Tor forms a dimer through an N-terminal helical solenoid with a complex topology

Abstract

The target of rapamycin (Tor) is a Ser/Thr protein kinase that regulates a range of anabolic and catabolic processes. Tor is present in two complexes, TORC1 and TORC2, in which the Tor-Lst8 heterodimer forms a common sub-complex. We have determined the cryo-electron microscopy (EM) structure of Tor bound to Lst8. Two Tor-Lst8 heterodimers assemble further into a dyad-symmetry dimer mediated by Tor-Tor interactions. The first 1,300 residues of Tor form a HEAT repeat-containing α-solenoid with four distinct segments: a highly curved 800-residue N-terminal 'spiral', followed by a 400-residue low-curvature 'bridge' and an extended 'railing' running along the bridge leading to the 'cap' that links to FAT region. This complex topology was verified by domain insertions and offers a new interpretation of the mTORC1 structure. The spiral of one TOR interacts with the bridge of another, which together form a joint platform for the Regulatory Associated Protein of TOR (RAPTOR) regulatory subunit.

Figure 1. Fold of the KmTor monomer.

The ribbon diagram of the KmTor monomer is illustrated above a bar showing the domain organisation of KmTor. Domain coloring of the bar matches the coloring of the ribbons. The dashed line (on the model) and a straight line (at the beginning of the bar showing the domain organization) indicate a portion of the KmTor N-terminus that is not present in the cryo-EM map.

Figure 2. Negative regulatory domain (NRD) of KmTor.

(a) The model of the KmTor active site with the superimposed KmTor–Lst8 cryo-EM density. Domain coloring of the density matches coloring of the ribbons (only KmTor kinase domain and KmLst8 are shown). The active site is marked by an ADP (red) that was modelled using the human mTOR FATKIN crystal structure (PDB ID 4JSV). (b) A close-up view of the NRD (dark blue) between helices kα9b and kα10. The NRD, the Lst8-binding element (LBE, yellow) and the FRB restrict access to the active site. (c) View of the KmTor–Lst8 interface in the model (grey) with the KmTor–Lst8 cryo-EM density superimposed (for display, densities in the map were normalised by subtracting the mean density in the map and dividing by the standard deviation of the densities. The map shown was contoured at 6 times the standard deviation, 6σ). The Lst8-binding element (LBE) and the Lst8-binding loop (LBL) of KmTor form interactions with the Lst8. For comparison, the same elements from human mTOR–mLST8 (PDB ID 4JSV, yellow) are superimposed on the KmTor–Lst8.

Figure 3. The interface between the two KmTor–Lst8 heterodimers in the assembly.

The KmTor dimer assembly is mediated by interaction between HEAT-repeats. The primary contacts form between the helices in the spiral (Nα25, Nα27 and Nα29) of one KmTor with helices in the bridge (Nα44, Nα46, Nα48 and Nα50) of the other. This interaction leads to a KmTor dimer that has two-fold rotational symmetry with the dyad axis between the two HRD domains (α24, α26 and α28) of the FATKINs where secondary contacts form. The cryo-EM density for one KmTor–Lst8 heterodimer is shown, contoured at 9σ everywhere except for the railing density, which is contoured at 4σ.

Figure 4. The FATKINs rest on a cradle made up of the N-terminal solenoids.

The spiral (green) and bridge (magenta) domains associate with each other in the dimer of KmTor–Lst8 heterodimers to form a cradle on which the FATKINs rest. KmTor–Lst8 cryo-EM density is shown for the N-terminal solenoid region that precedes the FATKIN. The HRD that forms the interface between the FATKINs in the dimer is colored red and the FRB is colored light purple. The cryo-EM density was contoured as in Fig. 3.

Figure 5. RAPTOR in the mTORC1 complex engages both mTOR molecules.

Each of the two KmTor–Lst8 heterodimers are uniformly colored and shown as ribbons superimposed on the cryo-EM density for mTORC1 (EMDB-3212). Our topology of Tor implies that the RAPTOR in the mTORC1 complex engages both mTOR molecules through interaction with the spiral of one mTOR and the bridge of the dimer-related mTOR (interface enclosed in the dotted square).