Target of rapamycin (TOR), a conserved protein kinase and central controller of cell growth, functions in two structurally and functionally distinct complexes: TORC1 and TORC2. Dysregulation of mammalian TOR (mTOR) signaling is implicated in pathologies that include diabetes, cancer, and neurodegeneration. We resolved the architecture of human mTORC1 (mTOR with subunits Raptor and mLST8) bound to FK506 binding protein (FKBP)-rapamycin, by combining cryo-electron microscopy at 5.9 angstrom resolution with crystallographic studies of Chaetomium thermophilum Raptor at 4.3 angstrom resolution. The structure explains how FKBP-rapamycin and architectural elements of mTORC1 limit access to the recessed active site. Consistent with a role in substrate recognition and delivery, the conserved amino-terminal domain of Raptor is juxtaposed to the kinase active site.

Polo-like kinases (PLK) are eukaryotic regulators of cell cycle progression, mitosis and cytokinesis; PLK4 is a master regulator of centriole duplication. Here, we demonstrate that the SCL/TAL1 interrupting locus (STIL) protein interacts via its coiled-coil region (STIL-CC) with PLK4 in vivo. STIL-CC is the first identified interaction partner of Polo-box 3 (PB3) of PLK4 and also uses a secondary interaction site in the PLK4 L1 region. Structure determination of free PLK4-PB3 and its STIL-CC complex via NMR and crystallography reveals a novel mode of Polo-box–peptide interaction mimicking coiled-coil formation. In vivo analysis of structure-guided STIL mutants reveals distinct binding modes to PLK4-PB3 and L1, as well as interplay of STIL oligomerization with PLK4 binding. We suggest that the STIL-CC/PLK4 interaction mediates PLK4 activation as well as stabilization of centriolar PLK4 and plays a key role in centriole duplication.DOI: http://dx.doi.org/10.7554/eLife.07888.001

The protein kinase mammalian target of rapamycin (mTOR) is the central regulator of cell growth. Aberrant mTOR signaling is linked to cancer, diabetes, and neurological disorders. mTOR exerts its functions in two distinct multiprotein complexes, mTORC1 and mTORC2. Here, we report a 3.2-Å resolution cryo-EM reconstruction of mTORC2. It reveals entangled folds of the defining Rictor and the substrate-binding SIN1 subunits, identifies the carboxyl-terminal domain of Rictor as the source of the rapamycin insensitivity of mTORC2, and resolves mechanisms for mTORC2 regulation by complex destabilization. Two previously uncharacterized small-molecule binding sites are visualized, an inositol hexakisphosphate (InsP6) pocket in mTOR and an mTORC2-specific nucleotide binding site in Rictor, which also forms a zinc finger. Structural and biochemical analyses suggest that InsP6 and nucleotide binding do not control mTORC2 activity directly but rather have roles in folding or ternary interactions. These insights provide a firm basis for studying mTORC2 signaling and for developing mTORC2-specific inhibitors.

The mammalian target of rapamycin (mTOR) is a key protein kinase controlling cellular metabolism and growth. It is part of the two structurally and functionally distinct multiprotein complexes mTORC1 and mTORC2. Dysregulation of mTOR occurs in diabetes, cancer and neurological disease. We report the architecture of human mTORC2 at intermediate resolution, revealing a conserved binding site for accessory proteins on mTOR and explaining the structural basis for the rapamycin insensitivity of the complex.

The phosphatidylinositol 3-kinase related protein kinases (PIKKs) are key to the regulation of a variety of eukaryotic cellular processes including DNA repair and growth regulation. While these massive proteins had long resisted structural analysis, recent advances in electron cryo-microscopy have now facilitated structural analysis of the major examples of PIKKs, including mTOR, DNA-PK, ATM, ATR and TRAPP/Tra1. In these PIKKs, the carboxy-terminal kinase domains and their proximal regions are structurally conserved. The structural organization of their extensive amino-terminal repeat regions, however, as well as their oligomeric organization and their interactions with accessory proteins, differ markedly amongst PIKKs. This architectural divergence provides the structural basis for the complex regulatory roles and functional diversity of PIKKs.

The vertebrate-specific DEP domain-containing mTOR interacting protein (DEPTOR), an oncoprotein or tumor suppressor, has important roles in metabolism, immunity, and cancer. It is the only protein that binds and regulates both complexes of mammalian target of rapamycin (mTOR), a central regulator of cell growth. Biochemical analysis and cryo-EM reconstructions of DEPTOR bound to human mTOR complex 1 (mTORC1) and mTORC2 reveal that both structured regions of DEPTOR, the PDZ domain and the DEP domain tandem (DEPt), are involved in mTOR interaction. The PDZ domain binds tightly with mildly activating effect, but then acts as an anchor for DEPt association that allosterically suppresses mTOR activation. The binding interfaces of the PDZ domain and DEPt also support further regulation by other signaling pathways. A separate, substrate-like mode of interaction for DEPTOR phosphorylation by mTOR complexes rationalizes inhibition of non-stimulated mTOR activity at higher DEPTOR concentrations. The multifaceted interplay between DEPTOR and mTOR provides a basis for understanding the divergent roles of DEPTOR in physiology and opens new routes for targeting the mTOR-DEPTOR interaction in disease.

Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control.

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