Stepwise activation mechanism of the scramblase nhTMEM16 revealed by cryo-EM
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.
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.
Cryo-EM structure of calcium-bound nhTMEM16 lipid scramblase in DDMProtein Data Bank, 6QM5.
Cryo-EM structure of calcium-free nhTMEM16 lipid scramblase in DDMProtein Data Bank, 6QM6.
Cryo-EM structure of calcium-bound nhTMEM16 lipid scramblase in DDMElectron Microscopy Data Bank, EMD-4588.
Cryo-EM structure of calcium-free nhTMEM16 lipid scramblase in DDMElectron Microscopy Data Bank, EMD-4589.
Cryo-EM structure of calcium-bound nhTMEM16 lipid scramblase in nanodisc (open state)Electron Microscopy Data Bank, EMD-4592.
Cryo-EM structure of calcium-bound nhTMEM16 lipid scramblase in nanodisc (intermediate state)Electron Microscopy Data Bank, EMD-4593.
Cryo-EM structure of calcium-bound nhTMEM16 lipid scramblase in nanodisc (closed state)Electron Microscopy Data Bank, EMD-4594.
Cryo-EM structure of calcium-free nhTMEM16 lipid scramblase in nanodiscElectron Microscopy Data Bank, EMD-4587.
Article and author information
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.
- Kenton Jon Swartz, National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States
- Received: December 12, 2018
- Accepted: February 8, 2019
- Accepted Manuscript published: February 20, 2019 (version 1)
- Accepted Manuscript updated: February 21, 2019 (version 2)
- Version of Record published: March 12, 2019 (version 3)
© 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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