Abstract
Cellular growth and proliferation are primarily dictated by the mechanistic target of rapamycin complex 1 (mTORC1), which balances nutrient availability against the cell’s anabolic needs. Central to the activity of mTORC1 is the RagA–RagC GTPase heterodimer, which under favorable conditions recruits the complex to the lysosomal surface to promote its activity. The RagA–RagC heterodimer has a unique architecture in that both subunits are active GTPases. To promote mTORC1 activity, the RagA subunit is loaded with GTP and the RagC subunit is loaded with GDP, while the opposite nucleotide-loading configuration inhibits this signaling pathway. Despite its unique molecular architecture, how the Rag GTPase heterodimer maintains the oppositely loaded nucleotide state remains elusive. Here, we applied structure–function analysis approach to the crystal structures of the Rag GTPase heterodimer and identified a key hydrogen bond that stabilizes the GDP-loaded state of the Rag GTPases. This hydrogen bond is mediated by the backbone carbonyl of Asn30 in the nucleotide-binding domain of RagA or Lys84 of RagC and the hydroxyl group on the side chain of Thr210 in the C-terminal roadblock domain of RagA or Ser266 of RagC, respectively. Eliminating this interdomain hydrogen bond abolishes the ability of the Rag GTPase to maintain its functional state, resulting in a distorted response to amino acid signals. Our results reveal that this long-distance interdomain interaction within the Rag GTPase is required for the maintenance and regulation of the mTORC1 nutrient-sensing pathway.
Highlights
Cellular growth and proliferation are primarily dictated by the mechanistic target of rapamycin complex 1, which balances nutrient availability against the cell’s anabolic needs
The mechanistic target of rapamycin complex 1 pathway is responsible for monitoring the availability of nutrients and growth factors and determining if sufficient materials are available to proceed with cellular growth (1, 2)
When cellular amino acid concentrations are high, the RagA subunit is loaded with GTP and the RagC subunit is loaded with GDP, which recruits mechanistic target of rapamycin complex 1 (mTORC1) to the lysosomal surface (3), where its kinase activity is stimulated by another small GTPase, Rheb, which is only active when growth factors are abundant (5, 6)
Summary
To identify structural elements that maintain the nucleotideloading states of the Rag GTPases, we compared the crystal structures of the Rag homolog in yeast, Gtr1p–Gtr2p, at different nucleotide-loading states (9, 10), as these two highresolution structures presented the clearest atomic details of the dramatic conformational changes of the Switch I region upon binding to GTP versus GDP (Fig. 1A, upper panel). The binding can be differentiated by SDS-PAGE as Rag subunits have distinct molecular weights and migrate at different positions (Fig. 2B) Using this approach, we measured the binding affinity of GTP and GDP to the mutants. Because the interdomain hydrogen bond we identified above can only form when a Rag subunit is loaded with GDP, we considered whether it might participate in maintaining the oppositely nucleotide-loaded state. To test this hypothesis, we carried out GTP hydrolysis experiments as detailed below. For WT Rag GTPases, kcat in a multiple-turnover setup is 15fold higher than that in a single-turnover setup, suggesting when both subunits bind GTP, the Rag heterodimer tends to
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