1. Structural Biology and Molecular Biophysics

Architecture of the human mTORC2 core complex

  1. Edward Stuttfeld
  2. Christopher H S Aylett
  3. Stefan Imseng
  4. Daniel Boehringer
  5. Alain Scaiola
  6. Evelyn Sauer
  7. Michael N Hall  Is a corresponding author
  8. Timm Maier  Is a corresponding author
  9. Nenad Ban  Is a corresponding author
  1. University of Basel, Switzerland
  2. ETH Zürich, Switzerland
https://doi.org/10.7554/eLife.33101
Share this article
Cite this article
  1. Edward Stuttfeld
  2. Christopher H S Aylett
  3. Stefan Imseng
  4. Daniel Boehringer
  5. Alain Scaiola
  6. Evelyn Sauer
  7. Michael N Hall
  8. Timm Maier
  9. Nenad Ban
(2018)
Architecture of the human mTORC2 core complex
eLife 7:e33101.
https://doi.org/10.7554/eLife.33101
  2. Download BibTeX
  3. Download .RIS

Abstract

The mammalian target of rapamycin (mTOR) is a key protein kinase controlling cellular metabolism and growth. It is part of the two structurally and functionally distinct multiprotein complexes mTORC1 and mTORC2. Dysregulation of mTOR occurs in diabetes, cancer and neurological disease. We report the architecture of human mTORC2 at intermediate resolution, revealing a conserved binding site for accessory proteins on mTOR and explaining the structural basis for the rapamycin insensitivity of the complex.

Data availability

The following data sets were generated

Article and author information

Author details

  1. Edward Stuttfeld

    Biozentrum, University of Basel, Basel, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3932-9076

  2. Christopher H S Aylett

    Institute for Molecular Biology and Biophysics, ETH Zürich, Zürich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.

  3. Stefan Imseng

    Biozentrum, University of Basel, Basel, Switzerland
    Competing interests
    The authors declare that no competing interests exist.

  4. Daniel Boehringer

    Institute for Molecular Biology and Biophysics, ETH Zürich, Zürich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.

  5. Alain Scaiola

    Institute for Molecular Biology and Biophysics, ETH Zürich, Zürich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.

  6. Evelyn Sauer

    Biozentrum, University of Basel, Basel, Switzerland
    Competing interests
    The authors declare that no competing interests exist.

  7. Michael N Hall

    Biozentrum, University of Basel, Basel, Switzerland
    For correspondence
    m.hall@unibas.ch
    Competing interests
    The authors declare that no competing interests exist.

  8. Timm Maier

    Biozentrum, University of Basel, Basel, Switzerland
    For correspondence
    timm.maier@unibas.ch
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7459-1363

  9. Nenad Ban

    Institute for Molecular Biology and Biophysics, ETH Zürich, Zürich, Switzerland
    For correspondence
    ban@mol.biol.ethz.ch
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9527-210X

Funding

Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (51NF40_141735_NCCR)

  • Nenad Ban

Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (310030_159492)

  • Michael N Hall

European Research Council (609883)

  • Michael N Hall

Sir Henry Dale Fellowship (206212/Z/17/Z)

  • Christopher H S Aylett

Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (138262)

  • Nenad Ban

Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (159696)

  • Timm Maier

Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (310030B_163478)

  • Nenad Ban

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

Copyright

© 2018, Stuttfeld 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

  • 5,152
    views
  • 1,027
    downloads
  • 64
    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. Edward Stuttfeld
  2. Christopher H S Aylett
  3. Stefan Imseng
  4. Daniel Boehringer
  5. Alain Scaiola
  6. Evelyn Sauer
  7. Michael N Hall
  8. Timm Maier
  9. Nenad Ban
(2018)
Architecture of the human mTORC2 core complex
eLife 7:e33101.
https://doi.org/10.7554/eLife.33101

Share this article

https://doi.org/10.7554/eLife.33101

Categories and tags

Research organism

Further reading

    1. Structural Biology and Molecular Biophysics

    Ligand-coupled conformational changes in a cyclic nucleotide-gated ion channel revealed by time-resolved transition metal ion FRET

    Pierce Eggan, Sharona E Gordon, William N Zagotta
    Research Article

    Cyclic nucleotide-binding domain (CNBD) ion channels play crucial roles in cellular-signaling and excitability and are regulated by the direct binding of cyclic adenosine- or guanosine-monophosphate (cAMP, cGMP). However, the precise allosteric mechanism governing channel activation upon ligand binding, particularly the energetic changes within domains, remains poorly understood. The prokaryotic CNBD channel SthK offers a valuable model for investigating this allosteric mechanism. In this study, we investigated the conformational dynamics and energetics of the SthK C-terminal region using a combination of steady-state and time-resolved transition metal ion Förster resonance energy transfer (tmFRET) experiments. We engineered donor-acceptor pairs at specific sites within a SthK C-terminal fragment by incorporating a fluorescent noncanonical amino acid donor and metal ion acceptors. Measuring tmFRET with fluorescence lifetimes, we determined intramolecular distance distributions in the absence and presence of cAMP or cGMP. The probability distributions between conformational states without and with ligand were used to calculate the changes in free energy (ΔG) and differences in free energy change (ΔΔG) in the context of a simple four-state model. Our findings reveal that cAMP binding produces large structural changes, with a very favorable ΔΔG. In contrast to cAMP, cGMP behaved as a partial agonist and only weakly promoted the active state. Furthermore, we assessed the impact of protein oligomerization and ionic strength on the structure and energetics of the conformational states. This study demonstrates the effectiveness of time-resolved tmFRET in determining the conformational states and the ligand-dependent energetics of the SthK C-terminal region.

    1. Structural Biology and Molecular Biophysics

    A cryo-electron tomography study of ciliary rootlet organization

    Chris van Hoorn, Andrew P Carter
    Research Article

    Ciliary rootlets are striated bundles of filaments that connect the base of cilia to internal cellular structures. Rootlets are critical for the sensory and motile functions of cilia. However, the mechanisms underlying these functions remain unknown, in part due to a lack of structural information of rootlet organization. In this study, we obtain 3D reconstructions of membrane-associated and purified rootlets from mouse retina using cryo-electron tomography. We show that flexible protrusions on the rootlet surface, which emanate from the cross-striations, connect to intracellular membranes. In purified rootlets, the striations were classified into amorphous (A)-bands, associated with accumulations on the rootlet surface, and discrete (D)-bands corresponding to punctate lines of density that run through the rootlet. These striations connect a flexible network of longitudinal filaments. Subtomogram averaging suggests the filaments consist of two intertwined coiled coils. The rootlet’s filamentous architecture, with frequent membrane-connecting cross-striations, lends itself well for anchoring large membranes in the cell.

    1. Structural Biology and Molecular Biophysics

    Role of the αC-β4 loop in protein kinase structure and dynamics

    Jian Wu, Nisha A Jonniya ... Susan S Taylor
    Research Article

    Although the αC-β4 loop is a stable feature of all protein kinases, the importance of this motif as a conserved element of secondary structure, as well as its links to the hydrophobic architecture of the kinase core, has been underappreciated. We first review the motif and then describe how it is linked to the hydrophobic spine architecture of the kinase core, which we first discovered using a computational tool, local spatial Pattern (LSP) alignment. Based on NMR predictions that a mutation in this motif abolishes the synergistic high-affinity binding of ATP and a pseudo substrate inhibitor, we used LSP to interrogate the F100A mutant. This comparison highlights the importance of the αC-β4 loop and key residues at the interface between the N- and C-lobes. In addition, we delved more deeply into the structure of the apo C-subunit, which lacks ATP. While apo C-subunit showed no significant changes in backbone dynamics of the αC-β4 loop, we found significant differences in the side chain dynamics of K105. The LSP analysis suggests disruption of communication between the N- and C-lobes in the F100A mutant, which would be consistent with the structural changes predicted by the NMR spectroscopy.