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A locally closed conformation of a bacterial pentameric proton-gated ion channel

Nature Structural & Molecular Biology volume 19, pages 642–649 (2012)Cite this article

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Abstract

Pentameric ligand-gated ion channels mediate signal transduction through conformational transitions between closed-pore and open-pore states. To stabilize a closed conformation of GLIC, a bacterial proton-gated homolog from Gloeobacter violaceus whose open structure is known, we separately generated either four cross-links or two single mutations. We found all six mutants to be in the same 'locally closed' conformation using X-ray crystallography, sharing most of the features of the open form but showing a locally closed pore as a result of a concerted bending of all of its M2 helices. The mutants adopt several variant conformations of the M2-M3 loop, and in all cases an interacting lipid that is observed in the open form disappears. A single cross-linked mutant is functional, according to electrophysiology, and the locally closed structure of this mutant indicates that it has an increased flexibility. Further cross-linking, accessibility and molecular dynamics data suggest that the locally closed form is a functionally relevant conformation that occurs during allosteric gating transitions.

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Figure 1: X-ray structure of the loop2-24′ mutant in the LC1 conformation.
Figure 2: The M2-M3 loop conformation in the different locally closed forms.
Figure 3: Electrophysiology of the cysteine mutants.
Figure 4: In silico characterization of the locally closed state determined by molecular dynamics and normal modes.
Figure 5: Probing the M2-M3 motion by cysteine accessibility and cross-linking.

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Acknowledgements

This work was supported by the European Commission grant 'NeuroCypres' to M.D. and P.-J.C., and the Louis D. Foundation from the Institut de France (to P.-J.C.). Molecular simulations were performed using high performance computing resources from Grand Equipement National de Calcul Intensif (GENCI)–Institut du Développement et des Ressources en Informatique Scientifique (IDRIS) (grant 2010-072292 to M.D.). The authors thank the staff at Soleil (PXl) and European Synchotron Radiation Facility (ESRF) (especially at beamlines ID29, ID14-4) for their help and their useful advice during X-ray Data Collection. We thank T. Allen for helping us optimize the setup of the simulation system. We thank J.-P. Changeux, S. Edelstein, A. Taly and D. Digregorio for critical reading of the manuscript.

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Author notes

  1. Marie S Prevost and Ludovic Sauguet: These authors contributed equally to this work.

Authors and Affiliations

  1. Institut Pasteur, Groupe Récepteurs-Canaux, Paris, France

    Marie S Prevost, Ludovic Sauguet, Hugues Nury, Catherine Van Renterghem, Christèle Huon & Pierre-Jean Corringer

  2. Le Centre National de la Recherche Scientifique (CNRS), Unité de Recherche Associée (URA) 2182, Paris, France

    Marie S Prevost, Ludovic Sauguet, Hugues Nury, Catherine Van Renterghem, Christèle Huon & Pierre-Jean Corringer

  3. University Pierre et Marie Curie (UPMC), Cellule Pasteur UPMC, Paris, France

    Marie S Prevost

  4. Institut Pasteur, Unité de Dynamique Structurale des Macromolécules, Paris, France

    Ludovic Sauguet, Hugues Nury, Frederic Poitevin & Marc Delarue

  5. CNRS, Unités Mixtes de Recherche (UMR) 3528, Paris, France.,

    Ludovic Sauguet, Hugues Nury, Frederic Poitevin & Marc Delarue

  6. University Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Paris, France.,

    Frederic Poitevin

  7. Laboratoire de Biochimie Théorique, CNRS, UPR 9080, University Paris Diderot, Sorbonne Paris Cité, Paris, France.,

    Marc Baaden

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Contributions

M.S.P. carried out mutagenesis, electrophysiology, immunofluorescence and biochemistry with the help of C.V.R. and C.H. L.S. and H.N. carried out protein production, crystal growth, data collection and model refinement. L.S. and H.N. performed structure analysis with the help of F.P. and M.S.P. F.P. carried out the normal mode analysis. M.B. carried out molecular dynamic simulations, which were analyzed by M.B. and F.P. M.D. and P.-J.C. supervised the work. M.S.P. and P.-J.C. wrote the manuscript with the help of the other authors.

Corresponding authors

Correspondence to Marc Delarue or Pierre-Jean Corringer.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Tables 1–3 (PDF 2387 kb)

Supplementary Video 1

MinActionPath plausible trajectory between the Open and LC conformation at the Cα level of description, according to Supplementary Fig. 5. The movie shows the TMD alone in upper view. (MPG 2829 kb)

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Prevost, M., Sauguet, L., Nury, H. et al. A locally closed conformation of a bacterial pentameric proton-gated ion channel. Nat Struct Mol Biol 19, 642–649 (2012). https://doi.org/10.1038/nsmb.2307

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