Structural and Functional Characterization of the mammalian Target of Rapamycin Complex 2

Proteins are the fundamental units of life. They take part in any process within the cells and their regulation is essential to adapt to different environmental and intracellular conditions. Cells integrate a large variety of inputs and in turn need to rapidly respond and generate outputs to control key mechanisms such as metabolism, growth and proliferation. Multisubunit protein complexes have evolved to sense and integrate these stimuli. The atypical protein kinase mTOR (mammalian target of rapamycin) is the master regulator of cell growth and proliferation. The association with other proteins enables mTOR to sense intracellular inputs, integrate these signals and respond by phosphorylation of downstream proteins that control cell physiology. Dysregulated mTOR signaling is linked to cancer, and to metabolic and neurodegenerative diseases. mTOR functions in two structurally and functionally distinct signaling complexes, mTORC1 and mTORC2.
This thesis provides high-quality structural information on human mTORC2, containing the protein subunits mTOR, mLST8, Rictor and SIN1 determined by cryo-electron microscopy at 3.2 Å resolution.
The work resolves the enigmatic structure and interplay of the core mTORC2 subunits Rictor and SIN1. Contrary to previous hypotheses, it is the Rictor C-terminal domain that blocks the rapamycin binding site and causes rapamycin insensitivity of mTORC2. We demonstrate how intrinsically disordered parts of SIN1 integrate into Rictor and wrap around mLST8 to position mTORC2 substrates.
We rationalize modes of mTORC2 regulation via control of complex stability and visualize novel ligand binding sites for nucleotides in Rictor and for inositol hexakisphosphate in mTOR. In summary, the results presented in this thesis provide a completely new framework to analyze mTORC2 regulation and its function. These studies open the route for further analyzing interactions with signaling proteins and membranes and pave the way for the development of specific mTORC2 inhibitors.