Inhibited KdpFABC transitions into an E1 off-cycle state

  1. Jakob M Silberberg
  2. Charlott Stock
  3. Lisa Hielkema
  4. Robin A Corey
  5. Jan Rheinberger
  6. Dorith Wunnicke
  7. Victor RA Dubach
  8. Phillip J Stansfeld
  9. Inga Hänelt  Is a corresponding author
  10. Cristina Paulino  Is a corresponding author
  1. Goethe University Frankfurt, Germany
  2. Aarhus University, Denmark
  3. University of Groningen, Netherlands
  4. University of Oxford, United Kingdom
  5. University of Warwick, United Kingdom

Abstract

KdpFABC is a high-affinity prokaryotic K+ uptake system that forms a functional chimera between a channel-like subunit (KdpA) and a P-type ATPase (KdpB). At high K+ levels, KdpFABC needs to be inhibited to prevent excessive K+ accumulation to the point of toxicity. This is achieved by a phosphorylation of the serine residue in the TGES162 motif in the A domain of the pump subunit KdpB (KdpBS162-P). Here, we explore the structural basis of inhibition by KdpBS162 phosphorylation by determining the conformational landscape of KdpFABC under inhibiting and non-inhibiting conditions. Under turnover conditions, we identified a new inhibited KdpFABC state that we termed E1P tight, which is not part of the canonical Post-Albers transport cycle of P-type ATPases. It likely represents the biochemically described stalled E1P state adopted by KdpFABC upon KdpBS162 phosphorylation. The E1P tight state exhibits a compact fold of the three cytoplasmic domains and is likely adopted when the transition from high-energy E1P states to E2P states is unsuccessful. This study represents a structural characterization of a biologically relevant off-cycle state in the P-type ATPase family and supports the emerging discussion of P-type ATPase regulation by such states.

Data availability

The three-dimensional cryo-EM densities and corresponding modelled coordinates generated have been deposited in the Electron Microscopy Data Bank and the Protein Data Bank under the accession numbers summarized in Table 4. The depositions include maps calculated with higher b-factors, both half-maps and the mask used for the final FSC calculation.

The following data sets were generated

Article and author information

Author details

  1. Jakob M Silberberg

    Institute of Biochemistry, Goethe University Frankfurt, Frankfurt, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1721-8666
  2. Charlott Stock

    Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5471-3696
  3. Lisa Hielkema

    Department of Structural Biology, University of Groningen, Groningen, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
  4. Robin A Corey

    Department of Biochemistry, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1820-7993
  5. Jan Rheinberger

    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:" 0000-0002-9901-2065
  6. Dorith Wunnicke

    Institute of Biochemistry, Goethe University Frankfurt, Frankfurt, Germany
    Competing interests
    The authors declare that no competing interests exist.
  7. Victor RA Dubach

    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:" 0000-0002-1657-7184
  8. Phillip J Stansfeld

    Department of Chemistry, University of Warwick, Coventry, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  9. Inga Hänelt

    Institute of Biochemistry, Goethe University Frankfurt, Frankfurt, Germany
    For correspondence
    haenelt@biochem.uni-frankfurt.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1495-3163
  10. 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 (Veni grant 722.017.001)

  • Cristina Paulino

Biotechnology and Biological Sciences Research Council (BB/R002517/1)

  • Phillip J Stansfeld

Biotechnology and Biological Sciences Research Council (BB/S003339/1)

  • Phillip J Stansfeld

State of Hesse (LOEWE Schwerpunkt TRABITA)

  • Jakob M Silberberg

Wellcome Trust (208361/Z/17/Z)

  • Robin A Corey

Nederlandse Organisatie voor Wetenschappelijk Onderzoek (Start-Up grant 740.018.016)

  • Cristina Paulino

Deutsche Forschungsgemeinschaft (Emmy Noether grant HA6322/3-1)

  • Inga Hänelt

Deutsche Forschungsgemeinschaft (Heisenberg program HA6322/5-1)

  • Inga Hänelt

Aventis Foundation (Life Science Bridge Award)

  • Inga Hänelt

Uniscientia Foundation

  • Inga Hänelt

Wellcome Trust (208361/Z/17/Z)

  • Phillip J Stansfeld

Medical Research Council (MR/S009213/1)

  • Phillip J Stansfeld

Biotechnology and Biological Sciences Research Council (BB/P01948X/1)

  • Phillip J Stansfeld

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

Copyright

© 2022, Silberberg 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. Jakob M Silberberg
  2. Charlott Stock
  3. Lisa Hielkema
  4. Robin A Corey
  5. Jan Rheinberger
  6. Dorith Wunnicke
  7. Victor RA Dubach
  8. Phillip J Stansfeld
  9. Inga Hänelt
  10. Cristina Paulino
(2022)
Inhibited KdpFABC transitions into an E1 off-cycle state
eLife 11:e80988.
https://doi.org/10.7554/eLife.80988

Share this article

https://doi.org/10.7554/eLife.80988

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