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Structure of the nutrient-sensing hub GATOR2

Nature volume 607, pages 610–616 (2022)Cite this article

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Abstract

Mechanistic target of rapamycin complex 1 (mTORC1) controls growth by regulating anabolic and catabolic processes in response to environmental cues, including nutrients1,2. Amino acids signal to mTORC1 through the Rag GTPases, which are regulated by several protein complexes, including GATOR1 and GATOR2. GATOR2, which has five components (WDR24, MIOS, WDR59, SEH1L and SEC13), is required for amino acids to activate mTORC1 and interacts with the leucine and arginine sensors SESN2 and CASTOR1, respectively3,4,5. Despite this central role in nutrient sensing, GATOR2 remains mysterious as its subunit stoichiometry, biochemical function and structure are unknown. Here we used cryo-electron microscopy to determine the three-dimensional structure of the human GATOR2 complex. We found that GATOR2 adopts a large (1.1 MDa), two-fold symmetric, cage-like architecture, supported by an octagonal scaffold and decorated with eight pairs of WD40 β-propellers. The scaffold contains two WDR24, four MIOS and two WDR59 subunits circularized via two distinct types of junction involving non-catalytic RING domains and α-solenoids. Integration of SEH1L and SEC13 into the scaffold through β-propeller blade donation stabilizes the GATOR2 complex and reveals an evolutionary relationship to the nuclear pore and membrane-coating complexes6. The scaffold orients the WD40 β-propeller dimers, which mediate interactions with SESN2, CASTOR1 and GATOR1. Our work reveals the structure of an essential component of the nutrient-sensing machinery and provides a foundation for understanding the function of GATOR2 within the mTORC1 pathway.

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Fig. 1: Structure of human GATOR2.
Fig. 2: MIOS interacts with WDR24 and WDR59 via dimerization of CTDs.
Fig. 3: α-Solenoid junctions, supported by β-propellers of SEH1L and SEC13, complete the GATOR2 scaffold.
Fig. 4: The GATOR2 β-propellers receive and transmit amino acid availability.

Data availability

The data that support the findings of this study are available from the corresponding authors and the Whitehead Institute (sabadmin@wi.mit.edu) upon request. Cryo-EM maps were deposited in the Electron Microscopy Data Bank under accession number EMD-26519. The atomic model of GATOR2 was deposited in the PDB under accession number 7UHY. Plasmids generated in this study are available from Addgene.

References

  1. Liu, G. Y. & Sabatini, D. M. mTOR at the nexus of nutrition, growth, ageing and disease. Nat. Rev. Mol. Cell Biol. 21, 183–203 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Condon, K. J. & Sabatini, D. M. Nutrient regulation of mTORC1 at a glance. J. Cell Sci. 132, jcs222570 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Bar-Peled, L. et al. A tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1. Science 340, 1100–1106 (2013).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  4. Wolfson, R. L. et al. Sestrin2 is a leucine sensor for the mTORC1 pathway. Science 351, 43–48 (2016).

    Article  ADS  CAS  PubMed  Google Scholar 

  5. Chantranupong, L. et al. The CASTOR proteins are arginine sensors for the mTORC1 pathway. Cell 165, 153–164 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Brohawn, S. G., Leksa, N. C., Spear, E. D., Rajashankar, K. R. & Schwartz, T. U. Structural evidence for common ancestry of the nuclear pore complex and vesicle coats. Science 322, 1369–1373 (2008).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  7. Sancak, Y. et al. The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science 320, 1496–1501 (2008).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  8. Rogala, K. B. et al. Structural basis for the docking of mTORC1 on the lysosomal surface. Science 366, 468–475 (2019).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  9. Anandapadamanaban, M. et al. Architecture of human Rag GTPase heterodimers and their complex with mTORC1. Science 366, 203–210 (2019).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  10. Wolfson, R. L. et al. KICSTOR recruits GATOR1 to the lysosome and is necessary for nutrients to regulate mTORC1. Nature 543, 438–442 (2017).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  11. Peng, M., Yin, N. & Li, M. O. SZT2 dictates GATOR control of mTORC1 signalling. Nature 543, 433–437 (2017).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  12. Shen, K. et al. Architecture of the human GATOR1 and GATOR1–Rag GTPases complexes. Nature 556, 64–69 (2018).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  13. Metzger, M. B., Pruneda, J. N., Klevit, R. E. & Weissman, A. M. RING-type E3 ligases: master manipulators of E2 ubiquitin-conjugating enzymes and ubiquitination. Biochim. Biophys. Acta 1843, 47–60 (2014).

    Article  CAS  PubMed  Google Scholar 

  14. Li, Z., Jaroszewski, L., Iyer, M., Sedova, M. & Godzik, A. FATCAT 2.0: towards a better understanding of the structural diversity of proteins. Nucleic Acids Res. 48, W60–W64 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Bellon, S. F., Rodgers, K. K., Schatz, D. G., Coleman, J. E. & Steitz, T. A. Crystal structure of the RAG1 dimerization domain reveals multiple zinc-binding motifs including a novel zinc binuclear cluster. Nat. Struct. Biol. 4, 586–591 (1997).

    Article  CAS  PubMed  Google Scholar 

  16. Dokudovskaya, S. et al. A conserved coatomer-related complex containing Sec13 and Seh1 dynamically associates with the vacuole in Saccharomyces cerevisiae. Mol. Cell. Proteomics 10, M110.006478 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Plechanovová, A., Jaffray, E. G., Tatham, M. H., Naismith, J. H. & Hay, R. T. Structure of a RING E3 ligase and ubiquitin-loaded E2 primed for catalysis. Nature 489, 115–120 (2012).

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

  18. Hsia, K.-C., Stavropoulos, P., Blobel, G. & Hoelz, A. Architecture of a coat for the nuclear pore membrane. Cell 131, 1313–1326 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Whittle, J. R. R. & Schwartz, T. U. Structure of the Sec13–Sec16 edge element, a template for assembly of the COPII vesicle coat. J. Cell Biol. 190, 347–361 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Fath, S., Mancias, J. D., Bi, X. & Goldberg, J. Structure and organization of coat proteins in the COPII cage. Cell 129, 1325–1336 (2007).

    Article  CAS  PubMed  Google Scholar 

  21. Chen, J. et al. SAR1B senses leucine levels to regulate mTORC1 signalling. Nature 596, 281–284 (2021).

    Article  ADS  CAS  PubMed  Google Scholar 

  22. Rout, M. P. & Field, M. C. The evolution of organellar coat complexes and organization of the eukaryotic cell. Annu. Rev. Biochem. 86, 637–657 (2017).

    Article  CAS  PubMed  Google Scholar 

  23. Hunter, M. R., Scourfield, E. J., Emmott, E. & Graham, S. C. VPS18 recruits VPS41 to the human HOPS complex via a RING–RING interaction. Biochem. J. 474, 3615–3626 (2017).

    Article  CAS  PubMed  Google Scholar 

  24. Cai, W., Wei, Y., Jarnik, M., Reich, J. & Lilly, M. A. The GATOR2 component Wdr24 regulates TORC1 activity and lysosome function. PLoS Genet. 12, e1006036 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Saxton, R. A., Chantranupong, L., Knockenhauer, K. E., Schwartz, T. U. & Sabatini, D. M. Mechanism of arginine sensing by CASTOR1 upstream of mTORC1. Nature 536, 229–233 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  26. Gu, X. et al. SAMTOR is an S-adenosylmethionine sensor for the mTORC1 pathway. Science 358, 813–818 (2017).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  27. Jain, B. P. & Pandey, S. WD40 repeat proteins: signalling scaffold with diverse functions. Protein J. 37, 391–406 (2018).

    Article  CAS  PubMed  Google Scholar 

  28. Baek, M. et al. Accurate prediction of protein structures and interactions using a three-track neural network. Science 373, 871–876 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  29. Zhou, Y., Wang, C., Xiao, Q. & Guo, L. Crystal structures of arginine sensor CASTOR1 in arginine-bound and ligand free states. Biochem. Biophys. Res. Commun. 508, 387–391 (2018).

    Article  PubMed  CAS  Google Scholar 

  30. Saxton, R. A. et al. Structural basis for leucine sensing by the Sestrin2–mTORC1 pathway. Science 351, 53–58 (2015).

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

  31. Cristea, I. M. & Chait, B. T. Conjugation of magnetic beads for immunopurification of protein complexes. Cold Spring Harb. Protoc. 2011, pdb.prot5610 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Boussif, O. et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl Acad. Sci. USA 92, 7297–7301 (1995).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  33. Cuéllar, J. et al. Structural and functional analysis of the role of the chaperonin CCT in mTOR complex assembly. Nat. Commun. 10, 2865 (2019).

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

  34. Mastronarde, D. N. SerialEM: a program for automated tilt series acquisition on Tecnai microscopes using prediction of specimen position. Microsc. Microanal. 9, 1182–1183 (2003).

    Article  ADS  Google Scholar 

  35. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Zivanov, J., Nakane, T. & Scheres, S. H. W. A Bayesian approach to beam-induced motion correction in cryo-EM single-particle analysis. IUCrJ 6, 5–17 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Zhang, K. Gctf: real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  38. Wagner, T. et al. SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo-EM. Commun. Biol. 2, 218 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  40. 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. IUCrJ 7, 253–267 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Scheres, S. H. Beam-induced motion correction for sub-megadalton cryo-EM particles. eLife 3, e03665 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  42. Wilkinson, M. E., Kumar, A. & Casañal, A. Methods for merging data sets in electron cryo‐microscopy. Acta Crystallogr. D Struct. Biol. 75, 782–791 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  PubMed  Google Scholar 

  44. Punjani, A. & Fleet, D. J. 3D variability analysis: resolving continuous flexibility and discrete heterogeneity from single particle cryo-EM. J. Struct. Biol. 213, 107702 (2021).

    Article  CAS  PubMed  Google Scholar 

  45. Liebschner, D. et al. Macromolecular structure determination using X‐rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D Struct. Biol. 75, 861–877 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Sanchez-Garcia, R. et al. DeepEMhancer: a deep learning solution for cryo-EM volume post-processing. Commun. Biol. 4, 874 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  48. Bharucha, N. et al. Sec16 influences transitional ER sites by regulating rather than organizing COPII. Mol. Biol. Cell 24, 3406–3419 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Brohawn, S. G. & Schwartz, T. U. Molecular architecture of the Nup84–Nup145C–Sec13 edge element in the nuclear pore complex lattice. Nat. Struct. Mol. Biol. 16, 1173–1177 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Debler, E. W. et al. A fence-like coat for the nuclear pore membrane. Mol. Cell 32, 815–826 (2008).

    Article  CAS  PubMed  Google Scholar 

  51. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D Struct. Biol. 74, 531–544 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  PubMed  Google Scholar 

  54. Barad, B. A. et al. EMRinger: side chain–directed model and map validation for 3D cryo-electron microscopy. Nat. Methods 12, 943–946 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Goddard, T. D. et al. UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018).

    Article  CAS  PubMed  Google Scholar 

  56. Ghose, A. K., Viswanadhan, V. N. & Wendoloski, J. J. Prediction of hydrophobic (lipophilic) properties of small organic molecules using fragmental methods: an analysis of ALOGP and CLOGP methods. J. Phys. Chem. 102, 3762–3772 (1998).

    Article  CAS  Google Scholar 

  57. Chatzigoulas, A. & Cournia, Z. Predicting protein–membrane interfaces of peripheral membrane proteins using ensemble machine learning. Brief Bioinform. 23, bbab518 (2022).

  58. Linke, K. et al. Structure of the MDM2/MDMX RING domain heterodimer reveals dimerization is required for their ubiquitylation in trans. Cell Death Differ. 15, 841–848 (2008).

    Article  CAS  PubMed  Google Scholar 

  59. Liew, C. W., Sun, H., Hunter, T. & Day, C. L. RING domain dimerization is essential for RNF4 function. Biochem. J. 431, 23–29 (2010).

    Article  CAS  PubMed  Google Scholar 

  60. Mace, P. D. et al. Structures of the cIAP2 RING domain reveal conformational changes associated with ubiquitin-conjugating enzyme (E2) recruitment. J. Biol. Chem. 283, 31633–31640 (2008).

    Article  CAS  PubMed  Google Scholar 

  61. Brzovic, P. S., Rajagopal, P., Hoyt, D. W., King, M.-C. & Klevit, R. E. Structure of a BRCA1–BARD1 heterodimeric RING–RING complex. Nat. Struct. Biol. 8, 833–837 (2001).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank all members of the Sabatini laboratory for helpful insights; K. Linde-Garelli for assistance with protein purifications; M. Baek, I. Anishchenko and D. Baker for structural predictions; the high-performance computing team at Whitehead Institute (C. Andrew, P. McCabe, R. Taylor, P. Macfarlane and W. Pena) for the installation and maintenance of data-processing servers; and R. Levinson for the forearm shake illustration in Fig. 2b. This work was supported by grants to D.M.S. from the US NIH (R01 CA103866, R01 CA129105 and R01 AI47389), the Department of Defense (W81XWH-21-1-0260 and TS200035), the Lustgarten Foundation and the Leo Foundation, and fellowship support from the NIH to M.L.V. (T32 GM007753 and F30 CA228229), K.B.R. (K99 CA255926), L.C. (F31 CA180271), R.A.S. (F31 GM121093) and from the MIT School of Science Fellowship in Cancer Research to M.L.V. and from the Ludwig Center at MIT Graduate Student Fellowship to X.G. K.B.R. was also supported by the Tuberous Sclerosis Association and the Charles A. King Trust.

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Author notes

  1. These authors contributed equally: Max L. Valenstein, Kacper B. Rogala

Authors and Affiliations

  1. Whitehead Institute for Biomedical Research, Cambridge, MA, USA

    Max L. Valenstein, Kacper B. Rogala, Pranav V. Lalgudi, Xin Gu, Robert A. Saxton, Lynne Chantranupong, Jonas Kolibius & Jan-Philipp Quast

  2. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA

    Max L. Valenstein, Pranav V. Lalgudi, Edward J. Brignole, Xin Gu, Robert A. Saxton & Lynne Chantranupong

  3. Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, MA, USA

    Kacper B. Rogala

  4. Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA, USA

    Kacper B. Rogala

  5. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, USA

    Kacper B. Rogala

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

    Kacper B. Rogala

  7. MIT.nano, Massachusetts Institute of Technology, Cambridge, MA, USA

    Edward J. Brignole

Author notes

  1. Unaffiliated: David M. Sabatini

    • David M. Sabatini
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Contributions

M.L.V., K.B.R. and D.M.S. formulated the research plan and interpreted experimental results with assistance from P.V.L. M.L.V. and P.V.L. purified the proteins. K.B.R. determined the cryo-EM map for GATOR2 and built the structural model with M.L.V. and P.V.L. E.J.B. assisted in cryo-EM data collection. M.L.V. and P.V.L. designed and performed biochemical experiments with assistance from X.G. L.C. generated GATOR2-knockout cell lines. R.A.S., J.K. and J.-P.Q. contributed to the GATOR2 truncation experiments. M.L.V. and D.M.S. wrote the manuscript with contributions from K.B.R. and P.V.L. All authors edited the manuscript.

Corresponding authors

Correspondence to Max L. Valenstein or Kacper B. Rogala.

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Competing interests

D.M.S. is a shareholder of Navitor Pharmaceuticals, which is targeting the amino-acid-sensing pathway upstream of mTORC1 for therapeutic benefit.

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Extended data figures and tables

Extended Data Fig. 1 GATOR2 integrity and composition are insensitive to amino acid availability.

(a) Amino acids do not regulate the size of the GATOR2 complex. Elution fractions from size-exclusion chromatography of transfected GATOR2 or endogenously-tagged GATOR2 were analyzed by SDS-PAGE. Gels were stained with Coomassie Blue or SYPRO Ruby, as indicated. n.s. indicates a non-specific co-purifying contaminant. Lane labels indicate elution volumes from TSKgel G4000SWxl column. (b) Analyses of endogenously tagged-WDR59 and -DEPDC5 expression in cell lysates. (c) Analysis of mTORC1 signaling in cell lysates from which endogenously-tagged GATOR2 was purified in (a). (d) Amino acids do not regulate the subunit composition or stoichiometry of endogenous GATOR2. Peak elution fractions from size-exclusion chromatography of endogenously-tagged GATOR2 were analyzed by SDS-PAGE and the gel was stained with SYPRO Ruby. (e) The subunit composition of GATOR2 and the interaction between GATOR2 and GATOR1 or KICSTOR are not regulated by amino acids. Anti-FLAG immunoprecipitates were prepared from HEK293T cells with the indicated genotype and were analyzed by immunoblotting for the indicated proteins. Data in (a)-(e) are representative of two independent experiments. For gel source data, see Supplementary Figure 1.

Extended Data Fig. 2 Modular Architecture of GATOR2.

(a) Two orthogonal views of the molecular model of GATOR2. (b), (c), (d) Molecular models (top) and corresponding domain schematics (bottom) of three distinct building blocks of GATOR2: MIOS-SEH1L (b), WDR24-SEH1L (c), and WDR59-SEC13 (d). Domains not resolved in our cryo-EM structure are drawn with increased transparency for reference. (e) Each core GATOR2 subunit (MIOS, WDR24, WDR59) associates with either SEH1L or SEC13. Anti-FLAG immunoprecipitates were prepared from HEK293T cells that transiently expressed the indicated cDNAs and were analyzed by immunoblotting for the indicated proteins. HA, hemagglutinin. Data in (e) are representative of two independent experiments. For gel source data, see Supplementary Figure 1.

Extended Data Fig. 3 Surface properties of GATOR2.

(a) Three orthogonal views of GATOR2 are shown in surface representation and colored by: subunit identity (top), molecular lipophilicity potential (middle), coulombic electrostatic potential (bottom). Surface calculations were performed in ChimeraX55, with atomic lipophilicity values from Ghose et al.56 The most lipophilic surfaces are drawn as orange and the least lipophilic as cyan. Negatively charged surfaces are drawn as red, and positively as blue. GATOR2 is highly charged at its surface, with the only exposed lipophilic patches of significant size found near the donor β-blade of WDR59, and the β-propeller of WDR24 (see panels (b) and (c) for details). Furthermore, there are two major surface patches where either negative or positive charge dominates. The mostly-positive patch stretches from the side of the β-propeller of WDR24, through the WDR24-MIOS CTD, to MIOS glove β-propeller. The mostly-negative patch starts with the MIOS α-solenoid, passes through the MIOS-WDR59 CTD, and ends at the tip of SEC13. The center of MIOS β-propellers appears highly positively charged. (b) Detailed view of the lipophilic patch near the donor β-blade of WDR59. While it might appear that the surface of the WDR59 β-blade forms a lipophilic groove, that groove is in fact occupied by protein density extending from the α-solenoid of WDR24. This WDR24 density likely continues to the top of the SEC13WDR59 β-propeller (green dashed line), where we found two better-resolved stretches of amino-acid sequence (colored in gray). Yet, for these particular sections of GATOR2, we were unable to build a high-confidence structural model or to unambiguously deduce the amino-acid sequence. (c) Detailed view of the highly lipophilic and charged surface of the WDR24 β-propeller. The WDR24 β-propeller contains strong patches of negatively and positively charged surface. There are additional sporadic sections of highly lipophilic surface. Residues predicted by the DREAMM algorithm as membrane-penetrating are drawn as spheres57.

Extended Data Fig. 4 GATOR2 CTDs contain RING domains.

(a) Sequence alignment of the homologous C-terminal regions of MIOS, WDR24, and WDR59. Zinc-coordinating residues are highlighted in yellow. Zinc-coordinating residues in the ZnF are indicated by red dots. Zinc-coordinating residues in the RING domain are indicated with blue dots. (b) Side-by-side comparison of the structurally-aligned MIOS, WDR24, and WDR59 CTDs. (c) Structural comparison of the MIOS, WDR24, and WDR59 RING domains with the E3 ligase RNF4 RING domain (PDB ID: 4AP4)17. (d), (e), and (f) Detailed views of the experimental map and the corresponding molecular model surrounding the zinc coordination sites within the MIOS CTD ZnF (d) and RING domain, (e) and (f). (g) Schematic of zinc ion coordination in the MIOS CTD. The MIOS CTD coordinates a total of four zinc ions with 15 amino-acid side chains — similarly to the CTDs in WDR24 and WDR59. (h) Cartoon representation of the zinc ion coordination in a canonical RING domain associated with E3 ubiquitin ligase activity. The RING domain coordinates two zinc ions with eight side chains.

Extended Data Fig. 5 CTD-CTD junctions link core GATOR2 proteins.

(a) Schematic representations (top) and structural comparison (bottom of the MIOS-WDR24 and MIOS-WDR59 CTD-CTD junctions. Auxiliary interactions between CTDs and SEH1L or SEC13 contribute to the interface. This mode of interaction is distinct from that reported for dimeric RING E3 ligases58,59,60,61. (b) View of the interface formed between the βC4 strands of the MIOS and WDR24 CTDs. Additional hydrophobic side chains from the MIOS and WDR24 βC1 strands, the MIOS αC2 helix and the WDR24 αC1 helix contribute to the interaction. (c) View of the interface formed between the βC4 strands of the MIOS and WDR59 CTDs. Additional hydrophobic side chains from the MIOS and WDR59 βC1 strands, the MIOS αC2 helix and the WDR59 αC1 helix contribute to the interaction. (d) The CTDs of MIOS and WDR59 are sufficient to mediate the MIOS-WDR59 interaction. Anti-FLAG immunoprecipitates were prepared from WDR24-deficient HEK293T cells that transiently expressed the indicated cDNAs and were analyzed by immunoblotting for the indicated proteins. (e) The CTDs of MIOS and WDR24 are sufficient to mediate the MIOS-WDR24 interaction. Anti-FLAG immunoprecipitates were prepared from WDR59-deficient HEK293T cells that transiently expressed the indicated cDNAs and were analyzed as in (d). (f) The WDR24 CTD is necessary for the MIOS-WDR24 interaction. Anti-FLAG immunoprecipitates were prepared from WDR59-deficient HEK293T cells that transiently expressed the indicated cDNAs and were analyzed as in (d). (g) The MIOS CTD is necessary for the MIOS-WDR24 interaction. Anti-FLAG immunoprecipitates were prepared from WDR59-deficient HEK293T cells that transiently expressed the indicated cDNAs and were analyzed as in (d). (h) The WDR59 CTD is necessary for the MIOS-WDR59 interaction. Anti-FLAG immunoprecipitates were prepared from WDR24-deficient HEK293T cells that transiently expressed the indicated cDNAs and were analyzed as in (d). (i) The MIOS CTD is necessary for the MIOS-WDR59 interaction. Anti-FLAG immunoprecipitates were prepared from WDR24-deficient HEK293T cells that transiently expressed the indicated cDNAs and were analyzed as in (d). Data in (d)-(i) are representative of two independent experiments. For gel source data, see Supplementary Figure 1.

Extended Data Fig. 6 GATOR2 CTDs are required for complex assembly and function.

(a) Size-exclusion chromatography profiles for wild-type GATOR2 (WT, black) or GATOR2 without individual CTDs: WDR24 CTD (WDR24 ∆CTD, green), WDR59 CTD (WDR59 ∆CTD, red), or MIOS CTD (MIOS ∆CTD, blue). The y-axis indicates normalized absorbance at 280 nm. Estimated molecular weights are indicated above each GATOR2 peak. (b) Elution fractions from size-exclusion chromatography of wild-type GATOR2 or GATOR2 without the MIOS CTD (MIOS ∆CTD), the WDR24 CTD (WDR24 ∆CTD), and the WDR59 CTD (WDR59 ∆CTD), corresponding to panel (a). The gel image of wild-type GATOR2 elution fractions was reproduced from Extended Data Fig. 1a. Lane labels indicate elution volume from TSKgel G4000SWxl column. (c), (d) The CTDs of WDR24 and WDR59 appear to be dispensable for the assembly of GATOR2 when analyzed by immunoprecipitation and immunoblotting. Anti-FLAG immunoprecipitates were prepared from WDR24-deficient (c) or WDR59-deficient (d) HEK293T cells that transiently expressed the indicated cDNAs and were analyzed by immunoblotting for the indicated proteins. (e) The MIOS CTD is required for MIOS to immunoprecipitate WDR24, WDR59 and SEC13. Anti-FLAG immunoprecipitates were prepared from MIOS-deficient HEK293T cells that transiently expressed the indicated cDNAs and were analyzed as in (c). (f), (g), and (h) The CTDs of MIOS (f), WDR24 (g), and WDR59 (h) are necessary for mTORC1 activation and for the GATOR2-GATOR1 and GATOR2-KICSTOR interactions. Anti-FLAG and anti-HA immunoprecipitates were prepared from MIOS-deficient (f), WDR24-deficient (g), or WDR59-deficient (h) HEK293T cells that transiently expressed the indicated cDNAs and were analyzed as in (c). HA, hemagglutinin. Data in (a)-(h) are representative of two independent experiments. For gel source data, see Supplementary Figure 1.

Extended Data Fig. 7 GATOR2 RING domains are incompatible with E2 binding.

Structural alignment between GATOR2 CTD-CTD junctions and the RNF4-UBCH5A complex (PDB ID: 4AP4)17. Individual CTD pairs are drawn (left) and superimposed with RNF4-UBCH5A (right) as follows: (a) WDR24-MIOS, (b) MIOS-WDR24, (c) MIOS-WDR59, and (d) WDR59-MIOS.

Extended Data Fig. 8 GATOR2 complex does not exhibit E3 ubiquitin ligase activity.

(a), (b) In vitro ubiquitination assay does not demonstrate GATOR2-dependent ubiquitin chain formation or modification of GATOR1. GATOR1 (500 nM) was incubated with a panel of E2 enzymes in the absence (a) or presence of GATOR2 (250 nM; b). Reactions were incubated at 37 °C for 1 h and analyzed by immunoblotting for the indicated proteins. (c) Coomassie-stained SDS-PAGE analysis of GATOR1 and GATOR2 complexes assayed in (a) and (b). (d) Ubiquitin chains formed in the presence of GATOR2 and UBE2D3 are not affected by GATOR2 inhibitors Sestrin2 or CASTOR1. UBE4B was used as a positive control for E3-dependent ubiquitin chain forming activity. Reactions were analyzed as in (a). (e) GATOR2 does not promote ubiquitin discharge from E2-Ub conjugates. Ubiquitin charged-UBE2D3 (UBE2D3-Ub) was incubated in the presence of 10 mM lysine and UBE4B, HOIP catalytic domain, or GATOR2 (100 nM each) at 22 °C for the indicated amounts of time. Samples were harvested in SDS-PAGE buffer without reducing agent, except where otherwise indicated, to preserve the E2-Ub thioester linkage. (f) Pharmacological inhibition of the ubiquitination cascade does not affect activation of mTORC1 by amino acids. HEK293T cells were treated with 0.5 µM TAK-243 for a total of 4 h. Cells were starved of amino acids for 60 min and restimulated with amino acids for 15 min prior to harvest. Samples were harvested in SDS-PAGE sample buffer without reducing agent to preserve the E2-Ub thioester linkage and analyzed by immunoblotting. (g) NPRL2 lysine residues are not required for activation and regulation of mTORC1 by amino acids. NPRL2-deficient cells stably expressing the indicated constructs were starved of amino acids for 60 min and restimulated with amino acids for 15 min prior to harvest and were analyzed by immunoblotting for the indicated proteins. HA, hemagglutinin. (h) NPRL3 lysine residues are not required for activation and regulation of mTORC1 by amino acids. NPRL3-deficient cells stably expressing the indicated constructs were starved of amino acids for 60 min and restimulated with amino acids for 15 min prior to harvest and were analyzed as in (g). Data in (a), (b), (d)-(h) are representative of two independent experiments. For gel source data, see Supplementary Figure 1.

Extended Data Fig. 9 WDR24-WDR59 α-solenoid junction is required for GATOR2 assembly and function.

(a) Two views of the WDR24-WDR59 α-solenoid junction. (b) Mutations in the WDR24 α-solenoid domain disrupt the WDR24-WDR59 interaction. Anti-FLAG immunoprecipitates were prepared from MIOS-deficient HEK293T cells that transiently expressed the indicated cDNAs and were analyzed by immunoblotting for the indicated proteins. HA, hemagglutinin. (c) The WDR24-WDR59 interaction is required for activation of mTORC1 and the GATOR2-GATOR1 and GATOR2-KICSTOR interactions. Anti-FLAG and anti-HA immunoprecipitates were prepared from WDR24-deficient HEK293T cells that transiently expressed the indicated cDNAs and were analyzed as in (b). (d) Mutation of the WDR59 solenoidal domain disrupts the WDR24-WDR59 interaction. Anti-FLAG immunoprecipitates were prepared from MIOS-deficient HEK293T cells that transiently expressed the indicated cDNAs and were analyzed as in (b). (e) The WDR24-WDR59 interaction is required for activation of mTORC1 and the GATOR2-GATOR1 and GATOR2-KICSTOR interactions. Anti-FLAG and anti-HA immunoprecipitates were prepared from WDR24-deficient HEK293T cells that transiently expressed the indicated cDNAs and were analyzed as in (b). Data in (b)–(e) are representative of two independent experiments. For gel source data, see Supplementary Figure 1.

Extended Data Fig. 10 MIOS-MIOS α-solenoid homodimerization is required for GATOR2 assembly and function.

(a) Structure of the MIOS-MIOS α-solenoid junction. The two interacting MIOS protomers are colored as either darker (MIOSA) or lighter blue (MIOSB). (b) Mutagenesis of the interface between the two MIOS α-solenoids (MIOS A560E) disrupts MIOS homodimerization. Anti-FLAG immunoprecipitates were prepared from WDR24-deficient HEK293T cells that transiently expressed the indicated cDNAs and were analyzed by immunoblotting for the indicated proteins. (c) Size-exclusion chromatography traces comparing the elution profile of GATOR2 containing wild-type MIOS with GATOR2 containing MIOS A560E. The trace for wild-type GATOR2 was reproduced from Extended Data Fig. 6a. (d) SDS-PAGE analysis of elution fractions from size-exclusion chromatography in (c). The gel image of wild-type GATOR2 elution fractions was reproduced from Extended Data Fig. 1a. Lane labels indicate elution volume from TSKgel G4000SWxl column. (e) Integrity of the MIOS α-solenoid junction is necessary for mTORC1 activation and the GATOR2-GATOR1 and GATOR2-KICSTOR interactions. Anti-FLAG and anti-HA immunoprecipitates were prepared from MIOS-deficient HEK293T cells that transiently expressed the indicated cDNAs and were analyzed as in (b). HA, hemagglutinin. Data in (b)–(e) are representative of two independent experiments. For gel source data, see Supplementary Figure 1.

Extended Data Fig. 11 Core GATOR2 proteins incorporate SEH1L and SEC13 by blade donation.

(a) Structure (left) and schematic (right) of the SEH1LWDR24 β-propeller completed by the donated WDR24 β-blade. (b) Structure (left) and schematic (right) of the SEH1LMIOS β-propeller completed by the donated MIOS β-blade. (c) Structure (left) and schematic (right) of the SEC13WDR59 β-propeller completed by the donated WDR59 β-blade. (d) Mutation of the donated WDR24 β-blade disrupts association of WDR24 with SEH1L. Anti-FLAG immunoprecipitates were prepared from MIOS-deficient HEK293T cells that transiently expressed the indicated cDNAs and were analyzed by immunoblotting for the indicated proteins. HA, hemagglutinin. (e) Mutation of the donated MIOS β-blade disrupts association of MIOS with SEH1L. Anti-FLAG immunoprecipitates were prepared from HEK293T cells that transiently expressed the indicated cDNAs and were analyzed as in (d). (f) Mutation of the donated WDR59 β-blade disrupts association of WDR59 with SEC13. Anti-FLAG immunoprecipitates were prepared from MIOS-deficient HEK293T cells that transiently expressed the indicated cDNAs and were analyzed as in (d). (g) Depletion of SEH1L or SEC13 destabilizes GATOR2 proteins and disrupts complex formation. SEH1L or SEC13 were depleted by CRISPR/Cas9 in a HEK293T clone expressing endogenously tagged 3xFLAG-WDR59. Anti-FLAG immunoprecipitates were prepared and analyzed as in (d). (h), (i) Depletion of SEH1L (h) or SEC13 (i) renders mTORC1 signaling resistant to amino acid availability. HEK293T cells of the indicated genotype were starved of amino acids for 60 min and restimulated with amino acids for 15 min prior to harvest and analysis by immunoblotting. Data in (d)–(i) are representative of two independent experiments. For gel source data, see Supplementary Figure 1.

Extended Data Fig. 12 Functional analysis of the core GATOR2 β-propellers.

(a) The β-propellers of MIOS, WDR59, and WDR24 are required for interactions between GATOR2 and CASTOR1, GATOR1 and KICSTOR, and Sestrin2, respectively. Anti-FLAG immunoprecipitates were prepared from MIOS-deficient, WDR59-deficient or WDR24-deficient HEK293T cells that transiently expressed the indicated cDNAs and were analyzed by immunoblotting for the indicated proteins. CASTOR1 D304A and Sestrin2 E451Q mutants do not bind arginine or leucine, respectively, and bind GATOR2 irrespective of amino acid availability. HA, hemagglutinin. (b) Schematic comparing WDR24 Nβ and SEH1L β-propeller organization in wild-type and β-single chain (βsc) constructs. (c) Schematic comparing WDR59 Nβ and SEC13 β-propeller organization in wild-type and β-single chain (βsc) constructs. The fused construct contains the intervening RWD domain. (d) The GATOR2 single chain β-propeller pairs of MIOS-SEH1L, WDR59-SEC13, and WDR24-SEH1L are sufficient to interact with CASTOR1, GATOR1 and KICSTOR, and Sestrin2, respectively. Anti-FLAG immunoprecipitates were prepared from MIOS-deficient HEK293T cells that transiently expressed the indicated cDNAs and were analyzed as in (a). (e) GATOR2 mediates the interaction between Sestrin2 and CASTOR1, indicating that they can co-occupy the same GATOR2 particle. Anti-FLAG immunoprecipitates were prepared from MIOS-deficient HEK293T cells that transiently expressed the indicated cDNAs and were analyzed as in (a). (f) Sestrin2 and CASTOR1, but not SAR1B, interact with GATOR2 and suppress mTORC1 signaling. Anti-FLAG and anti-HA immunoprecipitates were prepared from HEK293T cells that transiently expressed the indicated cDNAs and were analyzed as in (a). (g) GATOR2 interacts with Sestrin2 and CASTOR1, but not SAR1B. Anti-FLAG immunoprecipitates were prepared from HEK293T cells that transiently expressed the indicated cDNAs and were analyzed as in (a). (h) GATOR2 does not immunoprecipitate the Rag GTPases independent of GATOR1. Anti-FLAG immunoprecipitates were prepared from wild-type of DEPDC5-deficient HEK293T cells that transiently expressed the indicated cDNAs and were analyzed as in (a). (i) MIOS Nβ is not absolutely required for activation of mTORC1 or the GATOR2-GATOR1 and GATOR2-KICSTOR interactions. Anti-FLAG and anti-HA immunoprecipitates were prepared from MIOS-deficient HEK293T cells that transiently expressed the indicated cDNAs and were analyzed as in (a). (j) WDR59 Nβ is necessary for activation of mTORC1 and the GATOR2-GATOR1 and GATOR2-KICSTOR interactions. Anti-FLAG and anti-HA immunoprecipitates were prepared from WDR59-deficient HEK293T cells that transiently expressed the indicated cDNAs and were analyzed as in (a). Data in (a), (d)-(j) are representative of two independent experiments. For gel source data, see Supplementary Figure 1.

Supplementary information

Supplementary Information

This file includes Supplementary Figs. 1–9, including the uncropped blots, and Supplementary Table 1.

Reporting Summary

Video 1

| Structure of human GATOR2. The experimental cryo-EM map (left) and the corresponding molecular model (right) of the GATOR2 complex, with individual layers of the structure featured.

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Valenstein, M.L., Rogala, K.B., Lalgudi, P.V. et al. Structure of the nutrient-sensing hub GATOR2. Nature 607, 610–616 (2022). https://doi.org/10.1038/s41586-022-04939-z

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