1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics
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A single power stroke by ATP binding drives substrate translocation in a heterodimeric ABC transporter

  1. Erich Stefan
  2. Susanne Hofmann
  3. Robert Tampé  Is a corresponding author
  1. Goethe-University Frankfurt, Germany
Research Article
  • Cited 10
  • Views 1,653
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Cite this article as: eLife 2020;9:e55943 doi: 10.7554/eLife.55943

Abstract

ATP-binding cassette (ABC) transporters constitute the largest family of primary active transporters, responsible for many physiological processes and human maladies. However, the mechanism how chemical energy of ATP facilitates translocation of chemically diverse compounds across membranes is poorly understood. Here, we advance the quantitative mechanistic understanding of the heterodimeric ABC transporter TmrAB, a functional homolog of the transporter associated with antigen processing (TAP) by single-turnover analyses at single-liposome resolution. We reveal that a single conformational switch by ATP binding drives unidirectional substrate translocation. After this power stroke, ATP hydrolysis and phosphate release launch the return to the resting state, which facilitates nucleotide exchange and a new round of substrate binding and translocation. In contrast to hitherto existing steady-state assays, our single-turnover approach uncovers the power stroke in substrate translocation and the tight chemomechanical coupling in these molecular machines.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1, 2, 3, 4, and 5.

Article and author information

Author details

  1. Erich Stefan

    Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Frankfurt, Germany
    Competing interests
    The authors declare that no competing interests exist.
  2. Susanne Hofmann

    Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Frankfurt, Germany
    Competing interests
    The authors declare that no competing interests exist.
  3. Robert Tampé

    Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Frankfurt, Germany
    For correspondence
    tampe@em.uni-frankfurt.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0403-2160

Funding

Deutsche Forschungsgemeinschaft (SFB 807 and Ta157/12-1)

  • Robert Tampé

European Commission (ERC_Ad 789121)

  • Robert Tampé

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

Reviewing Editor

  1. Sonja V Albers, University of Freiburg, Germany

Publication history

  1. Received: February 11, 2020
  2. Accepted: April 20, 2020
  3. Accepted Manuscript published: April 21, 2020 (version 1)
  4. Version of Record published: May 7, 2020 (version 2)

Copyright

© 2020, Stefan 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. Further reading

Further reading

    1. Biochemistry and Chemical Biology
    Erich Stefan et al.
    Research Article Updated

    ATP-binding cassette (ABC) transporters constitute the largest family of primary active transporters involved in a multitude of physiological processes and human diseases. Despite considerable efforts, it remains unclear how ABC transporters harness the chemical energy of ATP to drive substrate transport across cell membranes. Here, by random nonstandard peptide integrated discovery (RaPID), we leveraged combinatorial macrocyclic peptides that target a heterodimeric ABC transport complex and explore fundamental principles of the substrate translocation cycle. High-affinity peptidic macrocycles bind conformationally selective and display potent multimode inhibitory effects. The macrocycles block the transporter either before or after unidirectional substrate export along a single conformational switch induced by ATP binding. Our study reveals mechanistic principles of ATP binding, conformational switching, and energy transduction for substrate transport of ABC export systems. We highlight the potential of de novo macrocycles as effective inhibitors for membrane proteins implicated in multidrug resistance, providing avenues for the next generation of pharmaceuticals.

    1. Biochemistry and Chemical Biology
    Qiang Liu et al.
    Research Article Updated

    The multimodal sensory channel transient receptor potential vanilloid-3 (TRPV3) is expressed in epidermal keratinocytes and implicated in chronic pruritus, allergy, and inflammation-related skin disorders. Gain-of-function mutations of TRPV3 cause hair growth disorders in mice and Olmsted syndrome in humans. Nevertheless, whether and how TRPV3 could be therapeutically targeted remains to be elucidated. We here report that mouse and human TRPV3 channel is targeted by the clinical medication dyclonine that exerts a potent inhibitory effect. Accordingly, dyclonine rescued cell death caused by gain-of-function TRPV3 mutations and suppressed pruritus symptoms in vivo in mouse model. At the single-channel level, dyclonine inhibited TRPV3 open probability but not the unitary conductance. By molecular simulations and mutagenesis, we further uncovered key residues in TRPV3 pore region that could toggle the inhibitory efficiency of dyclonine. The functional and mechanistic insights obtained on dyclonine-TRPV3 interaction will help to conceive therapeutics for skin inflammation.