a framework for understanding subtype-specific actions of short-chain α-neurotoxins and inspire strategies for design of new snake antivenoms.

AB - Bites by elapid snakes (e.g. cobras) can result in life-threatening paralysis caused by venom neurotoxins blocking neuromuscular nicotinic acetylcholine receptors. Here, we determine the cryo-EM structure of the muscle-type Torpedo receptor in complex with ScNtx, a recombinant short-chain α-neurotoxin. ScNtx is pinched between loop C on the principal subunit and a unique hairpin in loop F on the complementary subunit, thereby blocking access to the neurotransmitter binding site. ScNtx adopts a binding mode that is tilted toward the complementary subunit, forming a wider network of interactions than those seen in the long-chain α-Bungarotoxin complex. Certain mutations in ScNtx at the toxin-receptor interface eliminate inhibition of neuronal α7 nAChRs, but not of human muscle-type receptors. These observations explain why ScNtx binds more tightly to muscle-type receptors than neuronal receptors. Together, these data offer a framework for understanding subtype-specific actions of short-chain α-neurotoxins and inspire strategies for design of new snake antivenoms.

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The molecular mechanism of snake short-chain α-neurotoxin binding to muscle-type nicotinic acetylcholine receptors

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