ABSTRACT
Nutrients, such as amino acids and glucose, signal through the Rag GTPases to activate mTORC1. The GATOR1 protein complex-comprising DEPDC5, NPRL2 and NPRL3-regulates the Rag GTPases as a GTPase-activating protein (GAP) for RAGA; loss of GATOR1 desensitizes mTORC1 signalling to nutrient starvation. GATOR1 components have no sequence homology to other proteins, so the function of GATOR1 at the molecular level is currently unknown. Here we used cryo-electron microscopy to solve structures of GATOR1 and GATOR1-Rag GTPases complexes. GATOR1 adopts an extended architecture with a cavity in the middle; NPRL2 links DEPDC5 and NPRL3, and DEPDC5 contacts the Rag GTPase heterodimer. Biochemical analyses reveal that our GATOR1-Rag GTPases structure is inhibitory, and that at least two binding modes must exist between the Rag GTPases and GATOR1. Direct interaction of DEPDC5 with RAGA inhibits GATOR1-mediated stimulation of GTP hydrolysis by RAGA, whereas weaker interactions between the NPRL2-NPRL3 heterodimer and RAGA execute GAP activity. These data reveal the structure of a component of the nutrient-sensing mTORC1 pathway and a non-canonical interaction between a GAP and its substrate GTPase.
Subject(s)
Cryoelectron Microscopy , GTPase-Activating Proteins/metabolism , GTPase-Activating Proteins/ultrastructure , Monomeric GTP-Binding Proteins/metabolism , Monomeric GTP-Binding Proteins/ultrastructure , Multiprotein Complexes/metabolism , Multiprotein Complexes/ultrastructure , Amino Acids/deficiency , GTPase-Activating Proteins/antagonists & inhibitors , GTPase-Activating Proteins/chemistry , Guanosine Triphosphate/metabolism , Humans , Hydrolysis , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Mechanistic Target of Rapamycin Complex 1/metabolism , Models, Molecular , Monomeric GTP-Binding Proteins/chemistry , Multiprotein Complexes/antagonists & inhibitors , Multiprotein Complexes/chemistry , Protein Binding , Protein Domains , Protein Multimerization , Protein Subunits/chemistry , Protein Subunits/metabolism , Repressor Proteins/chemistry , Repressor Proteins/metabolism , Repressor Proteins/ultrastructure , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/metabolism , Tumor Suppressor Proteins/ultrastructureABSTRACT
mTOR complex I (mTORC1) is a central growth regulator that senses amino acids through a pathway that converges on the Rag GTPases, an obligate heterodimer of two related GTPases. Despite their central role in amino acid sensing, it is unknown why the Rag GTPases are heterodimeric and whether their subunits communicate with each other. Here, we find that the binding of guanosine triphosphate (GTP) to one subunit inhibits the binding and induces the hydrolysis of GTP by the other. This intersubunit communication pushes the Rag GTPases into either of two stable configurations, which represent active "on" or "off" states that interconvert via transient intermediates. Subunit coupling confers on the mTORC1 pathway its capacity to respond rapidly to the amino acid level. Thus, the dynamic response of mTORC1 requires intersubunit communication by the Rag GTPases, providing a rationale for why they exist as a dimer and revealing a distinct mode of control for a GTP-binding protein.
