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. 2019 Oct 25;366(6464):468-475.
doi: 10.1126/science.aay0166. Epub 2019 Oct 10.

Structural Basis for the Docking of mTORC1 on the Lysosomal Surface

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Free PMC article

Structural Basis for the Docking of mTORC1 on the Lysosomal Surface

Kacper B Rogala et al. Science. .
Free PMC article

Abstract

The mTORC1 (mechanistic target of rapamycin complex 1) protein kinase regulates growth in response to nutrients and growth factors. Nutrients promote its translocation to the lysosomal surface, where its Raptor subunit interacts with the Rag guanosine triphosphatase (GTPase)-Ragulator complex. Nutrients switch the heterodimeric Rag GTPases among four different nucleotide-binding states, only one of which (RagA/B•GTP-RagC/D•GDP) permits mTORC1 association. We used cryo-electron microscopy to determine the structure of the supercomplex of Raptor with Rag-Ragulator at a resolution of 3.2 angstroms. Our findings indicate that the Raptor α-solenoid directly detects the nucleotide state of RagA while the Raptor "claw" threads between the GTPase domains to detect that of RagC. Mutations that disrupted Rag-Raptor binding inhibited mTORC1 lysosomal localization and signaling. By comparison with a structure of mTORC1 bound to its activator Rheb, we developed a model of active mTORC1 docked on the lysosome.

Conflict of interest statement

Competing interests: The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Purification, assembly, and structure determination of the Raptor-Rag-Ragulator supercomplex.
(A) Gel filtration profile and corresponding SDS–polyacrylamide gel electrophoresis of the reconstituted Raptor-Rag-Ragulator supercomplex as visualized with Coomassie Blue staining. The fully assembled complex (peak 1) partially overlaps with two subcomplexes: Rag-Ragulator (peak 1 tail) and Ragulator (peak 2). (B) Representative two-dimensional class averages of the Raptor-Rag-Ragulator supercomplex. (C) Cryo-EM structure of the supercomplex, determined to 3.2 Å resolution. Two orthogonal views of the experimental electron density (left) are shown next to corresponding views of the molecular model (right). (D) Domain organization of all components that make up the supercomplex.
Figure 2.
Figure 2.. Raptor-RagA interaction.
(A) Three helices (α24, α26, α29) in the HEAT-repeat domain of Raptor directly engage the switch-I face of GTP-loaded RagA. (B) Description of mutations introduced in the RagA-binding helices of Raptor. (C) Mutations in these helices render Raptor expressed via cDNA transient transfection unable to co-immunoprecipitate the endogenous RagA-RagC heterodimer. (D) Description of mutations introduced in the Raptor-binding region of RagA. (E) In RagA-RagB DKO HEK-293T cells, the transiently expressed RagA mutants in (D) cannot bind endogenous Raptor but have an intact capacity to bind to RagC and Ragulator, as assessed by its p18 subunit. Flag-metap2 was used as a control protein.
Figure 3.
Figure 3.. Raptor-RagC interaction.
(A) The binding interface between the Raptor α-solenoid and Rag GTPases. (B) The GTPase switch machineries of Rags are positioned at the opposite ends of the heterodimer. Note that in our structure, the switch machinery of RagC is largely disordered, and the circled region marks the space it would have occupied if it was fully resolved. (C) The Raptor claw is a 22–amino acid loop that enters the inter-Rag space depending on the nucleotide state of RagC. The claw forms interactions with both RagA and RagC, and its residue E935 engages RagC-loaded GDP directly. (D) The E935-GDP interaction is driven by hydrogen bonding between the E935 side chain and N2 and N3 of the guanine of GDP.
Figure 4.
Figure 4.. The dynamics of the Rag-Raptor interaction.
(A) Loading of GTP by RagC would trigger its switch I to rigidify and clash with the Raptor claw. The structure of the GTP-loaded GTPase domain of RagC (PDB ID 3LLU) was superimposed with our cryo-EM structure of RagC•GDP. (B) The organization of the switches in RagC (top) and Arf6 (bottom) [PDB ID: 1E0S, 2J5X (37, 38)]. Note that although the switches change their positions in GTP- versus GDP-loaded states, the core of the structure does not move. Even though a large proportion of the switches in our GDP-loaded RagC structure are disordered, we observed that the interswitch changes its register by two residues, in a manner similar to Arf GTPases. (C) The movement of the interswitch during the GDP-to-GTP transition would cause its loop to clash with the CRD pocket (circled area). Instead, the interswitch repositions itself such that it engages with the more central part of the CRD. The disordered interswitch strand β2 of RagC•GDP is drawn with a dashed line. The surface of the CRD is colored according to electrostatic potential (see the color key). (D) The shifting of the GTPase domains in the Rag heterodimer during GTP-GDP binding exchanges. GTPase domains loaded with GDP are positioned away from the central axis of the Rag heterodimer, and their interswitches are retracted. Loading of GTP causes the interswitch to extend and press the CRD pocket such that the entire GTPase domain becomes repositioned closer to the Rag central axis. The models were created by superimposing RagA•GTP with RagC•GDP through their CRDs (specifically by matching their β7 and α9). (E) Description of the Raptor claw mutants used in (F). (F) Elimination of the Raptor claw or mutations in its critical Rag-interacting regions prevent Raptor from coimmunoprecipitating the RagA-RagC heterodimer. Longer exposures of the RagA and RagC immunoblots did not reveal any signal for Raptor. Flag-metap2 was used as a negative control protein.
Figure 5.
Figure 5.. mTORC1 on the lysosomal surface.
(A) Model of a Rheb-activated mTORC1 dimer bound to two lysosomal-targeting Rag-Ragulator complexes. The Rag-Ragulator complex clamps mTORC1 to the surface of the lysosome. Note that both Ragulator and Rheb carry lipid-modified terminal residues that tether the supercomplex to the membrane. Our cryo-EM structure of the Raptor-Rag-Ragulator complex was superimposed with that of Rheb-bound mTORC1 [PDB ID 6BCU (31)]. (B) Stable expression in RagA-RagB DKO HEK-293T cells of RagA mutants that cannot bind Raptor (Fig. 2, D and E) does not restore mTORC1 activity, as assayed by the phosphorylation of S6K1 in amino acid–replete cells. Note that although the levels of the stably expressed wild-type and mutant Flag-RagA are relatively even as assessed with the anti-Flag antibody, the recognition of the RA2 and RA3 RagA mutants by the RagA antibody (monoclonal; CST D8B5) is reduced, which suggests that the mutated residues affect the epitope recognized by this antibody, which has not been disclosed. (C) Unlike expression of wild-type RagA, expression in RagA-RagB DKO HEK-293T cells of the Y31A RagA (RA1) mutant that cannot bind Raptor does not rescue the amino acid–induced colocalization of mTOR with lysosomes, as marked by the lysosomal protein LAMP2. (D) In cells hypomorphic for Raptor, transient expression of wild-type Raptor promotes S6K1 phosphorylation, whereas that of Raptor mutants defective in the RagA-Raptor interaction (αx3 and α26) does not. Mutants that cannot bind the CRD of RagC (CRD1) or lacking the claw (Clw1) activate S6K1 phosphorylation to a small degree. See supplementary materials and fig. S6B for a description of the Raptor hypomorph cell line.

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