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[Preprint]. 2025 May 13:rs.3.rs-5073364.
doi: 10.21203/rs.3.rs-5073364/v1.

Structural basis for mTORC1 regulation by the CASTOR1-GATOR2 complex

Rachel M Jansen  1   2 , Clément Maghe  1   2 , Karla Tapia  1 , Selina Wu  1 , Serim Yang  1   2 , Xuefeng Ren  1   2 , Roberto Zoncu  1   2 , James H Hurley  1   2   3
Affiliations

Structural basis for mTORC1 regulation by the CASTOR1-GATOR2 complex

Rachel M Jansen et al. Res Sq. .

Update in

Abstract

Mechanistic target of rapamycin complex 1 (mTORC1) is a nutrient-responsive master regulator of metabolism. Amino acids control the recruitment and activation of mTORC1 at the lysosome via the nucleotide loading state of the heterodimeric Rag GTPases. Under low nutrients, including arginine (Arg), the GTPase activating protein (GAP) complex, GATOR1, promotes GTP hydrolysis on RagA/B, inactivating mTORC1. GATOR1 is regulated by the cage-like GATOR2 complex and cytosolic amino acid sensors. To understand how the Arg-sensor CASTOR1 binds to GATOR2 to disinhibit GATOR1 under low cytosolic Arg, we determined the cryo-EM structure of GATOR2 bound to CASTOR1 in the absence of Arg. Two MIOS WD40 domain β-propellers of the GATOR2 cage engage with both subunits of a single CASTOR1 homodimer. Each propeller binds to a negatively charged MIOS-binding interface on CASTOR1 that is distal to the Arg pocket. The structure shows how Arg-triggered loop ordering in CASTOR1 blocks the MIOS-binding interface, switches off its binding to GATOR2, and so communicates to downstream mTORC1 activation.

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Conflict of interest statement

Competing Interests: J.H.H. is a co-founder and shareholder of Casma Therapeutics, recieves research funding from Hoffmann-La Roche, and has consulted for Corsalex. R.Z. is a cofounder and shareholder of Frontier Medicines, science advisory board member for Nine Square Therapeutics and receives research funding from Genentech.

Figures

Extended Fig.1:
Extended Fig.1:. Purification for GATOR2 and CASTOR1.
(a) Chromatogram and gel for GATOR2 purification. (b) Chromatogram and gel for CASTOR1 purification. (c) Chromatogram and gel for Sestrin2 purification. (d) Chromatogram and gel for GATOR1 purification.
Extended Fig.2:
Extended Fig.2:. Data Processing Pipeline for GATOR2-CASTOR1 complex.
(a) Representative micrograph (b) Representative 2D classes (c) Data processing workflow (d) Overall map for GATOR2-CASTOR1 (e) FSC graph (f) Orientation plot.
Extended Fig. 3:
Extended Fig. 3:. Local Refinement for GATOR2-CASTOR1.
(a-g) Local refinement for different sections of complex. Including mask (shown in cyan), FSC graph and resulting map.
Extended Fig. 4:
Extended Fig. 4:. Local resolution estimation.
(a) Full complex map (b) CASTOR1-MIOS interface and (c) additional local refinement maps for the complex.
Extended Fig. 5:
Extended Fig. 5:. Map to model fit.
(a) CASTOR1 at CASTOR1-MIOS interface, (b) MIOS at CASTOR1-MIOS interface (c) CASTOR1 residues near arginine binding pocket.
Extended Fig. 6:
Extended Fig. 6:. GATOR2 Cage Symmetry.
Comparison of cage symmetry for (a) GATOR2 unbound and (b) GATOR2-CASTOR1 complex. For each complex the individual monomers are reflected over the symmetry axis. Regions distal to the alignments region are enlarged for visualization.
Extended Fig. 7:
Extended Fig. 7:. GATOR2 WDR59-MIOS CTD-CTD Junctions.
(a) Comparison of the WDR59-MIOS junctions (black dash circle) on GATOR2-CASTOR1 complex and GATOR2 unbound. (b) Close up view of the changes to the WDR59-MIOS CTD junctions. GATOR2 unbound (grey) overlayed with the GATOR2-CASTOR1 (blue and red).
Extended Fig. 8.
Extended Fig. 8.. Data Processing Pipeline for GATOR2-CASTOR1-Sestrin2.
(a) Representative micrograph (b) Representative 2D classes (c) Data processing workflow (d) Overall map for GATOR2-CASTOR1, FSC graph and orientation plot. (e) Data processing for GATOR1 and representative 2D classes of isolated GATOR1 complex particles.
Extended Fig. 9:
Extended Fig. 9:. GATOR2-CASTOR1- Sestrin2 interaction.
(a) GATOR2-CASTOR1 structure docked into cryo-EM map of GATOR2-CASTOR1-Sestrin2. (b) Close up of GATOR2-CASTOR1-Sestrin2 cryo-EM density fitted with Sestrin2-WDR24-SEH1L-SEH1L-MIOS AlphaFold model (c) Full Sestrin2-WDR24-SEH1L-SEH1L-MIOS AlphaFold model (ipTM = 0.69). Close up of interface between WDR24 (green) and Sestrin2 (orange) in AlphaFold model in (d) ribbon view and (e) surface view colored by electrostatic potential. pLDDT for Arg 228 is 0.89. (f) HEK-293T cells transiently expressing HA-tagged SESTRIN2 along with the indicated FLAG-tagged WDR24 constructs or FLAG-tagged METAP2 as a control were starved of leucine for 50 minutes. Where indicated, leucine was added to the lysates during immunoprecipitation. FLAG-immunoprecipitates were generated and analyzed by immunoblotting for the indicated proteins.
Extended Fig. 10:
Extended Fig. 10:. qPCR confirmation shCASTOR1.
qPCR against CASTOR1 performed in HEK293T transduced with a shRNA targeting Luciferase (shLUC) or a shRNA targeting CASTOR1. Data were normalized using ACTB and HPRT1 as housekeeping genes.
Fig. 1:
Fig. 1:. Cryo-EM structure of GATOR2-CASTOR1 complex.
(a) Domain organization of subunits within the GATOR2-CASTOR1 structure. Composite map and reconstructed model for the GATOR2-CASTOR1 complex viewing from the (b) front face (c) side view (d) back face. Focused maps for different portions of the complex were combined to generate a composite map containing the highest resolution information for each subunit.
Fig. 2:
Fig. 2:. CASTOR1 triggers a structural rearrangement in GATOR2 complex.
Comparison of the front and back faces of the (a) GATOR2-CASTOR1 complex and (b) GATOR2apo complex. CASTOR1 is removed for visualization in the GATOR2-CASTOR1 complex. Changes in the MIOS subunits are highlighted in boxes below complex. Key junctions connecting the inner cage are indicated.
Fig. 3:
Fig. 3:. CASTOR1 interacts with MIOS through negatively charged pocket.
(a) Overview of GATOR2-CASTOR1 complex. Front face MIOS subunits (blue) interact with CASTOR1 (yellow). (b) Close up view of CASTOR1 interaction with MIOS β-propellers. (c) Blade diagram for a front face MIOS β-propeller highlighting CASTOR1 interacting loops. Close up of the CASTOR1-MIOS interaction shown with (d) CASTOR1 surface view and MIOS ribbon view (e) CASTOR1 surface colored based on electrostatic potential (f) Ribbon view highlighting specific residues in MIOS loops residues 110–114 and 134–140 (blue) interacting with CASTOR1 residues (yellow). (g) HEK-293T cells transiently expressing the indicated FLAG-tagged WT and MIOS binding interface (MBI)-mutant CASTOR1 constructs, or FLAG-tagged METAP2 as a control, were starved of arginine for 50 minutes and, where indicated, restimulated for 10 minutes. FLAG-immunoprecipitates were generated and analyzed by immunoblotting for the indicated proteins. (h) HEK-293T cells transiently expressing CASTOR1-HA and either FLAG-tagged WT MIOS, FLAG-tagged MBI-mutant MIOS constructs or FLAG-tagged METAP2 as a control. Cells were starved of arginine for 50 minutes and, where indicated, restimulated for 10 minutes. HA-immunoprecipitates were generated and analyzed by immunoblotting for the indicated proteins. (i) CASTOR1 knockdown HEK-293T cells transiently expressing the indicated FLAG-tagged WT and MBI-mutant CASTOR1 constructs, or FLAG-tagged METAP2 as a control, were starved of arginine for 50 minutes and, where indicated, restimulated for 10 minutes. Anti-HA-immunoprecipitates were prepared and analyzed by immunoblotting for the indicated proteins and phospho-proteins.
Fig. 4:
Fig. 4:. CASTOR1 interaction with arginine triggers closing of GATOR2-interacting pocket.
(a) Diagram of CASTOR1 interaction with MIOS β-propellers and location of arginine pocket and MIOS binding interface. (b) Electrostatic surface cartoon of CASTOR1apo and close up of GATOR2-interact pocket. Key residues in CASTOR1 that form pocket are indicated. (c) Electrostatic surface cartoon of CASTOR1Arg and close up of GATOR2-interact pocket. Key residues in CASTOR1 that block pocket are indicated. (d) HEK-293T cells transiently expressing the indicated FLAG-tagged WT and MIOS releasing loop (MRL)-mutant CASTOR1 constructs, or FLAG-tagged METAP2 as a control, were starved of arginine for 50 minutes and, where indicated, restimulated for 10 minutes. FLAG-immunoprecipitates were generated and analyzed by immunoblotting for the indicated proteins. (e) CASTOR1 knockdown HEK-293T cells transiently expressing the indicated FLAG-tagged WT and MRL-mutant CASTOR1 constructs, or FLAG-tagged METAP2 as a control, were starved of arginine for 50 minutes and, where indicated, restimulated for 10 minutes. Anti-HA-immunoprecipitates were prepared and analyzed by immunoblotting for the indicated proteins and phospho-proteins. (f) Overlay of CASTOR1apo (yellow) and CASTOR1Arg (cyan). Rotation in ACT2 and ACT4 α-helices enlarged for visualization. (g) Surface view of CASTOR1apo and CASTOR1Arg arginine binding pocket. CASTOR1apo is modelled with arginine in binding pocket. (h) Ribbon view of arginine binding pocket in CASTOR1apo and CASTOR1Arg. (i) Overall model for arginine-dependent CASTOR1 interaction with GATOR2.
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