Structural delineation of the mTOR pathway
Cell growth and proliferation are fundamental processes tightly regulated through complex networks of signalling pathways that respond to environmental cues. The serine/threonine kinase mechanistic target of rapamycin (mTOR) pathway is a crucial nutrient and energy-sensitive pathway that responds to the intracellular energy state. Through multi-subunit complexes, mTOR senses the amino acid and oxygen levels within the cell and integrates extracellular signals to control cell size and proliferation. In addition, it responds to growth factors to regulate cell survival and cytoskeletal organisation. Deregulation of the mTOR pathway is associated with human disorders like obesity, diabetes and cancer. Although several aspects of the pathway are clear, the lack of high-resolution structures prevents a complete understanding of complex protein assembly, function and regulation. In this context, the EU-funded MTOR_COMPLEXES (Structural and biophysical characterization of the human mTOR kinase and its signaling complexes) project set out to obtain structural information of mTORC1 and mTORC2 signalling complexes. To achieve this, researchers utilised a multi-gene Baculovirus expression system to produce the various components of mTORC. Considerable effort went into the optimisation of the expression and purification conditions, with scientists testing different affinity purification resins and size-exclusion chromatography methods. The purified mTORC1 was catalytically active and underwent biophysical characterisation by static light scattering. To proceed with precise structure determination, scientists employed an approach that combines cross-linking mass spectrometry with negative stain and cryo-electron microscopy. Results confirmed the dimer nature of mTORC1 and unveiled a head-to-tail arrangement of the two components. Overall, the MTOR_COMPLEXES study successfully set up a process for generating the fully assembled mTOR complexes. This is an important prerequisite for successful structure determination at higher resolution in the future. Considering the implication of the mTOR pathway in human disease, such structural information is essential for designing pharmaceutical strategies.
