Speaker: Roger Williams, MRC Laboratory of Molecular Biology, Cambridge, UK
Eukaryotic cells maintain a balance of anabolic and catabolic pathways that are tuned to meet the demands of changing environments. The eIF2alpha kinase GCN2 is activated by amino acid starvation, while the protein kinase mTORC1 is activated when amino acids are replete. These nutrition-sensing pathways are linked through their influence on autophagy and lysosomal sorting. The VPS34-containing complexes are key components of these sorting pathways. Our work on GCN2, mTORC1 and VPS34 complexes is aimed at understanding the structural mechanisms that regulate these processes in control of growth, pathogenesis and cancer. The mTORC1 and VPS34 complexes are regulated by small G proteins that assemble on membrane surfaces.
Heterodimeric Rag GTPases are key to activating the mTORC1 pathway in response to amino acids. We used X-ray crystallography to determine the structures of active forms of human Rag heterodimers and hydrogen/deuterium exchange mass spectrometry (HDX-MS) to map the mTORC1/Rag heterodimer interface and dynamics in solution. Our cryo-EM structure of a complex of mTORC1 with an active Rag heterodimer explains how oncogenic mutants activate the Rag heterodimer, how nucleotide binding is communicated between partners in the heterodimer, and why only one of the four states of the heterodimer activates mTORC1. Cryo-EM structures suggest that the primary role of Rag GTPase binding is to recruit mTORC1 to lysosomal membranes, in contrast to RHEB that activates the complex by an allosteric mechanism.
While mTORC1 is activated by amino acid abundance and promotes protein translation, nutrient stress activates GCN2, which initiates the Integrated Stress Response (ISR) and inhibits general translation. We have reconstituted this process in vitro, using purified components. This system shows that human GCN2 is potently stimulated by ribosomes. Using HDX-MS we showed that GCN2 recognises domain II of the uL10 subunit of the ribosomal P-stalk. The conserved 14 residue C-terminal tails (CTTs) of the P1 and P2 P-stalk proteins are also essential for GCN2 activation, and both the HisRS-like and kinase domains of GCN2 change conformation on binding the P-stalk complex. We propose that the P-stalk could link GCN2 activation to translational stress, leading to initiation of ISR.