Architecture of the human GATOR1 and GATOR1–Rag GTPases complexes

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. Cryo-electron microscopy and biochemical analyses of the GATOR1 protein complex reveal that two binding modes underpin its ability to regulate Rag GTPases as a GTPase-activating protein for RAGA. Restraining the RAGA GAP mTORC1 is a master growth regulator that senses nutrient signals such as amino acids and glucose to coordinate many cellular processes. mTORC1 is regulated by the Rag GTPases, which are in turn regulated by several protein complexes including GATOR1, which serves as a GTPase-activating protein (GAP) for RAGA. Here, David Sabatini and colleagues use cryo-electron microscropy to determine the structures of GATOR1 and of GATOR1 bound to the Rag GTPases, and perform biochemical experiments to investigate GAP function. They find that GATOR1 adopts an extended architecture with the NPRL2 subunit linking the two other subunits DEPDC5 and NPRL3. Unexpectedly, the GATOR1–Rag GTPases structure represents an inhibitory state, where an interaction of DEPDC5 with RAGA inhibits GATOR1 GAP activity. Instead, a weaker interaction of the NPRL2–NPRL3 heterodimer with RAGA executes GAP function. Such an inhibitory mechanism has not been seen before for other GAPs.