Structural studies on the target of rapamycin complex 1

Proteins are the functional units executing the genetic program of living cells. Protein activity has to be modulated and adapted to environmental and intracellular conditions throughout the life cycle of every cell. In higher eukaryotes, complex multidomain proteins and multisubunit complexes have evolved to integrate large numbers of input signals to control key steps in metabolism, growth and proliferation. Structural studies of authentic eukaryotic multidomain proteins are required to understand their emergent properties resulting from interdomain and intersubunit crosstalk and conformational dynamics. However, due to problems in sample preparation and characterization, resolving the structures of large eukaryotic protein assemblies remains a considerable challenge. This thesis provides novel structural and mechanistic insights to three highly relevant eukaryotic protein systems, based on integrative multimethod approaches to tackle the inherent complexity of each case.
Mammalian target of rapamycin (mTOR) is the master regulator of growth and proliferation; it senses nutrient and growth signals and in response mediates the switch between anabolism and catabolism. Dysregulation of mTOR signaling is implicated in metabolic diseases and cancer, and mTOR is an established drug target. mTOR is comprised in two structurally and functionally distinct signaling complexes, mTORC1 and mTORC2. In Chapter 2, we determine the structure of the human mTORC1, containing the protein subunits mTOR, Raptor and mLST8, bound to FKBP12, by combining cryo-electron microscopy of the assembled complex at 5.9 Å resolution and crystallographic studies of the 149 kDa Raptor from Chaetomium thermophilum at 4.3 Å resolution. The core scaffold of the complex is formed by mTOR; the Raptor N-terminal conserved (RNC) domain is bound in vicinity to the mTOR catalytic site, suggesting a key role of the RNC in substrate recognition and delivery.
Polo-like kinase 4 (PLK4) is a central controller of centriole duplication. Chapter 3 identifies a mechanism for PLK4 regulation by the partner protein STIL by using biochemical mapping, kinase assays, super resolution microscopy, isothermal calorimetry in combination with structural studies of the interaction of the PLK4- polobox 3 (PB3) domain with a coiled-coil region of STIL (STIL-CC). NMR
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spectroscopy provides a solution structure of the isolated PLK4-PB3 and crystallographic structure determination reveals the mode of complex formation of PLK4-PB3 and STIL-CC. Mutations in STIL-CC abrogate the interaction to PB3 and diminish centriole duplication in cells, demonstrating the relevance of the PLK4-STIL interaction for centriole duplication.
Acetyl-CoA carboxylase (ACC) catalyzes the conversion of acetyl-CoA to malonyl- CoA, providing the building blocks for fatty acid synthesis. Eukaryotic ACCs are large multidomain proteins, that comprise a unique 120 kDa regulatory central domain (CD) besides the N- and C-terminal catalytic domains biotin carboxylase (BC) and carboxyl transferase (CT). In chapter 4 we determine the structure of the human and yeast CD and provide intermediate resolution crystal structures of up to nearly full-length ACC from Chaetomium thermophilum. In combination with functional assays, these data reveal the structural basis for phosphorylation-dependent control of yeast ACC activity.
In summary, the results presented in this thesis provide new structural and mechanistic insights into crucial eukaryote-specific regulatory properties of large multidomain proteins and protein complexes. These studies open important routes for further dissecting functional mechanisms by targeted biochemical and biophysical approaches. In particular for mTORC1, the current results provide a basis for analyzing the interactions with signaling partner proteins. Interdomain crosstalk and regulated protein conformational dynamics in these systems are closely linked to disease. Targeting interdomain interactions may serve as a relevant strategy for therapeutic intervention, e. g. in cancer therapy. The detailed depiction of intact assemblies of ACC and mTORC1 provides the structural groundworks for such approaches.