The mechanistic target of rapamycin complex 1 (mTORC1) anchors a conserved signalling pathway that regulates growth in response to nutrient availability1-5. Amino acids activate mTORC1 through the Rag GTPases, which are regulated by GATOR, a supercomplex consisting of GATOR1, KICSTOR and the nutrient-sensing hub GATOR2 (refs. 6-9). GATOR2 forms an octagonal cage, with its distinct WD40 domain β-propellers interacting with GATOR1 and the leucine sensors Sestrin1 and Sestrin2 (SESN1 and SESN2) and the arginine sensor CASTOR1 (ref. 10). The mechanisms through which these sensors regulate GATOR2 and how they detach from it upon binding their cognate amino acids remain unknown. Here, using cryo-electron microscopy, we determined the structures of a stabilized GATOR2 bound to either Sestrin2 or CASTOR1. The sensors occupy distinct and non-overlapping binding sites, disruption of which selectively impairs the ability of mTORC1 to sense individual amino acids. We also resolved the apo (leucine-free) structure of Sestrin2 and characterized the amino acid-induced structural rearrangements within Sestrin2 and CASTOR1 that trigger their dissociation from GATOR2. Binding of either sensor restricts the dynamic WDR24 β-propeller of GATOR2, a domain essential for nutrient-dependent mTORC1 activation. These findings reveal the allosteric mechanisms that convey amino acid sufficiency to GATOR2 and the ensuing structural changes that lead to mTORC1 activation.

Broad Institute of MIT and Harvard Cambridge MA USA

Center for Genomic Medicine Massachusetts General Hospital Boston MA USA

Department of Biology Massachusetts Institute of Technology Cambridge MA USA

Department of Chemical and Systems Biology Stanford University School of Medicine Stanford CA USA

Department of Medicine Massachusetts General Hospital Boston MA USA

Department of Structural Biology Stanford University School of Medicine Stanford CA USA

Department of Surgery Massachusetts General Hospital Boston MA USA

Harvard Medical School Boston MA USA

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Boston Branch Cambridge MA USA

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Prague Czech Republic

Stanford Cancer Institute Stanford University School of Medicine Stanford CA USA

Whitehead Institute for Biomedical Research Cambridge MA USA

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Liu, G. Y. & Sabatini, D. M. mTOR at the nexus of nutrition, growth, ageing and disease. PubMed DOI PMC

Kim, J. & Guan, K.-L. mTOR as a central hub of nutrient signalling and cell growth. PubMed DOI

Valvezan, A. J. & Manning, B. D. Molecular logic of mTORC1 signalling as a metabolic rheostat. PubMed DOI PMC

Melick, C. H. & Jewell, J. L. Regulation of mTORC1 by upstream stimuli. PubMed DOI PMC

González, A. & Hall, M. N. Nutrient sensing and TOR signaling in yeast and mammals. PubMed DOI PMC

Sancak, Y. et al. The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. PubMed DOI PMC

Bar-Peled, L. et al. A tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1. PubMed DOI PMC

Wolfson, R. L. et al. KICSTOR recruits GATOR1 to the lysosome and is necessary for nutrients to regulate mTORC1. PubMed DOI PMC

Valenstein, M. L. et al. Rag–Ragulator is the central organizer of the physical architecture of the mTORC1 nutrient-sensing pathway. PubMed DOI PMC

Valenstein, M. L. et al. Structure of the nutrient-sensing hub GATOR2. PubMed DOI PMC

Linde-Garelli, K. Y. & Rogala, K. B. Structural mechanisms of the mTOR pathway. PubMed DOI

Sancak, Y. et al. Ragulator–Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. PubMed DOI PMC

Anandapadamanaban, M. et al. Architecture of human Rag GTPase heterodimers and their complex with mTORC1. PubMed DOI PMC

Rogala, K. B. et al. Structural basis for the docking of mTORC1 on the lysosomal surface. PubMed DOI PMC

Shen, K. et al. Architecture of the human GATOR1 and GATOR1–Rag GTPases complexes. PubMed DOI PMC

Wolfson, R. L. et al. Sestrin2 is a leucine sensor for the mTORC1 pathway. PubMed DOI PMC

Chantranupong, L. et al. The CASTOR proteins are arginine sensors for the mTORC1 pathway. PubMed DOI PMC

Zhou, Y., Wang, C., Xiao, Q. & Guo, L. Crystal structures of arginine sensor CASTOR1 in arginine-bound and ligand free states. PubMed DOI

Saxton, R. A., Chantranupong, L., Knockenhauer, K. E., Schwartz, T. U. & Sabatini, D. M. Mechanism of arginine sensing by CASTOR1 upstream of mTORC1. PubMed PMC

Gai, Z. et al. Structural mechanism for the arginine sensing and regulation of CASTOR1 in the mTORC1 signaling pathway. PubMed DOI PMC

Saxton, R. A. et al. Structural basis for leucine sensing by the Sestrin2–mTORC1 pathway. PubMed DOI PMC

Saxton, R. A., Knockenhauer, K. E., Schwartz, T. U. & Sabatini, D. M. The apo-structure of the leucine sensor Sestrin2 is still elusive. PubMed DOI PMC

Tafur, L. et al. Cryo-EM structure of the SEA complex. PubMed DOI PMC

Agarwal, V. & McShan, A. C. The power and pitfalls of AlphaFold2 for structure prediction beyond rigid globular proteins. PubMed DOI PMC

Gu, X. et al. SAMTOR is an PubMed DOI PMC

Jiang, C. et al. Ring domains are essential for GATOR2-dependent mTORC1 activation. PubMed DOI PMC

Liu, G. Y., Jouandin, P., Bahng, R. E., Perrimon, N. & Sabatini, D. M. An evolutionary mechanism to assimilate new nutrient sensors into the mTORC1 pathway. PubMed DOI PMC

Cangelosi, A. L. et al. Zonated leucine sensing by Sestrin–mTORC1 in the liver controls the response to dietary leucine. PubMed DOI PMC

Yang, C., Sun, X. & Wu, G. New insights into GATOR2-dependent interactions and its conformational changes in amino acid sensing. PubMed DOI PMC

Boussif, O. et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. PubMed DOI PMC

Tsun, Z.-Y. et al. The folliculin tumor suppressor is a GAP for the RagC/D GTPases that signal amino acid levels to mTORC1. PubMed DOI PMC

Abraham, M. J. et al. GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. DOI

Nnyigide, O. S., Nnyigide, T. O., Lee, S.-G. & Hyun, K. Protein Repair and Analysis Server: a web server to repair PDB structures, add missing heavy atoms and hydrogen atoms, and assign secondary structures by amide interactions. PubMed DOI

Huang, J. et al. CHARMM36m: an improved force field for folded and intrinsically disordered proteins. PubMed DOI PMC

Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W. & Klein, M. L. Comparison of simple potential functions for simulating liquid water. DOI

Sousa da Silva, A. W. & Vranken, W. F. ACPYPE — antechamber Python parser interface. PubMed DOI PMC

Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. PubMed DOI

[No authors listed.] Reliability and reproducibility checklist for molecular dynamics simulations. PubMed PMC

Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. PubMed DOI PMC

Zivanov, J., Nakane, T. & Scheres, S. H. W. A Bayesian approach to beam-induced motion correction in cryo-EM single-particle analysis. PubMed DOI PMC

Burt, A. et al. An image processing pipeline for electron cryo‐tomography in RELION‐5. PubMed DOI PMC

Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. PubMed DOI PMC

Bepler, T., Kelley, K., Noble, A. J. & Berger, B. Topaz-Denoise: general deep denoising models for cryoEM and cryoET. PubMed DOI PMC

Bepler, T. et al. Positive-unlabeled convolutional neural networks for particle picking in cryo-electron micrographs. PubMed DOI PMC

Kimanius, D., Dong, L., Sharov, G., Nakane, T. & Scheres, S. H. W. New tools for automated cryo-EM single-particle analysis in RELION-4.0. PubMed DOI PMC

Zivanov, J., Nakane, T. & Scheres, S. H. W. Estimation of high-order aberrations and anisotropic magnification from cryo-EM data sets in RELION-3.1. PubMed DOI PMC

Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A.cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. PubMed DOI

Punjani, A., Zhang, H. & Fleet, D. J. Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction. PubMed DOI

Punjani, A. & Fleet, D. J. 3D variability analysis: resolving continuous flexibility and discrete heterogeneity from single particle cryo-EM. PubMed DOI

Sanchez-Garcia, R. et al. DeepEMhancer: a deep learning solution for cryo-EM volume post-processing. PubMed DOI PMC

Afonine, P. V. et al. New tools for the analysis and validation of cryo-EM maps and atomic models. PubMed PMC

Pettersen, E. F. et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. PubMed DOI PMC

Casañal, A., Lohkamp, B. & Emsley, P. Current developments in Coot for macromolecular model building of electron cryo‐microscopy and crystallographic data. PubMed DOI PMC

Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. PubMed DOI PMC

Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. PubMed PMC

Terwilliger, T. C. et al. Improved AlphaFold modeling with implicit experimental information. PubMed DOI PMC

Prisant, M. G., Williams, C. J., Chen, V. B., Richardson, J. S. & Richardson, D. C. New tools in MolProbity validation: CaBLAM for CryoEM backbone, UnDowser to rethink “waters,” and NGL Viewer to recapture online 3D graphics. PubMed DOI PMC

Liebschner, D. et al. Macromolecular structure determination using X‐rays, neutrons and electrons: recent developments in Phenix. PubMed DOI PMC

Laskowski, R. A. & Swindells, M. B. LigPlot+: multiple ligand–protein interaction diagrams for drug discovery. PubMed DOI