4.4 Å Resolution Cryo-EM structure of human mTOR Complex 1

Abstract

Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) integrates signals from growth factors, cellular energy levels, stress and amino acids to control cell growth and proliferation through regulating translation, autophagy and metabolism. Here we determined the cryo-electron microscopy structure of human mTORC1 at 4.4 Å resolution. The mTORC1 comprises a dimer of heterotrimer (mTOR-Raptor-mLST8) mediated by the mTOR protein. The complex adopts a hollow rhomboid shape with 2-fold symmetry. Notably, mTORC1 shows intrinsic conformational dynamics. Within the complex, the conserved N-terminal caspase-like domain of Raptor faces toward the catalytic cavity of the kinase domain of mTOR. Raptor shows no caspase activity and therefore may bind to TOS motif for substrate recognition. Structural analysis indicates that FKBP12-Rapamycin may generate steric hindrance for substrate entry to the catalytic cavity of mTORC1. The structure provides a basis to understand the assembly of mTORC1 and a framework to characterize the regulatory mechanism of mTORC1 pathway.

Keywords: cryo-electron microscopy; mTORC1; structure.

Figure 1

Size exclusion chromatogram of the human mTORC1 and kinase activity. (A) The gel-filtration was performed using a Superose 6 column (10/3004 GL, GE Healthcare). The peak fractions were subjected to SDS-PAGE and stained with Coomassie blue. (B and C) Phosphorylation of purified S6K1 (K100R) (B) and 4EBP1 (C) by mTORC1 in the presence or absence of Torin. The phosphorylation was detected by immunoblotting with antibodies targeting phospho-Thr-389 (top), Flag (middle), and mTOR (bottom) in (B), and antibodies targeting phospho-4EBP1 (top), 4EBP1 (middle), and mTOR (bottom) in (C). Below are the quantification of the immunoblots for B and C

Figure 2

Overall structure of human mTORC1. (A) Colored coded domain architecture of the three essential components of human mTORC1. The same color scheme is used in all structure figures. The inter- and intramolecular interactions are indicated as arrows. B) Ribbon representation of mTORC1 structure in four different views. The proteins and domains are indicated. Casp^Raptor represents Caspase-like domain of Raptor

Figure 3

The cryo-EM electron potential maps for the interfaces between M-HEAT and Core and between N-HEAT and M-HEAT (D) contoured at 5 sigma level. (A–C) is the different zoom-in view. The putative linkage between N-HEAT and M-HEAT is depicted as the red dot line, which is measured to be 20.4 Å. Because of the high quality of the cryo-EM map, it is clear that the density making up the M-HEAT region does not directly join with the FAT domain. (E and F) Comparison of cryo-EM map from this study at 4.4 Å (E) and previous study at 5.9 Å (F) resolution, respectively. Both of the maps are displayed in Chimera at the same threshold after normalization

Figure 4

Intramolecular and intermolecular interactions within mTORC1. (A) Closed-up view of the dimer formation of mTORC1. Ribbon representation of the mTORC1 is shown. The mTORC1 dimer is formed through the intermolecular interaction between the M-HEAT and the Core and that between the N-HEAT and the Core. (B) Closed-up view of the interaction between Raptor and mTOR. Raptor binds to the ridge region of the M-HEAT repeats in one mTOR and the convex side of the N-HEAT region in the other mTOR

Figure 5

Caspase-like domain of Raptor and FKBP12-mTORC1 interaction. (A) Superimposition of caspase-3 and Raptor in mTORC1 structures. Two structures are shown in ribbon representations with unnecessary regions omitted. Caspase-3 is colored in orange. The active site of kinase domain of mTOR and the active site of caspase-like domain of Raptor are indicated, respectively. Shown below is the closed-up view of the structural comparison between caspase-3 and caspase-like domain of Raptor. (B) Equal amount of purified caspase-3 and Raptor were incubated with 20 µmol/L Ac-DEVD-AMC in the presence or absence of caspase inhibitor Z-VAD-FMK at 37°C for 60 min. The activities were measured using a Spectrafluor Fluorescence Plate Reader with excitation at 400 nm and emission at 505 nm. Error bars, s.d. for triplicate experiments. (C) Superimposition of FRB^mTOR-FKBP and mTORC1 structures is shown in ribbon representations. (D) Effect of FKBP12-Rapamycin for mTORC1 assembly. Increased amount of FKBP12-Rapamycin incubated with mTORC1 (Flag-Raptor) immobilized on the Flag resin. Bound proteins were subjected to SDS-PAGE and stained with Coomassie blue