Architecture of the human mTORC2 core complex
- University of Basel, Switzerland
- ETH Zürich, Switzerland
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
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Human mTORC2 Core Complex (C2)Publicly available at the EBI EM-3927.
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Human mTORC2 core complex accessory factorsPublicly available at the EBI EM-3928.
Article and author information
Author details
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.
Reviewing Editor
- Nahum Sonenberg, McGill University, Canada
Publication history
- Received: October 25, 2017
- Accepted: February 8, 2018
- Accepted Manuscript published: February 9, 2018 (version 1)
- Version of Record published: March 5, 2018 (version 2)
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.
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Architecture of the human mTORC2 core complex
eLife 7:e33101.
https://doi.org/10.7554/eLife.33101
Further reading
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- Structural Biology and Molecular Biophysics
Undruggability of RAS proteins has necessitated alternative strategies for the development of effective inhibitors. In this respect, phosphorylation has recently come into prominence as this reversible post-translational modification attenuates sensitivity of RAS towards RAF. As such, in this study, we set out to unveil the impact of phosphorylation on dynamics of HRASWT and aim to invoke similar behavior in HRASG12D mutant by means of small therapeutic molecules. To this end, we performed molecular dynamics (MD) simulations using phosphorylated HRAS and showed that phosphorylation of Y32 distorted Switch I, hence the RAS/RAF interface. Consequently, we targeted Switch I in HRASG12D by means of approved therapeutic molecules and showed that the ligands enabled detachment of Switch I from the nucleotide-binding pocket. Moreover, we demonstrated that displacement of Switch I from the nucleotide-binding pocket was energetically more favorable in the presence of the ligand. Importantly, we verified computational findings in vitro where HRASG12D/RAF interaction was prevented by the ligand in HEK293T cells that expressed HRASG12D mutant protein. Therefore, these findings suggest that targeting Switch I, hence making Y32 accessible might open up new avenues in future drug discovery strategies that target mutant RAS proteins.
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- Structural Biology and Molecular Biophysics
Cryogenic electron tomography (cryo-ET) combined with subtomogram averaging, allows in situ visualization and structure determination of macromolecular complexes at subnanometre resolution. Cryogenic focused ion beam (cryo-FIB) micromachining is used to prepare a thin lamella-shaped sample out of a frozen-hydrated cell for cryo-ET imaging, but standard cryo-FIB fabrication is blind to the precise location of the structure or proteins of interest. Fluorescence-guided focused ion beam (FIB) milling at target locations requires multiple sample transfers prone to contamination, and relocation and registration accuracy is often insufficient for 3D targeting. Here, we present in situ fluorescence microscopy-guided FIB fabrication of a frozen-hydrated lamella to address this problem: we built a coincident three-beam cryogenic correlative microscope by retrofitting a compact cryogenic microcooler, custom positioning stage, and an inverted widefield fluorescence microscope (FM) on an existing FIB scanning electron microscope. We show FM controlled targeting at every milling step in the lamella fabrication process, validated with transmission electron microscope tomogram reconstructions of the target regions. The ability to check the lamella during and after the milling process results in a higher success rate in the fabrication process and will increase the throughput of fabrication for lamellae suitable for high-resolution imaging.
