Structural characterization of the protein kinase "target of rapamycin" and its interactions with regulators and substrates

• Applicant: Privatdozentin Dr. Sonja Alexandra Dames
• Subject Area: Structural Biology
• Term: from 2009 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 156863793

The "target of rapamycin" (TOR) is a protein kinase that regulates cellular growth by intercepting different signalling cascades. A stringent control of cell growth is mandatory for cells, organs, and organisms to achieve a characteristic size. Misregulation of cell growth can result in pathological changes of cell, organ, and organism size and in metabolic disorders, cancer and neurodegenerative diseases such Alzheimer's.TOR is a modular, about 280 kDa big protein. Besides a catalytic domain, TOR has additional functional regions that mediate interactions with other cellular components. The long-term goal of this project is the structural characterization of all domains of TOR and their interactions with regulators and substrates. An important issue within this project is the structural characterization of TOR-lipid and -protein interactions that account for the localization of TOR at different cellular membranes (ER, Golgi, mitochondria, lysosomes, plasma membrane) and in the nucleus. The exact localization of TOR determines which specific cellular processes are regulated. In the past time we characterized the membrane-binding properties of the redox-sensitive, C-terminal FATC domain and published already part of the data. The FKBP12-rapamycin binding (FRB) domain was suggested to mediate the regulation of TOR by the lipid phosphatidic acid (PA). Regarding this, we could show that the FRB domain undergoes strong conformational changes in the presence of membrane-mimetics with and without the regulator PA. To better understand the role of the FRB domain as conditional membrane anchor, its structure in the presence of membrane-mimetics shall be determined. In addition, we established purification protocols for several binding partners (Rheb, LST8, FKBP38) and the respective binding regions on TOR as well as expression systems for various constructs for the structural characterization of the kinase domain, interdomain contacts and interactions with further known complex partners and regulators. All of this work is the foundation for the proposed continuation of the project.

• DFG Programme: Research Grants