1. Structural Biology and Molecular Biophysics

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
https://doi.org/10.7554/eLife.80988
Share this article
Cite this article
  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
  2. Download BibTeX
  3. Download .RIS

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.

Metrics

  • 937
    views
  • 216
    downloads
  • 11
    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. 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

Categories and tags

Research organism

Further reading

    1. Computational and Systems Biology
    2. Structural Biology and Molecular Biophysics

    Discovery of a heparan sulfate binding domain in monkeypox virus H3 as an anti-poxviral drug target combining AI and MD simulations

    Bin Zheng, Meimei Duan ... Peng Zheng
    Research Article

    Viral adhesion to host cells is a critical step in infection for many viruses, including monkeypox virus (MPXV). In MPXV, the H3 protein mediates viral adhesion through its interaction with heparan sulfate (HS), yet the structural details of this interaction have remained elusive. Using AI-based structural prediction tools and molecular dynamics (MD) simulations, we identified a novel, positively charged α-helical domain in H3 that is essential for HS binding. This conserved domain, found across orthopoxviruses, was experimentally validated and shown to be critical for viral adhesion, making it an ideal target for antiviral drug development. Targeting this domain, we designed a protein inhibitor, which disrupted the H3-HS interaction, inhibited viral infection in vitro and viral replication in vivo, offering a promising antiviral candidate. Our findings reveal a novel therapeutic target of MPXV, demonstrating the potential of combination of AI-driven methods and MD simulations to accelerate antiviral drug discovery.

    1. Chromosomes and Gene Expression
    2. Structural Biology and Molecular Biophysics

    Surprising features of nuclear receptor interaction networks revealed by live-cell single-molecule imaging

    Liza Dahal, Thomas GW Graham ... Xavier Darzacq
    Research Article

    Type II nuclear receptors (T2NRs) require heterodimerization with a common partner, the retinoid X receptor (RXR), to bind cognate DNA recognition sites in chromatin. Based on previous biochemical and overexpression studies, binding of T2NRs to chromatin is proposed to be regulated by competition for a limiting pool of the core RXR subunit. However, this mechanism has not yet been tested for endogenous proteins in live cells. Using single-molecule tracking (SMT) and proximity-assisted photoactivation (PAPA), we monitored interactions between endogenously tagged RXR and retinoic acid receptor (RAR) in live cells. Unexpectedly, we find that higher expression of RAR, but not RXR, increases heterodimerization and chromatin binding in U2OS cells. This surprising finding indicates the limiting factor is not RXR but likely its cadre of obligate dimer binding partners. SMT and PAPA thus provide a direct way to probe which components are functionally limiting within a complex TF interaction network providing new insights into mechanisms of gene regulation in vivo with implications for drug development targeting nuclear receptors.

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

    Recognition and cleavage of human tRNA methyltransferase TRMT1 by the SARS-CoV-2 main protease

    Angel D'Oliviera, Xuhang Dai ... Jeffrey S Mugridge
    Research Article

    The SARS-CoV-2 main protease (Mpro or Nsp5) is critical for production of viral proteins during infection and, like many viral proteases, also targets host proteins to subvert their cellular functions. Here, we show that the human tRNA methyltransferase TRMT1 is recognized and cleaved by SARS-CoV-2 Mpro. TRMT1 installs the N2,N2-dimethylguanosine (m2,2G) modification on mammalian tRNAs, which promotes cellular protein synthesis and redox homeostasis. We find that Mpro can cleave endogenous TRMT1 in human cell lysate, resulting in removal of the TRMT1 zinc finger domain. Evolutionary analysis shows the TRMT1 cleavage site is highly conserved in mammals, except in Muroidea, where TRMT1 is likely resistant to cleavage. TRMT1 proteolysis results in reduced tRNA binding and elimination of tRNA methyltransferase activity. We also determined the structure of an Mpro-TRMT1 peptide complex that shows how TRMT1 engages the Mpro active site in an uncommon substrate binding conformation. Finally, enzymology and molecular dynamics simulations indicate that kinetic discrimination occurs during a later step of Mpro-mediated proteolysis following substrate binding. Together, these data provide new insights into substrate recognition by SARS-CoV-2 Mpro that could help guide future antiviral therapeutic development and show how proteolysis of TRMT1 during SARS-CoV-2 infection impairs both TRMT1 tRNA binding and tRNA modification activity to disrupt host translation and potentially impact COVID-19 pathogenesis or phenotypes.