1. Structural Biology and Molecular Biophysics

Structural basis of Ca2+-dependent activation and lipid transport by a TMEM16 scramblase

  1. Maria E Falzone
  2. Jan Rheinberger
  3. Byoung-Cheol Lee
  4. Thasin Peyear
  5. Linda Sasset
  6. Ashleigh M Raczkowski
  7. Edward T Eng
  8. Annarita Di Lorenzo
  9. Olaf S Andersen
  10. Crina M Nimigean
  11. Alessio Accardi  Is a corresponding author
  1. Weill Cornell Medical College, United States
  2. New York Structural Biology Center, United States
https://doi.org/10.7554/eLife.43229
Share this article
Cite this article
  1. Maria E Falzone
  2. Jan Rheinberger
  3. Byoung-Cheol Lee
  4. Thasin Peyear
  5. Linda Sasset
  6. Ashleigh M Raczkowski
  7. Edward T Eng
  8. Annarita Di Lorenzo
  9. Olaf S Andersen
  10. Crina M Nimigean
  11. Alessio Accardi
(2019)
Structural basis of Ca2+-dependent activation and lipid transport by a TMEM16 scramblase
eLife 8:e43229.
https://doi.org/10.7554/eLife.43229
  2. Download BibTeX
  3. Download .RIS

Abstract

The lipid distribution of plasma membranes of eukaryotic cells is asymmetric and phospholipid scramblases disrupt this asymmetry by mediating the rapid, nonselective transport of lipids down their concentration gradients. As a result, phosphatidylserine is exposed to the outer leaflet of membrane, an important step in extracellular signaling networks controlling processes such as apoptosis, blood coagulation, membrane fusion and repair. Several TMEM16 family members have been identified as Ca2+-activated scramblases but the mechanisms underlying their Ca2+-dependent gating and their effects on the surrounding lipid bilayer remain poorly understood. Here we describe three high-resolution cryo-electron microscopy structures of a fungal scramblase from Aspergillus fumigatus, afTMEM16, reconstituted in lipid nanodiscs. These structures reveal that Ca2+-dependent activation of the scramblase entails global rearrangement of the transmembrane and cytosolic domains. These structures, together with functional experiments, suggest that activation of the protein thins the membrane near the transport pathway to facilitate rapid transbilayer lipid movement.

Data availability

CryoEM data have been deposited in the EMPDB database under accession codes: 6E0H, 6DZ7, 6E1O, EMD-8931, EMD-8948, EMD-8959. Raw EM micrographs and un-masked maps are in the process of being deposited.Key parameters of the fluorescence time courses are detailed in the tables and figures and representative traces used in the figures have been provided as source data files. The raw fluorescence time courses are available upon request to the corresponding author.

The following data sets were generated

Article and author information

Author details

  1. Maria E Falzone

    Department of Biochemistry, Weill Cornell Medical College, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6738-7017

  2. Jan Rheinberger

    Department of Anesthesiology, Weill Cornell Medical College, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9901-2065

  3. Byoung-Cheol Lee

    Department of Anesthesiology, Weill Cornell Medical College, New York, United States
    Competing interests
    The authors declare that no competing interests exist.

  4. Thasin Peyear

    Department of Physiology and Biophysics, Weill Cornell Medical College, New York, United States
    Competing interests
    The authors declare that no competing interests exist.

  5. Linda Sasset

    Department of Pathology, Weill Cornell Medical College, New York, United States
    Competing interests
    The authors declare that no competing interests exist.

  6. Ashleigh M Raczkowski

    Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States
    Competing interests
    The authors declare that no competing interests exist.

  7. Edward T Eng

    Simons Electron Microscopy Center, New York Structural Biology Center, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8014-7269

  8. Annarita Di Lorenzo

    Department of Pathology, Weill Cornell Medical College, New York, United States
    Competing interests
    The authors declare that no competing interests exist.

  9. Olaf S Andersen

    Department of Physiology and Biophysics, Weill Cornell Medical College, New York, United States
    Competing interests
    The authors declare that no competing interests exist.

  10. Crina M Nimigean

    Department of Anesthesiology, Weill Cornell Medical College, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6254-4447

  11. Alessio Accardi

    Department of Biochemistry, Weill Cornell Medical College, New York, United States
    For correspondence
    ala2022@med.cornell.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6584-0102

Funding

National Institute of General Medical Sciences (R01GM106717)

  • Alessio Accardi

National Institute of Neurological Disorders and Stroke (R21NS10451)

  • Annarita Di Lorenzo

Irma T. Hirschl Trust

  • Alessio Accardi

Margaret and Herman Sokol Fellowship

  • Maria E Falzone

National Research Foundation of Korea (2013R1A6A3A03064407)

  • Byoung-Cheol Lee

Agouron Institute (F00316)

  • Edward T Eng

Simons Foundation (349247)

  • Edward T Eng

National Institute of General Medical Sciences (GM103310)

  • Edward T Eng

National Institute of General Medical Sciences (1R01GM124451-02)

  • Crina M Nimigean

Korean Ministry of Science (18-BR-01-02)

  • Byoung-Cheol Lee

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

Reviewing Editor

  1. László Csanády, Semmelweis University, Hungary

Version history

  1. Received: October 30, 2018
  2. Accepted: January 2, 2019
  3. Accepted Manuscript published: January 16, 2019 (version 1)
  4. Version of Record published: January 31, 2019 (version 2)
  5. Version of Record updated: February 1, 2019 (version 3)

Copyright

© 2019, Falzone 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.

Metrics

  • 5,162
    views
  • 1,010
    downloads
  • 89
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Maria E Falzone
  2. Jan Rheinberger
  3. Byoung-Cheol Lee
  4. Thasin Peyear
  5. Linda Sasset
  6. Ashleigh M Raczkowski
  7. Edward T Eng
  8. Annarita Di Lorenzo
  9. Olaf S Andersen
  10. Crina M Nimigean
  11. Alessio Accardi
(2019)
Structural basis of Ca2+-dependent activation and lipid transport by a TMEM16 scramblase
eLife 8:e43229.
https://doi.org/10.7554/eLife.43229

Share this article

https://doi.org/10.7554/eLife.43229

Categories and tags

Research organism

Further reading

    1. Structural Biology and Molecular Biophysics

    Plasticity of the proteasome-targeting signal Fat10 enhances substrate degradation

    Hitendra Negi, Aravind Ravichandran ... Ranabir Das
    Research Article

    The proteasome controls levels of most cellular proteins, and its activity is regulated under stress, quiescence, and inflammation. However, factors determining the proteasomal degradation rate remain poorly understood. Proteasome substrates are conjugated with small proteins (tags) like ubiquitin and Fat10 to target them to the proteasome. It is unclear if the structural plasticity of proteasome-targeting tags can influence substrate degradation. Fat10 is upregulated during inflammation, and its substrates undergo rapid proteasomal degradation. We report that the degradation rate of Fat10 substrates critically depends on the structural plasticity of Fat10. While the ubiquitin tag is recycled at the proteasome, Fat10 is degraded with the substrate. Our results suggest significantly lower thermodynamic stability and faster mechanical unfolding in Fat10 compared to ubiquitin. Long-range salt bridges are absent in the Fat10 structure, creating a plastic protein with partially unstructured regions suitable for proteasome engagement. Fat10 plasticity destabilizes substrates significantly and creates partially unstructured regions in the substrate to enhance degradation. NMR-relaxation-derived order parameters and temperature dependence of chemical shifts identify the Fat10-induced partially unstructured regions in the substrate, which correlated excellently to Fat10-substrate contacts, suggesting that the tag-substrate collision destabilizes the substrate. These results highlight a strong dependence of proteasomal degradation on the structural plasticity and thermodynamic properties of the proteasome-targeting tags.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics

    Special Issue: Allosteric regulation of kinase activity

    Amy H Andreotti, Volker Dötsch
    Editorial

    The articles in this special issue highlight how modern cellular, biochemical, biophysical and computational techniques are allowing deeper and more detailed studies of allosteric kinase regulation.

    1. Developmental Biology
    2. Structural Biology and Molecular Biophysics

    Structure and function of the ROR2 cysteine-rich domain in vertebrate noncanonical WNT5A signaling

    Samuel C Griffiths, Jia Tan ... Hsin-Yi Henry Ho
    Research Article Updated

    The receptor tyrosine kinase ROR2 mediates noncanonical WNT5A signaling to orchestrate tissue morphogenetic processes, and dysfunction of the pathway causes Robinow syndrome, brachydactyly B, and metastatic diseases. The domain(s) and mechanisms required for ROR2 function, however, remain unclear. We solved the crystal structure of the extracellular cysteine-rich (CRD) and Kringle (Kr) domains of ROR2 and found that, unlike other CRDs, the ROR2 CRD lacks the signature hydrophobic pocket that binds lipids/lipid-modified proteins, such as WNTs, suggesting a novel mechanism of ligand reception. Functionally, we showed that the ROR2 CRD, but not other domains, is required and minimally sufficient to promote WNT5A signaling, and Robinow mutations in the CRD and the adjacent Kr impair ROR2 secretion and function. Moreover, using function-activating and -perturbing antibodies against the Frizzled (FZ) family of WNT receptors, we demonstrate the involvement of FZ in WNT5A-ROR signaling. Thus, ROR2 acts via its CRD to potentiate the function of a receptor super-complex that includes FZ to transduce WNT5A signals.