Abstract

Scramblases catalyze the movement of lipids between both leaflets of a bilayer. Whereas the X-ray structure of the protein nhTMEM16 has previously revealed the architecture of a Ca2+-dependent lipid scramblase, its regulation mechanism has remained elusive. Here, we have used cryo-electron microscopy and functional assays to address this question. Ca2+-bound and Ca2+-free conformations of nhTMEM16 in detergent and lipid nanodiscs illustrate the interactions with its environment and they reveal the conformational changes underlying its activation. In this process, Ca2+-binding induces a stepwise transition of the catalytic subunit cavity, converting a closed cavity that is shielded from the membrane in the absence of ligand, into a polar furrow that becomes accessible to lipid headgroups in the Ca2+-bound state. Additionally, our structures demonstrate how nhTMEM16 distorts the membrane at both entrances of the subunit cavity, thereby decreasing the energy barrier for lipid movement.

Data availability

The three-dimensional cryo-EM density maps as well as the modelled coordinated have been deposited in the Electron Microscopy Data Bank and the Protein Data Bank, respectively. The deposition includes the cryo-EM maps, both half-maps, the mask used for final FSC calculation and the refined unmasked maps. The raw data (several TBs in size) can be provided upon request.

The following data sets were generated

Article and author information

Author details

  1. Valeria Kalienkova

    Department of Biochemistry, University of Zürich, Zürich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4143-6172
  2. Vanessa Clerico Mosina

    Department of Structural Biology, University of Groningen, Groningen, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8013-0144
  3. Laura Bryner

    Department of Biochemistry, University of Zürich, Zürich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  4. Gert T Oostergetel

    Department of Structural Biology, University of Groningen, Groningen, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
  5. Raimund Dutzler

    Department of Biochemistry, University of Zürich, Zürich, Switzerland
    For correspondence
    dutzler@bioc.uzh.ch
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2193-6129
  6. Cristina Paulino

    Department of Structural Biology, University of Groningen, Groningen, Netherlands
    For correspondence
    c.paulino@rug.nl
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7017-109X

Funding

Nederlandse Organisatie voor Wetenschappelijk Onderzoek (740.018.016)

  • Cristina Paulino

H2020 European Research Council (339116)

  • Raimund Dutzler

H2020 European Research Council (AnoBest)

  • Raimund Dutzler

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Kenton Jon Swartz, National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States

Version history

  1. Received: December 12, 2018
  2. Accepted: February 8, 2019
  3. Accepted Manuscript published: February 20, 2019 (version 1)
  4. Accepted Manuscript updated: February 21, 2019 (version 2)
  5. Version of Record published: March 12, 2019 (version 3)

Copyright

© 2019, Kalienkova et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Valeria Kalienkova
  2. Vanessa Clerico Mosina
  3. Laura Bryner
  4. Gert T Oostergetel
  5. Raimund Dutzler
  6. Cristina Paulino
(2019)
Stepwise activation mechanism of the scramblase nhTMEM16 revealed by cryo-EM
eLife 8:e44364.
https://doi.org/10.7554/eLife.44364

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https://doi.org/10.7554/eLife.44364

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