ABSTRACT
The mechanistic target of rapamycin complex 1 (mTORC1) controls cell growth and metabolism in response to nutrients, energy levels, and growth factors. It contains the atypical kinase mTOR and the RAPTOR subunit that binds to the Tor signalling sequence (TOS) motif of substrates and regulators. mTORC1 is activated by the small GTPase RHEB (Ras homologue enriched in brain) and inhibited by PRAS40. Here we present the 3.0 ångström cryo-electron microscopy structure of mTORC1 and the 3.4 ångström structure of activated RHEB-mTORC1. RHEB binds to mTOR distally from the kinase active site, yet causes a global conformational change that allosterically realigns active-site residues, accelerating catalysis. Cancer-associated hyperactivating mutations map to structural elements that maintain the inactive state, and we provide biochemical evidence that they mimic RHEB relieving auto-inhibition. We also present crystal structures of RAPTOR-TOS motif complexes that define the determinants of TOS recognition, of an mTOR FKBP12-rapamycin-binding (FRB) domain-substrate complex that establishes a second substrate-recruitment mechanism, and of a truncated mTOR-PRAS40 complex that reveals PRAS40 inhibits both substrate-recruitment sites. These findings help explain how mTORC1 selects its substrates, how its kinase activity is controlled, and how it is activated by cancer-associated mutations.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cryoelectron Microscopy , Mechanistic Target of Rapamycin Complex 1/chemistry , Mechanistic Target of Rapamycin Complex 1/ultrastructure , Ras Homolog Enriched in Brain Protein/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Amino Acid Motifs , Binding Sites , Biocatalysis , Catalytic Domain , Crystallography, X-Ray , Enzyme Activation , Humans , Mechanistic Target of Rapamycin Complex 1/agonists , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Models, Molecular , Mutation , Neoplasms/genetics , Protein Binding , Protein Domains , Ras Homolog Enriched in Brain Protein/chemistry , Ras Homolog Enriched in Brain Protein/ultrastructure , Regulatory-Associated Protein of mTOR/chemistry , Regulatory-Associated Protein of mTOR/metabolism , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Signal Transduction , Sirolimus/metabolism , Substrate Specificity , Tacrolimus Binding Protein 1A/metabolismABSTRACT
The mammalian target of rapamycin (mTOR), a phosphoinositide 3-kinase-related protein kinase, controls cell growth in response to nutrients and growth factors and is frequently deregulated in cancer. Here we report co-crystal structures of a complex of truncated mTOR and mammalian lethal with SEC13 protein 8 (mLST8) with an ATP transition state mimic and with ATP-site inhibitors. The structures reveal an intrinsically active kinase conformation, with catalytic residues and a catalytic mechanism remarkably similar to canonical protein kinases. The active site is highly recessed owing to the FKBP12-rapamycin-binding (FRB) domain and an inhibitory helix protruding from the catalytic cleft. mTOR-activating mutations map to the structural framework that holds these elements in place, indicating that the kinase is controlled by restricted access. In vitro biochemistry shows that the FRB domain acts as a gatekeeper, with its rapamycin-binding site interacting with substrates to grant them access to the restricted active site. Rapamycin-FKBP12 inhibits the kinase by directly blocking substrate recruitment and by further restricting active-site access. The structures also reveal active-site residues and conformational changes that underlie inhibitor potency and specificity.
