Screening of candidate substrates and coupling ions of transporters by thermostability shift assays

  1. Homa Majd
  2. Martin S King
  3. Shane M Palmer
  4. Anthony C Smith
  5. Liam DH Elbourne
  6. Ian T Paulsen
  7. David Sharples
  8. Peter JF Henderson
  9. Edmund RS Kunji  Is a corresponding author
  1. University of Cambridge, United Kingdom
  2. Macquarie University, Australia
  3. University of Leeds, United Kingdom

Abstract

Substrates of most transport proteins have not been identified, limiting our understanding of their role in physiology and disease. Traditional identification methods use transport assays with radioactive compounds, but they are technically challenging and many compounds are unavailable in radioactive form or are prohibitively expensive, precluding large-scale trials. Here, we present a high-throughput screening method that can identify candidate substrates from libraries of unlabeled compounds. The assay is based on the principle that transport proteins recognize substrates through specific interactions, which lead to enhanced stabilization of the transporter population in thermostability shift assays. Representatives of three different transporter (super)families were tested, which differ in structure as well as transport and ion coupling mechanisms. In each case, the substrates were identified correctly from a large set of chemically related compounds, including stereo-isoforms. In some cases, stabilization by substrate binding was enhanced further by ions, providing testable hypotheses on energy coupling mechanisms.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided in Dryad.

The following data sets were generated

Article and author information

Author details

  1. Homa Majd

    Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  2. Martin S King

    Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  3. Shane M Palmer

    Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  4. Anthony C Smith

    Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  5. Liam DH Elbourne

    Department of Molecular Sciences, Macquarie University, Sydney, Australia
    Competing interests
    The authors declare that no competing interests exist.
  6. Ian T Paulsen

    Department of Molecular Sciences, Macquarie University, Sydney, Australia
    Competing interests
    The authors declare that no competing interests exist.
  7. David Sharples

    Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  8. Peter JF Henderson

    Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  9. Edmund RS Kunji

    Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge, United Kingdom
    For correspondence
    ek@mrc-mbu.cam.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0610-4500

Funding

Medical Research Council (MC_UU_00015/1)

  • Homa Majd
  • Martin S King
  • Shane M Palmer
  • Anthony C Smith
  • Edmund RS Kunji

Cambridge Commonwealth, European and International Trust

  • Homa Majd

Leverhulme Trust (EM-2014 -045)

  • Peter JF Henderson

Biotechnology and Biological Sciences Research Council (MPSI BBS/B/14418)

  • David Sharples

Wellcome (JIF 062164/Z/00/Z)

  • David Sharples

University of Leeds

  • David Sharples

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

Reviewing Editor

  1. Volker Dötsch, J.W. Goethe-University, Germany

Version history

  1. Received: July 11, 2018
  2. Accepted: October 11, 2018
  3. Accepted Manuscript published: October 15, 2018 (version 1)
  4. Version of Record published: November 1, 2018 (version 2)

Copyright

© 2018, Majd 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. Homa Majd
  2. Martin S King
  3. Shane M Palmer
  4. Anthony C Smith
  5. Liam DH Elbourne
  6. Ian T Paulsen
  7. David Sharples
  8. Peter JF Henderson
  9. Edmund RS Kunji
(2018)
Screening of candidate substrates and coupling ions of transporters by thermostability shift assays
eLife 7:e38821.
https://doi.org/10.7554/eLife.38821

Share this article

https://doi.org/10.7554/eLife.38821

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