Structural basis for the dynamic regulation of mTORC1 by amino acids

Abstract

The mechanistic target of rapamycin complex 1 (mTORC1) anchors a conserved signalling pathway that regulates growth in response to nutrient availability^1-5. 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.

Fig. 1. Structures of the sc-GATOR2, sc-GATOR2–Sestrin2 and sc-GATOR2–CASTOR1 complexes.

a, Domain organization of GATOR2 and sc-GATOR2 components. The grey trapezoids indicate β-blade donation by WDR24, MIOS or WDR59 to complete the β-propellers of SEH1L or SEC13. b, sc-GATOR2 interacts with GATOR1, KICSTOR and the nutrient sensors. Anti-Flag immunoprecipitates (IPs) were prepared from MIOS-deficient HEK293T cells transiently expressing the indicated cDNAs and were analysed by immunoblotting for the indicated proteins. Data are representative of two independent experiments. HA, haemagglutinin; KO, knockout. c, Size-exclusion chromatography profiles of sc-GATOR2, sc-GATOR2–Sestrin2 and sc-GATOR2–CASTOR1 complexes. d, Coomassie blue-stained SDS–PAGE analysis of sc-GATOR2, sc-GATOR2–Sestrin2 and sc-GATOR2–CASTOR1 complexes. e, Cryo-EM structures of the human apo sc-GATOR2 (centre), sc-GATOR2–CASTOR1 (left) and sc-GATOR2–Sestrin2 (right) complexes. Views of the experimental maps are shown. Sestrin2 and CASTOR1 bind to distinct, non-overlapping sites on sc-GATOR2. For gel source data, see Supplementary Fig. 1.

Fig. 2. Structure of the GATOR2–Sestrin2 complex and its regulation by leucine.

a, Overview of the GATOR2–Sestrin2 co-complex. The top and bottom views along the C2 symmetry axis are presented. Each GATOR2 particle can bind two copies of Sestrin2. b, The GATOR2–Sestrin2 interface. Sestrin2 primarily interacts with the WDR24 and SEH1L(WDR24) β-propeller but makes additional contacts with the MIOS CTD. c,d, Views of the interfaces between WDR24 and Sestrin2. e, View of the interface between Sestrin2 and SEH1L(WDR24). f, View of the interface between Sestrin2 and the MIOS CTD. g, Validation of the GATOR2–Sestrin2 interface. Anti-Flag IPs were prepared from WDR24-deficient HEK293T cells transiently expressing the indicated cDNAs and were analysed by immunoblotting for the indicated proteins. h, Validation of the GATOR2–Sestrin2 interface. Anti-HA IPs were prepared from HEK293T cells transiently expressing the indicated cDNAs and deprived of all amino acids for 60 min and were analysed as in g. i–k, Leucine triggers a conformational change that results in the release of Sestrin2 from GATOR2. Comparison of the structures of leucine-bound Sestrin2 (pink; 5DJ4 rebuilt) with leucine-free, GATOR2-bound Sestrin2 (magenta). i, The binding of leucine to Sestrin2 promotes the formation of helices αL1 and αL2 atop Sestrin2. j, The binding of leucine to Sestrin2 repositions the ‘lid’ to close the leucine-binding pocket and allow for the formation of helix αL1. k, The formation of the Sestrin2 helix αL2 repositions critical residues, including Arg404 and Arg338, that repel GATOR2. Data are representative of two independent experiments (g,h). For gel source data, see Supplementary Fig. 1.

Fig. 3. Structure of the GATOR2–CASTOR1 complex and its regulation by arginine.

a, Overview of the GATOR2–CASTOR1 co-complex. The top and bottom views along the C2 symmetry axis are presented. CASTOR1 binds to a dimeric interface formed by the MIOS brace region. Additional copies of CASTOR1 interact with the MIOS gloves via a single binding site but are probably not physiologically relevant. b, The GATOR2–CASTOR1 interface. Each protomer of CASTOR1 contacts one of the MIOS β-propellers that constitute the brace. c,d, Views of the interfaces between MIOS and CASTOR1. e, Validation of the GATOR2–CASTOR1 interface. Anti-Flag IPs were prepared from MIOS-deficient HEK293T cells transiently expressing the indicated cDNAs and analysed by immunoblotting for the indicated proteins. f, Validation of the GATOR2–CASTOR1 interface. Anti-HA IPs were prepared from HEK293T cells transiently expressing the indicated cDNAs and deprived of all amino acids for 60 min and analysed as in e. g–i, Arginine triggers a conformational change that results in the release of CASTOR1 from GATOR2. Comparison of the structures of arginine-bound CASTOR1 (light coral; 5I2C rebuilt) with arginine-free, GATOR2-bound CASTOR1 (salmon). g, The binding of arginine to CASTOR1 repositions helix α7 and reorients helix αL3. h, The binding of arginine to CASTOR1 reconfigures the loop enclosing the arginine-binding pocket and displaces helix α7 towards GATOR2 by one-half turn. i, Arginine binding, and translation of the CASTOR1 helix α7, reorients helix α3 and the ‘release loop’, which clashes with and displaces CASTOR1 from GATOR2. Data are representative of two independent experiments (e,f). For gel source data, see Supplementary Fig. 1.

Fig. 4. The nutrient sensors restrain the dynamic WDR24 β-propeller.

a, Association of either CASTOR1 or Sestrin2 stabilizes the dynamic WDR24 β-propellers and promotes their attachment to the GATOR2 scaffold. b, Quantification of the WDR24 β-propeller position in apo GATOR2, sc-GATOR2–CASTOR1 and sc-GATOR2–Sestrin2 particles used for structural determination. Both Sestrin2 and CASTOR1 rigidify the WDR24 β-propeller, but Sestrin2 does so more effectively. Data are mean and s.d. c, Epitope tagging of the WDR24 N-terminal β-propeller impairs mTORC1 activation in a tag size-dependent manner. WDR24-deficient cells transiently expressing the indicated cDNAs were starved of all amino acids for 60 min and restimulated with all amino acids for 15 min before collection. Anti-HA IPs were prepared and analysed by immunoblotting for the indicated proteins. Data are representative of two independent experiments. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 1. Functional validation of the sc-GATOR2 constructs.

(a) Wild-type WDR24 and sc-WDR24-SEH1L comparably activate mTORC1 signaling. WDR24-deficient HEK293T cells transiently expressing the indicated cDNAs were starved of all amino acids for 60 min and restimulated with all amino acids for 15 min before harvest. Anti-HA immunoprecipitates were prepared and analyzed by immunoblotting for the indicated proteins. (b) Wild-type MIOS and sc-MIOS-SEH1L comparably activate mTORC1 signaling. MIOS-deficient HEK293T cells transiently expressing the indicated cDNAs were starved of all amino acids for 60 min and restimulated with all amino acids for 15 min before harvest. Anti-HA immunoprecipitates were prepared and analyzed as in (a). (c) Wild-type WDR59 and sc-WDR59-SEC13 comparably activate mTORC1 signaling. WDR59-deficient HEK293T cells transiently expressing the indicated cDNAs were starved of all amino acids for 60 min and restimulated with all amino acids for 15 min before harvest. Anti-HA immunoprecipitates were prepared and analyzed as in (a). (d) Wild-type GATOR2 and sc-GATOR2 comparably activate mTORC1 signaling. MIOS-deficient HEK293T cells transiently expressing the indicated cDNAs were starved of all amino acids for 60 min and restimulated with all amino acids for 15 min before harvest. Anti-HA immunoprecipitates were prepared and analyzed as in (a). (e) Leucine comparably regulates the interaction between Sestrin2 and wild-type GATOR2 or sc-GATOR2. WDR24-deficient HEK293T cells transiently expressing the indicated cDNAs were starved of leucine for 60 min and restimulated with leucine for 15 min before harvest. Anti-HA immunoprecipitates were prepared and analyzed as in (a). (f) Arginine comparably regulates the interaction between CASTOR1 and wild-type GATOR2 or sc-GATOR2. MIOS-deficient HEK293T cells transiently expressing the indicated cDNAs were starved of arginine for 60 min and restimulated with arginine for 15 min before harvest. Anti-FLAG immunoprecipitates were prepared and analyzed as in (a). (g) Wild-type and sc-GATOR2 comparably activate mTORC1 signaling. MIOS-deficient HEK293T cells transiently expressing the indicated cDNAs were starved of all amino acids for 60 min and restimulated with all amino acids for 15 min before harvest. Anti-HA immunoprecipitates were prepared and analyzed as in (a). (h) Size-exclusion chromatography profiles of wild-type and sc-GATOR2 complexes. (i) Coomassie blue-stained SDS–PAGE analysis of wild-type and sc-GATOR2 complexes. (j) Comparison of the structures of wild-type GATOR2 (PDB ID: 7UHY) and apo sc-GATOR2 (this study). The MIOS brace region is highly flexible and its position within the sc-GATOR2 structure reflects one possible conformation. Data in (a)-(g) are representative of two independent experiments. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 2. Additional features resolved in sc-GATOR2.

(a) Density representation of apo sc-GATOR2, with region of interest labeled. (b) Density representation of the unsharpened composite map indicating the position of the resolved WDR24 extension (chain D, residues 610–630). (c) Cartoon representation of the apo sc-GATOR2 model illustrating the reassignment of chains I and J from wild-type GATOR2 (PDB ID: 7UHY), which we were previously unable to assign to a GATOR2 component, to WDR59 (chain D of apo sc-GATOR2). (d) Cartoon representation of the region containing the resolved linker within sc-WDR59-SEC13. (e) Representation of the density corresponding to the resolved sc-WDR59-SEC13 linker. (f) Comparison of the sc-WDR59-SEC13 linker region from wild-type GATOR2 (PDB ID: 7UHY) and apo sc-GATOR2.

Extended Data Fig. 3. Validation of the GATOR2-Sestrin2 interaction.

(a) Validation of the GATOR2-Sestrin2 interface. Anti-FLAG immunoprecipitates (IPs) were prepared from WDR24-deficient HEK293T cells transiently expressing the indicated cDNAs and were analyzed by immunoblotting for the indicated proteins. (b) The GATOR2-Sestrin2 interaction is required for overexpression of Sestrin2 to inhibit mTORC1 signaling. Anti-FLAG immunoprecipitates (IPs) were prepared from HEK293T cells transiently expressing the indicated cDNAs and were analyzed as in (a). (c, d) Validation of the (c) Sestrin1-GATOR2 and (d) Sestrin3-GATOR2 interactions. Anti-FLAG immunoprecipitates (IPs) were prepared from WDR24-deficient HEK293T cells transiently expressing the indicated cDNAs and were analyzed as in (a). (e) Identification of WDR24 mutants that interfere with the GATOR2-Sestrin2 interaction but not activation of mTORC1 by nutrients. WDR24-deficient HEK293T cells stably expressing the indicated cDNAs were starved of all amino acids for 60 min and restimulated with all amino acids for 15 min before harvest. Cell lysates were analyzed as in (a). (f) The GATOR2-Sestrin2 interaction is required for mTORC1 to sense leucine, but not arginine, deprivation. WDR24-deficient HEK293T cells stably expressing the indicated cDNAs were starved of either leucine or arginine for 60 min and restimulated with leucine or arginine for 15 min before harvest. Cell lysates were analyzed as in (a). Data in (a)-(f) are representative of two independent experiments. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 4. Leucine-induced allostery is required to disrupt the GATOR2-Sestrin2 interaction.

(a) Interactions involving the Sestrin2 αL1 and αL2 helices are required for leucine-induced disruption of the GATOR2-Sestrin2 complex. Anti-FLAG immunoprecipitates were prepared from HEK293T cells transiently expressing the indicated cDNAs and deprived of all amino acids for 60 min. Where indicated, leucine (300 μM) was added directly to the immunoprecipitates, which, after re-washing, were analyzed by immunoblotting for the indicated proteins. (b) Sestrin2 Arg338 and Arg404 are required for the leucine-induced disruption of the GATOR2-Sestrin2 complex. Anti-FLAG immunoprecipitates were prepared from HEK293T cells transiently expressing the indicated cDNAs and deprived of all amino acids for 60 min. Immunoprecipitates were treated with leucine and were analyzed as in (a). Data in (a) and (b) are representative of two independent experiments. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 5. Validation of the GATOR2-CASTOR1 interaction.

(a) Validation of the CASTOR2-GATOR2 interaction. Anti-FLAG immunoprecipitates (IPs) were prepared from MIOS-deficient HEK293T cells transiently expressing the indicated cDNAs and were analyzed by immunoblotting for the indicated proteins. (b) The GATOR2-CASTOR1 interaction is required for overexpression of CASTOR1 to inhibit mTORC1 signaling. Anti-FLAG immunoprecipitates (IPs) were prepared from HEK293T cells transiently expressing the indicated cDNAs and were analyzed as in (a). (c) The MIOS N-terminal β-propeller is not required for amino acids to activate mTORC1 signaling. Anti-FLAG immunoprecipitates (IPs) were prepared from HEK293T cells transiently expressing the indicated cDNAs and were analyzed as in (a). (d) The GATOR2-CASTOR1 interaction is required for arginine deprivation to inhibit mTORC1 signaling. MIOS-deficient HEK293T cells transiently expressing the indicated cDNAs were starved of arginine for 60 min and restimulated with arginine for 15 min before harvest. Anti-HA immunoprecipitates were prepared and analyzed as in (a). (e) The GATOR2-CASTOR1 interaction is necessary for arginine deprivation, but not for leucine deprivation, to inhibit mTORC1 signaling. MIOS-deficient HEK293T cells transiently expressing the indicated cDNAs were starved of either leucine or arginine for 60 min and restimulated with leucine or arginine for 15 min before harvest. Anti-HA immunoprecipitates were prepared and analyzed as in (a). Data in (a)-(e) are representative of two independent experiments. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 6. Arginine-induced allostery is required to disrupt the GATOR2-CASTOR1 interaction.

(a) Disruption of the CASTOR1 loop β6-α3 and α3 helix stabilizes the GATOR2-CASTOR1 interaction. Anti-HA immunoprecipitates (IPs) were prepared from HEK293T cells transiently expressing the indicated cDNAs and deprived of all amino acids for 60 min and were analyzed by immunoblotting for the indicated proteins. (b) The CASTOR1 loop β6-α3 and α3 helix are required for arginine-induced disruption of the GATOR2-CASTOR1 complex. Anti-FLAG immunoprecipitates were prepared from HEK293T cells transiently expressing the indicated cDNAs and deprived of all amino acids for 60 min. Where indicated, arginine (400 μM) was added directly to the immunoprecipitates, which, after re-washing, were analyzed as in (a). Data in (a) and (b) are representative of two independent experiments. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 7. Molecular dynamics of the GATOR2-CASTOR1 interaction.

(a) Accessibility of the CASTOR1 active site. In both states, arginine-bound (PDB ID: 5I2C, yellow) and arginine-free (blue) CASTOR1, the arginine-binding loop (residues 269 – 280), which is highlighted in darker colors, fully covers the CASTOR1 surface area restricting the accessibility of soluble arginine towards the CASTOR1 active site. (b) Left: Simulated dynamics of the arginine-binding loop (residues 269 – 280) in the absence of arginine. The general flexibility is measured in r.m.s.d compared to the energy equilibrated input state (step 7) over a simulation run time of 1000 ns. Right: Simulated dynamics of the arginine-binding loop (residues 269 – 280) in the arginine-bound state. The general flexibility is measured in r.m.s.d compared to the energy equilibrated input state (step 7) over a simulation run time of 1000 ns. (c) Transient coordination states during the arginine unbinding reaction. Left: arginine binding mode obtained by crystallography (PDB ID: 5I2C). Middle and right: Examples for transient arginine coordination states with highlighted critical side chain resides during the simulated arginine-unbinding reaction. (d) Key interactions of the shown transient states determined by LigPlot. Hydrogen bonds are shown as green dotted lines, while the spoked arcs represent residues making nonbonded contacts with the arginine. (e) Protein surface exposition of the transient arginine coordination states. (f) Disruption of the CASTOR1 residues involved in transient arginine binding stabilizes the GATOR2-CASTOR1 interaction. Anti-HA immunoprecipitates (IPs) were prepared from HEK293T cells transiently expressing the indicated cDNAs and deprived of all amino acids for 60 min and analyzed by immunoblotting for the indicated proteins. (g) CASTOR1 residues involved in transient arginine binding are required for arginine-induced disruption of the GATOR2-CASTOR1 complex. Anti-FLAG immunoprecipitates were prepared from HEK293T cells transiently expressing the indicated cDNAs and deprived of all amino acids for 60 min. Where indicated, arginine (400 μM) was added directly to the immunoprecipitates, which, after re-washing, were analyzed as in (f). Data in (f) and (g) are representative of two independent experiments. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 8. CASTOR1 weakens the GATOR2-GATOR1 interaction.

(a) Association of CASTOR1 to GATOR2 disrupts the WDR59 zinc finger (ZnF). Comparison of the WDR59 C-terminal domain (CTD) from apo sc-GATOR2 and sc-GATOR2-CASTOR1. (b) Association of CASTOR1 to GATOR2 alters the shape of the GATOR2 scaffold. Comparison of the GATOR2 scaffold size and shape in apo sc-GATOR2 and sc-GATOR2-CASTOR1. (c) The WDR59 ZnF is required for full activation of mTORC1 signaling and GATOR2-GATOR1-KICSTOR assembly. Anti-FLAG and anti-HA immunoprecipitates were prepared from WDR59-deficient HEK293T cells transiently expressing the indicated cDNAs and analyzed by immunoblotting for the indicated proteins. (d) CASTOR1 selectively associates with GATOR2 and not the entire GATOR supercomplex. Anti-FLAG immunoprecipitates were prepared from HEK293T cells transiently expressing the indicated cDNAs and analyzed as in (c). Data in (c) and (d) are representative of two independent experiments. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 9. Quantification of the WDR24 β-propeller dynamics.

(a) Flowchart depicting the workflow to quantitatively determine the WDR24 β-propeller occupancy between the apo GATOR2 state and the inhibitor-bound sc-GATOR2-Sestrin2 and sc-GATOR2-CASTOR1 complexes. The solvent masks applied for particle subset selection in 3D classification are shown in green and applied focus masks are shown in blue. The shown particle subset maps are representative examples of the respective classification. (b) Epitope tagging of the WDR24 N-terminal β-propeller does not disrupt interactions between GATOR2-GATOR1-KICSTOR. Anti-MYC immunoprecipitates from WDR24-deficient HEK293T cells transiently expressing the indicated cDNAs were prepared and analyzed by immunoblotting for the indicated proteins. Data in (b) are representative of two independent experiments. For gel source data, see Supplementary Fig. 1.