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A lipid site shapes the agonist response of a pentameric ligand-gated ion channel

Nature Chemical Biology volume 15, pages 1156–1164 (2019)Cite this article

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Abstract

Phospholipids are key components of cellular membranes and are emerging as important functional regulators of different membrane proteins, including pentameric ligand-gated ion channels (pLGICs). Here, we take advantage of the prokaryote channel ELIC (Erwinia ligand-gated ion channel) as a model to understand the determinants of phospholipid interactions in this family of receptors. A high-resolution structure of ELIC in a lipid-bound state reveals a phospholipid site at the lower half of pore-forming transmembrane helices M1 and M4 and at a nearby site for neurosteroids, cholesterol or general anesthetics. This site is shaped by an M4-helix kink and a Trp–Arg–Pro triad that is highly conserved in eukaryote GABAA/C and glycine receptors. A combined approach reveals that M4 is intrinsically flexible and that M4 deletions or disruptions of the lipid-binding site accelerate desensitization in ELIC, suggesting that lipid interactions shape the agonist response. Our data offer a structural context for understanding lipid modulation in pLGICs.

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Fig. 1: High-resolution ELIC structure in a lipid-bound state.
Fig. 2: Lipid interactions at a highly conserved Pro kink in the M4 helix.
Fig. 3: Site-directed mutagenesis of the ELIC M4 helix accelerates desensitization.
Fig. 4: M4-helix truncation increases the dynamics of the lower half of the channel pore region.
Fig. 5: Alternative conformation of an unkinked M4 helix forming novel intersubunit interactions.
Fig. 6: Model for lipid modulation of channel desensitization.

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Data availability

The authors declare that the data supporting the findings of this study are available within the article and its supplementary information files. Atomic coordinates and structure factors have been deposited with the Protein Data Bank under accession numbers 6HJX, 6HJY and 6HK0 for ELIC 7′C+Nb72, ELIC Δ8+Nb72 and ELIC F16′S, respectively.

Code availability

Any code or mathematical algorithm used in this study are available from the corresponding authors upon request.

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Acknowledgements

We thank INSTRUCT-ERIC, part of the European Strategy Forum on Research Infrastructures and the Research Foundation-Flanders (FWO) for funding nanobody discovery (J.S.). We thank N. Buys and K. Willibal for technical assistance during nanobody discovery. SBO/IWT-project 1200261 and FWO-project G.0762.13 were awarded to J.S. and C.U. Additional support was from KU Leuven OT/13/095, C32/16/035 and C14/17/093 to C.U. We thank beamline staff at the Swiss Light Source and ESRF. J.E.B. was supported by a grant (113312) from the Natural Sciences and Engineering Research Council of Canada. G.B. was supported by research grant NIH P01GM55876-14A1. L.G.C. was supported by research grant NIH 2R01GM097159. Computational resources were provided by the National Science Foundation XSEDE program through allocation NSF-MCB110149 as well as the Rutgers Discovery Informatics Institute (G.B.).

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Authors and Affiliations

  1. Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada

    Camille M. Hénault & John E. Baenziger

  2. Laboratory for the Structure and Function of Biological Membranes, Center for Structural Biology and Bioinformatics, Université libre de Bruxelles, Brussels, Belgium

    Cedric Govaerts

  3. Laboratory of Structural Neurobiology, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium

    Radovan Spurny, Marijke Brams, Genevieve L. Evans & Chris Ulens

  4. Queensland Brain Institute, University of Queensland, Brisbane, Queensland, Australia

    Argel Estrada-Mondragon & Joseph Lynch

  5. HiQscreen, Vésenaz, Geneva, Switzerland

    Daniel Bertrand

  6. Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium

    Els Pardon & Jan Steyaert

  7. VIB-VUB Center for Structural Biology, VIB, Brussels, Belgium

    Els Pardon & Jan Steyaert

  8. Center for Computational and Integrative Biology, Rutgers University–Camden, Camden, NJ, USA

    Kristen Woods & Grace Brannigan

  9. Department of Physics, Rutgers University–Camden, Camden, NJ, USA

    Kristen Woods & Grace Brannigan

  10. Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, TTUHSC, Lubbock, TX, USA

    Benjamin W. Elberson & Luis G. Cuello

  11. University Grenoble Alpes, CNRS, IBS, Grenoble, France

    Hugues Nury

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Contributions

C.M.H. designed, performed and analyzed Xenopus electrophysiological recordings. C.G. performed structure building and refinement. R.S. designed and performed protein purification, crystallization, X-ray data collection and structure determination, building and refinement. M.B. designed and performed protein purification, crystallization, cysteine crosslinking, mutagenesis, RNA transcription and analyzed data. A.E.-M. designed, performed and analyzed VCF recordings. J.L. supervised voltage clamp recordings, data analysis and acquired funding. D.B. designed, performed and analyzed HiClamp recordings. E.P. designed, performed and analyzed all aspects of nanobody production. G.E. performed structure validation and data analysis. K.W. contributed to all aspects of MD simulations. B.W.E. contributed to lipid vesicle recordings. L.G.C. designed, performed and analyzed lipid vesicle recordings and contributed funding. G.B. designed, performed, analyzed MD simulations and acquired funding. H.N. performed X-ray structure building and refinement. J.S. contributed to all aspects of nanobody production and acquired funding. J.E.B. supervised the project, performed experimental design, data analysis and acquired funding. C.U. supervised the project, performed experimental design, contributed to all aspects of X-ray crystallography and acquired funding. J.E.B. and C.U. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to John E. Baenziger or Chris Ulens.

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Supplementary information

Supplementary information

Supplementary Tables 1–5 and Supplementary Figures 1–11.

Reporting Summary

Supplementary Video 1

The video is a morph illustrating the conformational change of M4 in ELIC (side view). The structure in red represents the alternate conformation of M4.

Supplementary Video 2

The video is a morph illustrating the conformational change of M4 in ELIC for the transmembrane domain only (side view, left; top view, right). The structure in red represents the alternate conformation of M4.

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Hénault, C.M., Govaerts, C., Spurny, R. et al. A lipid site shapes the agonist response of a pentameric ligand-gated ion channel. Nat Chem Biol 15, 1156–1164 (2019). https://doi.org/10.1038/s41589-019-0369-4

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