1. Structural Biology and Molecular Biophysics

Cryo-EM structures of the autoinhibited <em>E. coli</em> ATP synthase in three rotational states

  1. Meghna Sobti
  2. Callum Smits
  3. Andrew SW Wong
  4. Robert Ishmukhametov
  5. Daniela Stock
  6. Sara Sandin
  7. Alastair G Stewart  Is a corresponding author
  1. The Victor Chang Cardiac Research Institute, Australia
  2. Nanyang Technological University, Singapore
  3. University of Oxford, United Kingdom
https://doi.org/10.7554/eLife.21598
Share this article
Cite this article
  1. Meghna Sobti
  2. Callum Smits
  3. Andrew SW Wong
  4. Robert Ishmukhametov
  5. Daniela Stock
  6. Sara Sandin
  7. Alastair G Stewart
(2016)
Cryo-EM structures of the autoinhibited <em>E. coli</em> ATP synthase in three rotational states
eLife 5:e21598.
https://doi.org/10.7554/eLife.21598
  2. Download BibTeX
  3. Download .RIS

Abstract

A molecular model that provides a framework for interpreting the wealth of functional information obtained on the <em>E. coli</em> F-ATP synthase has been generated using cryo-electron microscopy. Three different states that relate to rotation of the enzyme were observed, with the central stalk's &epsilon; subunit in an extended autoinhibitory conformation in all three states. The Fo motor comprises of seven transmembrane helices and a decameric c-ring and invaginations on either side of the membrane indicate the entry and exit channels for protons. The proton translocating subunit contains near parallel helices inclined by ~30&ordm; to the membrane, a feature now synonymous with rotary ATPases. For the first time in this rotary ATPase subtype, the peripheral stalk is resolved over its entire length of the complex, revealing the F1 attachment points and a coiled-coil that bifurcates towards the membrane with its helices separating to embrace subunit a from two sides.

Data availability

The following data sets were generated

Article and author information

Author details

  1. Meghna Sobti

    Molecular, Structural and Computational Biology Division, The Victor Chang Cardiac Research Institute, Darlinghurst, Australia
    Competing interests
    The authors declare that no competing interests exist.

  2. Callum Smits

    Molecular, Structural and Computational Biology Division, The Victor Chang Cardiac Research Institute, Darlinghurst, Australia
    Competing interests
    The authors declare that no competing interests exist.

  3. Andrew SW Wong

    NTU Institute of Structural Biology, Nanyang Technological University, Singapore, Singapore
    Competing interests
    The authors declare that no competing interests exist.

  4. Robert Ishmukhametov

    Department of Physics, Clarendon Laboratory, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  5. Daniela Stock

    Molecular, Structural and Computational Biology Division, The Victor Chang Cardiac Research Institute, Darlinghurst, Australia
    Competing interests
    The authors declare that no competing interests exist.

  6. Sara Sandin

    NTU Institute of Structural Biology, Nanyang Technological University, Singapore, Singapore
    Competing interests
    The authors declare that no competing interests exist.

  7. Alastair G Stewart

    Molecular, Structural and Computational Biology Division, The Victor Chang Cardiac Research Institute, Darlinghurst, Australia
    For correspondence
    a.stewart@victorchang.edu.au
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2070-6030

Funding

National Health and Medical Research Council (1004620)

  • Daniela Stock

National Health and Medical Research Council (1109961)

  • Daniela Stock

National Health and Medical Research Council (1090408)

  • Alastair G Stewart

National Health and Medical Research Council (1022143)

  • Daniela Stock

National Health and Medical Research Council (1047004)

  • Daniela Stock

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

Reviewing Editor

  1. Werner Kühlbrandt, Max Planck Institute of Biophysics, Germany

Version history

  1. Received: September 19, 2016
  2. Accepted: December 15, 2016
  3. Accepted Manuscript published: December 21, 2016 (version 1)
  4. Accepted Manuscript updated: December 22, 2016 (version 2)
  5. Version of Record published: January 5, 2017 (version 3)
  6. Version of Record updated: February 10, 2017 (version 4)

Copyright

© 2016, Sobti 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

  • 6,647
    views
  • 957
    downloads
  • 115
    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. Meghna Sobti
  2. Callum Smits
  3. Andrew SW Wong
  4. Robert Ishmukhametov
  5. Daniela Stock
  6. Sara Sandin
  7. Alastair G Stewart
(2016)
Cryo-EM structures of the autoinhibited <em>E. coli</em> ATP synthase in three rotational states
eLife 5:e21598.
https://doi.org/10.7554/eLife.21598

Share this article

https://doi.org/10.7554/eLife.21598

Categories and tags

Research organism

Further reading

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

    Cryo-EM reveals distinct conformations of E. coli ATP synthase on exposure to ATP

    Meghna Sobti, Robert Ishmukhametov ... Alastair G Stewart
    Research Advance Updated

    ATP synthase produces the majority of cellular energy in most cells. We have previously reported cryo-EM maps of autoinhibited E. coli ATP synthase imaged without addition of nucleotide (Sobti et al. 2016), indicating that the subunit ε engages the α, β and γ subunits to lock the enzyme and prevent functional rotation. Here we present multiple cryo-EM reconstructions of the enzyme frozen after the addition of MgATP to identify the changes that occur when this ε inhibition is removed. The maps generated show that, after exposure to MgATP, E. coli ATP synthase adopts a different conformation with a catalytic subunit changing conformation substantially and the ε C-terminal domain transitioning via an intermediate ‘half-up’ state to a condensed ‘down’ state. This work provides direct evidence for unique conformational states that occur in E. coli ATP synthase when ATP binding prevents the ε C-terminal domain from entering the inhibitory ‘up’ state.

    1. Structural Biology and Molecular Biophysics

    The mechanism of mammalian proton-coupled peptide transporters

    Simon M Lichtinger, Joanne L Parker ... Philip C Biggin
    Research Article

    Proton-coupled oligopeptide transporters (POTs) are of great pharmaceutical interest owing to their promiscuous substrate binding site that has been linked to improved oral bioavailability of several classes of drugs. Members of the POT family are conserved across all phylogenetic kingdoms and function by coupling peptide uptake to the proton electrochemical gradient. Cryo-EM structures and alphafold models have recently provided new insights into different conformational states of two mammalian POTs, SLC15A1, and SLC15A2. Nevertheless, these studies leave open important questions regarding the mechanism of proton and substrate coupling, while simultaneously providing a unique opportunity to investigate these processes using molecular dynamics (MD) simulations. Here, we employ extensive unbiased and enhanced-sampling MD to map out the full SLC15A2 conformational cycle and its thermodynamic driving forces. By computing conformational free energy landscapes in different protonation states and in the absence or presence of peptide substrate, we identify a likely sequence of intermediate protonation steps that drive inward-directed alternating access. These simulations identify key differences in the extracellular gate between mammalian and bacterial POTs, which we validate experimentally in cell-based transport assays. Our results from constant-PH MD and absolute binding free energy (ABFE) calculations also establish a mechanistic link between proton binding and peptide recognition, revealing key details underpining secondary active transport in POTs. This study provides a vital step forward in understanding proton-coupled peptide and drug transport in mammals and pave the way to integrate knowledge of solute carrier structural biology with enhanced drug design to target tissue and organ bioavailability.

    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.