Hybrid Structure of the RagA/C-Ragulator mTORC1 Activation Complex

The lysosomal membrane is the locus for sensing cellular nutrient levels, which are transduced to mTORC1 via the Rag GTPases and the Ragulator complex. The crystal structure of the five-subunit human Ragulator at 1.4 Å resolution was determined. Lamtor1 wraps around the other four subunits to stabilize the assembly. The Lamtor2:Lamtor3 dimer stacks upon Lamtor4:Lamtor5 to create a platform for Rag binding. Hydrogen-deuterium exchange was used to map the Rag binding site to the outer face of the Lamtor2:Lamtor3 dimer and to the N-terminal intrinsically disordered region of Lamtor1. EM was used to reconstruct the assembly of the full-length RagAGTP:RagCGDP dimer bound to Ragulator at 16 Å resolution, revealing that the G-domains of the Rags project away from the Ragulator core. The combined structural model shows how Ragulator functions as a platform for the presentation of active Rags for mTORC1 recruitment, and might suggest an unconventional mechanism for Rag GEF activity. [Display omitted] •Crystal structure of V-shaped five subunit human Ragulator complex•Binding mode of RagA/C GTPase dimer by HDX-MS and EM•GTPase domains of RagA/C point away from and do not touch Ragulator•Ragulator affects conformation of GDP binding site in RagA without direct contact Su et al. report the crystal structure of Ragulator, a pentameric GEF for RagA, and the EM structure of its complex with the RagA/C dimer that activates mTORC1. HDX-MS shows that Ragulator modulates the RagA GTP binding site despite no contact with its GTPase domain.