Structure of the human mTOR complex I and its implications for rapamycin inhibition

Abstract

The mammalian target of rapamycin complex 1 (mTORC1) regulates cell growth in response to the nutrient and energy status of the cell, and its deregulation is common in human cancers. Little is known about the overall architecture and subunit organization of this essential signaling complex. We have determined the three-dimensional (3D) structure of the fully assembled human mTORC1 by cryo-electron microscopy (cryo-EM). Our analyses reveal that mTORC1 is an obligate dimer with an overall rhomboid shape and a central cavity. The dimeric interfaces are formed by interlocking interactions between the mTOR and raptor subunits. Extended incubation with FKBP12-rapamycin compromises the structural integrity of mTORC1 in a stepwise manner, leading us to propose a model in which rapamycin inhibits mTORC1-mediated phosphorylation of 4E-BP1 and S6K1 through different mechanisms.

Figure 1

mTORC1 purification. (A) mTORC1 and FLAG-raptor were purified by tandem gel filtration chromatography. Their masses were estimated based on known molecular weight standards as indicated (Thyroglobulin (669 kDa), Ferritin (440 kDa), Aldolase (158 kDa) and Conalbumin (75 kDa)). (B) The gel filtration fraction corresponding to mTORC1 was analyzed by SDS-PAGE followed by silver and Coomassie staining as well as immunoblotting for indicated proteins. (C) Schematics of mTORC1 components illustrating the various predicted domains. (D) In vitro kinase assay showing that purified mTORC1 phosphorylates S6K1 and is inhibited by both rapamycin-FKBP12 (rapa) and Torin1. (E) EM of negatively stained mTORC1. A raw image of mTORC1 particles (circled) and a representative class average from the classification of 10,080 particles (inset). The scale bar represents 50 nm, and the side length of the panel showing the class average is 45 nm. “see also Figure S1”

Figure 2

Cryo-EM reconstruction of mTORC1 and its molecular organization. (A) Image of a vitrified specimen showing individual mTORC1 particles (circled). The scale bar represents 50 nm. (B) Different views of the 3D reconstruction of mTORC1 filtered to 26 Å, with the main structural features denoted. The scale bar represents 5 nm. (C) Molecular organization of mTORC1. Left panels: Representative class averages from antibody labeling experiments of mTORC1 (top) and schematic representations showing mTORC1 in white and the antibody in pale red (bottom). The side length of each panel is 45 nm. Right panel: Location of raptor, mLST8, and PRAS40 in the cryo-EM density map of mTORC1. “see also Figure S2”

Figure 3

3D reconstruction of raptor and molecular docking. (A) Silver-stained gel and immunoblot of the gel filtration fraction containing free raptor detected the presence of PRAS40. (B) EM image of negatively stained raptor (circled) and two representative class averages from the classification of 12,216 particles (bottom right insets). Class II particles contain an additional density (red arrow) compared to Class I particles, which likely represents PRAS40. The scale bar represents 25 nm, and the side length of the panels showing the class averages is 27 nm. (C) Different views of the raptor 3D reconstruction. The scale bar represents 2.5 nm. (D) Two copies of the raptor 3D reconstruction (gold) and two models of a representative WD40 domain (PDB code 3EMH, red) were placed into the cryo-EM density map (gray). The green asterisk depicts the location of PRAS40 as determined by antibody labeling. The blue dotted line represents the dimer interface. (E) The proposed locations of the N- and C-terminal domains (marked “N” and “C”) and the kinase domain of mTOR (purple star). The black lines labeled “I” and “II” delineate the two interaction interfaces formed by each mTOR molecule with the two raptor subunits. “see also Figure S3”

Figure 4

Effects of rapamycin-FKBP12 on mTORC1. (A) Representative class averages of untreated mTORC1 (left) and mTORC1 treated with 50 nM of rapamycin and 0.02 μg/μl GST-FKBP12 (middle), and a schematic representation showing the additional density in pale red (right). The side length of each panel is 45 nm. To the right, the location of the FRB domain with respect to the other components in the cryo-EM map of mTORC1 is shown. (B) and (C) Purified mTORC1 was treated with 50 nM of rapamycin and 0.02 μg/μl GST-FKBP12 or 100 nM Torin1. EM images of negatively stained samples were taken at the indicated time points. The inset in each image shows an enlarged view of the area marked by the white square. The scale bars represent 100 nm. (D) and (E) mTOR immunoprecipitates, prepared in lysis buffers containing 0.3% CHAPS or 1% Triton X-100, were subjected to in vitro kinase assays using 4E-BP1 or S6K1 as a substrate in the presence of 100 nM rapamycin and 0.02 μg/μl FKBP12 or 100 nM Torin1. Assays were then analyzed by immunoblotting for the indicated proteins and phosphorylation states. (F) A model depicting a potential mechanism of mTORC1 inhibition by FKBP12-rapamycin. “see also Figure S4”