Seminars in Cell & Developmental Biology 36 (2014) 91–101

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Review

The structural basis for mTOR function ´ Roger L. Williams ∗ Domagoj Baretic, Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK

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Article history: Available online 5 October 2014 Keywords: mTOR PI3K Rapamycin 4E-BP1 S6K1 PIKK

a b s t r a c t The phosphoinositide 3-kinase (PI3K) related protein kinases (PIKKs) are a family of protein kinases with a diverse range of vital cellular functions. Recent high-resolution crystal structures of the protein kinase mTOR suggest general architectural principles that are likely to be common to all of the PIKKs. Furthermore, the structures make clear the close relationship of the PIKKs to the PI3Ks. However, the structures also make clear the unique features of mTOR that enable its substrate specificity. The active site is deeply recessed and flanked by structural elements unique to the PIKKs, namely, the FRB domain, the LST8 binding element, and a C-terminal stretch of helices known as the FATC domain. The FRB has a conserved element in it that is part of a bipartite substrate recognition mechanism that is probably characteristic of all of the PIKKs. The FRB also binds the mTOR inhibitor rapamycin that has been referred to as an allosteric inhibitor, implying that this inhibitor is actually a competitive inhibitor of the protein substrate. This bipartite substrate-binding site also helps clarify how rapamycin can result in substratespecific inhibition. © 2014 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4.

5. 6.

The mTOR complexes: an overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 The mTOR protein substrate recognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 The mTOR kinase domain structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 mTOR catalytic center in comparison with PI3Ks and PKs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 4.1. The N-lobe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 4.2. The activation and the catalytic loop . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 4.3. Highly conserved C-terminus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 4.4. Regulatory arch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 4.5. Communicating substrate binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 4.6. Mechanism of catalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 Inhibiting mTOR kinase activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 Conclusion and perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

1. The mTOR complexes: an overview In mammals, the mTOR signaling pathway is mediated by the two complexes, mTORC1 and mTORC2 (holoenzymes) that differ in the regulatory subunits with which they associate. These subunits affect the localization, substrate specificity and activity of the mTOR

∗ Corresponding author. Tel.: +44 1223 267094. ´ E-mail addresses: [email protected] (D. Baretic), [email protected] (R.L. Williams). http://dx.doi.org/10.1016/j.semcdb.2014.09.024 1084-9521/© 2014 Elsevier Ltd. All rights reserved.

complexes in response to various environmental cues and stress conditions [1]. RAPTOR (the yeast orthologue KOG1) defines the mTOR complex 1 (mTORC1/TORC1) [2–4], and RICTOR (AVO3) is the determinant of mTORC2 (TORC2) [3,5]. LST8 (all eukaryotes) and DEPTOR (only vertebrates) are present in both TOR complexes 1 and 2 [6–8], however, they differ in a number of other RAPTOR/KOG1and RICTOR/AVO3-associated subunits [9,10]. For both RAPTOR and KOG1, the primary TOR binding region is in the N-terminal helical repeats [4,11]. A low-resolution electron microscopy model of the native yeast TOR1 catalytic subunit suggests a superhelical organization of the N-terminus (flat and tubular arm), however

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Fig. 1. The 26 A˚ resolution cryo-EM model of mTORC1 and its substrates - S6K1 and 4E-BP1. (A) The mTORC1 complex forms a dimer with a rhomboid shape, having dimensions ˚ It contains native components mTOR, mLST8, PRAS40 and recombinant FLAG-RAPTOR, expressed from HEK293 cells. Individual components were 290 A˚ × 210 A˚ × 135 A. identified by antibody labeling [model deposited as EMD-5197]. PRAS40 binds to RAPTOR through a TOS motif [14], however the subunit boundaries are not clear from the model (designated with dashed line). (B) Structural model of 4E-BP1 in complex with eIF4E and m7 G-cap (PDB ID: 1WKW) with floppy N- and C-termini drawn on the model (red dashed curves). The green hexagon at the N-terminus denotes the RAIP motif. Structural model of S6K1 (PDB ID: 4L46) with the C-terminus carrying the FRB binding motif (purple) and N-terminus with the TOS motif (residues FDIDL) depicted as disordered (black dashed curves). (C) The schematic model points out important contacts between mTOR-mLST8, RAPTOR and S6K1 during substrate selection and phosphorylation (zig-zag line represents part of the mTOR ␣-solenoid extending from FAT (TRD1), yellow star marks the active site and P in red is the phosphorylated residue Thr389 ).

the identity, boundaries and a spatial organization of the putative domains could not be easily discerned [11]. A 26 A˚ resolution EM model [12] shows RAPTOR as an essential component in forming the mTOR dimerization interface (Fig. 1A). The symmetry of the model reveals that RAPTOR has a capacity to interact with both the N-terminal helical region and the kinase domain at the C-terminus of the mTOR catalytic subunit, and these interactions greatly accelerate the catalysis. Yip and colleagues have found that RAPTOR resembles a ‘comma’ with a globular domain that could accommodate the predicted WD40 domains at its C-terminus. The model also shows two other accessory subunits that co-localize with mTOR, mLST8 and PRAS40 [13,14], on each of the mTORs in the dimer. PRAS40 is considered both as a mTORC1 substrate [15,16] and an inhibitor [13,17]. In contrast to mTORC1 [12], the EM model of the yeast TOR1·KOG1 complex does not indicate a dimeric complex [11]. The monomeric state of the yeast TOR1·KOG1 complex could be a consequence of its localization on the vacuole surface, however, this state persists throughout the purification. In contrast, mTORC1 is predominantly found in the cytoplasm [1]. Adami and colleagues have found that KOG1 associates with the N-terminal region of TOR1, using the KOG1 C-terminal domain predicted to consist of WD40 repeats. The EM analysis of the differential map indicates KOG1 is comma-shaped, structurally resembling the reported shape of RAPTOR [12].

The sole mTOR catalytic subunit is a member of the phosphoinositide 3-kinase (PI3K) related protein kinases (PIKK), with a kinase domain similar to the PI3Ks. N-terminal to the well conserved mTOR kinase domain is a long array of the helical repeats, which is a structural motif shared among all PIKKs [18–21]. Only recently, the crystal structure of the mTOR kinase domain in a complex with mLST8, at 3.2 A˚ resolution [22] revealed the remarkable features of the two-lobe catalytic core present in both mTOR complexes 1 and 2. The FRB (FK506-rapamycin binding) domain, the LBE (LST8 binding element), the k␣AL (activation loop helix) and the FATC (FRAPP, ATM, TOR at C-terminus) domain are all PIKKspecific features that are not present in the bilobal kinase domain of PI3Ks (Fig. 2). In addition, the mTOR kinase domain crystal structure contains a portion of the N-terminal helical repeats comprising the FAT domain, which is thought to be important for the kinase domain structural integrity and activity. 2. The mTOR protein substrate recognition Activated mTORC1 regulates translation of mRNAs encoding for proteins with crucial biological functions. It regulates cap-dependent translation utilizing two major substrates: (1) the negative regulator of initiation, eIF4E-binding protein 1 (4E-BP1) and (2) a positive regulator of elongation, the 40S ribosomal protein S6 kinase 1 (S6K1). The mTORC1 mechanism of substrate

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Fig. 2. Structure of the mTOR kinase domain (residues 1385–2549) in a complex with mLST8 [22]. For clarity, mTOR domains are depicted in the top right corner. Prepared in PyMOL (PDB ID 4JSN).

selection is complex. Phosphorylation of 4E-BP1 and S6K1 is dependent on their general TOR signaling motif (TOS) [23,24], which is a 5-amino acid motif (5 FDIDL9 [human S6K1], 114 FEMDI118 [human 4E-BP1]) in the disordered termini of both S6K1[25] and 4E-BP1[26] (Fig. 1B). A mutation of the RAPTOR N-terminal conserved domain eliminates binding to the TOS motif, without affecting RAPTOR binding to mTOR. [27]. In addition, there seems to be a second, the substrate-specific motif. In human 4E-BP1 this is thought to be an 13 RAIP16 motif [28] and in S6K1 the motif that is located close to Thr389 (part of the hydrophobic motif, HM), near the C-terminus where it can be recognized by the FRB domain [22] (see Section 5). In 4E-BP1, a RAIP motif mutant can still be phosphorylated in vitro both at the Thr37 /Thr46 and Ser65 /Thr70 pairs, while the lack of a functional TOS motif affects only Ser65 /Thr70 phosphorylation [29]. Both the TOS motif and the FRB binding regions of S6K1 are necessary for Thr389 phosphorylation [22,23]. The presence of rapamycin negatively affects phosphorylation of Ser65 /Thr70 (but not Thr37 /Thr46 ) in 4E-BP1 and Thr389 in S6K1 [16,30]. Because it was shown that mTORC1 recruits substrate by recognizing its FRB-binding motif and the RAPTOR-binding motif (TOS), a bipartite recruitment mechanism is suggested for at least a subset of mTOR substrates (e.g., S6K1). This mechanism might be important for discriminating the mTORC1 specific substrates from those for mTORC2 and for properly positioning the targeted phosphorylation residues within the restricted active site. Structural analysis of the mTOR complexes [12,22], together with rapamycin studies [3,12]

indicate that RAPTOR sits on the long helical solenoid likely positioning its N-terminal domain close to the active site from where it selects and admits the substrate (Fig. 1C). 3. The mTOR kinase domain structure mTOR contains a canonical two-lobe kinase domain (KD) spanning about 550 C-terminal residues, with three characteristic insertions: FATC (∼30 residues), LBE (∼40 residues) and FRB (∼100 residues) (Fig. 2). The LBE and FRB protrude from the KD on the opposite sides of the catalytic cleft. Whereas the base of LBE interacts with the portion of the catalytic center in the C-lobe, including the FATC, its apical surface tightly interacts with the accessory protein mLST8. The FRB is a four-helix bundle adjacent to the beginning of the N-lobe kinase domain and acts as a gatekeeper [22], because it participates in recruiting substrate (e.g., S6K1) while generally restricting access to the catalytic cleft. Immediately N-terminal to the KD, is an array of helical repeats comprising the FAT domain, which extends toward the N-terminus (Fig. 2). The FAT domain is composed of 28 ␣-helices arranged as a solenoid that clips onto the kinase domain (Fig. 3A). The solenoid is right-handed and, according to the packing angles between helices and the resulting overall topology, it is composed of TPR and HEAT (Huntingtin elongation factor 3 [EF3], regulatory A subunit 65 kDa in protein phosphatase 2A [PP2A], TOR1) family repeats. The FAT domain contains three distinct Tetratrico peptide repeats (TPR)

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Fig. 3. Structural organization of the FAT domain in mTOR. (A) Top view of the FAT domain clipping to the kinase domain through the HRD and TRD1 modules. FAT helices making contacts with the kinase domain are indicated. mLST8 is omitted from the structure for clarity. (B) Interactions of TRD1 with the C-lobe. Potential hydrogen bonds are marked with red dotted lines and selected hydrophobic interactions are indicated by orange dots. Hydrophobic pockets are indicated by black arcs, e.g., a hydrophobic pocket within which His1398 stacks on top of Trp2304 . (C) Close view of HRD contacts with the N- and C-lobes. Prominent interactions labeled as in B. The RHEB-independent activating mutation of residue Glu2419 (in interaction with Arg1905 ) is marked in blue [34]. (D) The positions of mTOR activating mutations are shown as spheres. Sites colored blue are mutations found in yeast TOR1, TOR2 and mTOR arising from in vitro selection [33–35]. Magenta spheres show cancer-associated mutations found in mTOR [36–39]. The legend depicts mutated residue numbers colored according to the structure. Blue asterisks mark cancer mutations that were earlier found in yeast TOR genes.

domains, named TRD 1, 2 and 3 (varying in number of repeats within the domain from 1.5 to 4) followed by a single three-HEAT repeats domain (HRD). The canonical HEAT repeats and TPRs contain a pair of anti-parallel helices per repeating unit. However, adjacent HEAT repeats are only slightly tilted against each other, while adjacent TPR repeats are often rotated [31]. This results in a curved, double layer of ␣-helices in HEAT repeat regions (HRD)

and a single-layer of superhelical ␣-helices in the TPR regions (TRD 1–3) (Fig. 3A). The HRD first curves along one side of the KD making the extensive contacts with both the N- and the C-lobe, then the FAT domain takes a sharp turn away from the kinase domain via a superhelix comprised of TRD3 and TRD2 to finally return back to the other side of the KD with TRD1 in close proximity to the C-lobe (Fig. 3A). The

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HRD and TRD1 residues making contacts with the kinase domain are the most conserved segments of the FAT domain among all PIKKs [19]. The HRD is the only portion of the FAT domain that has a structural homologue in the helical domain of the PI3Ks [32]. The interactions between the FAT and the KD are established through a network of hydrogen bonds between residues surrounding the hydrophobic pockets (Fig. 3B and C). These contacts seem to be important for the kinase domain structural integrity and activity, and are common features of all PIKKs. This is supported by various hyperactivating mutations that have been characterized in mammalian and yeast TOR [33–36]. Three distinct clusters of activating mutations are located within the kinase domain and in the interface between the kinase domain and the FAT domain (Fig. 3D). Many mutations are at the end of the catalytic cleft and in helices k␣3a, k␣3b, k␣9 and k␣10, which center on the helix k␣9b. It is believed that mutations in this area make the end of the catalytic cleft less protected by either weakening interactions between tightly packed helices (e.g., L2427T in k␣9b) or by diminishing the kinase domain interactions with the HRD (e.g., E2419K in k␣9a can no longer make a salt bridge with Arg1905 in the HRD). Either way, mTOR becomes substantially more active toward the physiological substrates (4E-BP1 and S6K1). A second cluster of mutations can be found at the base of the FRB domain (tip of k␣1 and k␤5 [N-lobe ␤-sheet]). Some of the mutations, like I2017T [k␣1] [33] probably increase mTOR kinase activity by contributing to the flexibility of the rigid FRB domain, thereby opening access to the active site. Finally clusters of mutations also are found in the FAT domain at both its N-terminus (TRD1 and TRD2) and C-terminus (HRD), where conserved residues couple the FAT domain to the kinase domain. Mutations such as K1438P [TRD1], W1449R [TRD2] or I1973F [HRD] probably cause mTOR hyperactivity by destabilizing FAT/kinase domain interactions. These mutations suggest that FAT domain maintains the structural integrity of the kinase domain. A number of hyperactivating cancer mutations of the MTOR gene found in different human tissues (most frequently in lung, endometrium and gut) [37–39] coincide with clusters of hyperactivating mutations found in genetic screens in yeast and mammalian cells (shown in Fig. 3D). While the majority of the mutations detected in human cancers do not affect mTORC1 assembly, some TRD2 mutations, like C1483F or a nutrient-insensitive L1460P [27] negatively affect binding of DEPTOR [39], an inhibitor of the mTOR kinase activity [8]. 4. mTOR catalytic center in comparison with PI3Ks and PKs Like in the PI3Ks and other protein kinases (PKs), the N-lobe of the mTOR kinase domain is smaller, generally composed of a fivestranded ␤-sheet, associated with a few ␣-helices, while the larger C-lobe is predominantly ␣-helical (Fig. 4A). The cleft between the two lobes forms the catalytic site and the ATP-binding region. In mTOR, the KD N-lobe is structurally more similar to PI3Ks than to protein kinases. In PI3Ks and mTOR the N-lobe packs against the HEAT repeats of the helical and FAT domains, respectively, whereas in the canonical protein kinases (e.g., PKA) the N-lobe frequently interacts directly with regulatory partners, and therefore is structurally more divergent [40]. 4.1. The N-lobe Among kinase families, a prominent conserved element of the active site is the P-loop near the N-terminus of the conserved N-lobe ␤-sheet. Residues from this loop accommodate ATP by interacting with its phosphate groups and these interactions are conserved among mTOR, PI3Ks and PKs. In the PKs, the P-loop is enriched

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with conserved glycine residues (in PKA Gly50 , Gly52 and Gly55 [41]). In mTOR, the P-loop (residues 2163–2168) contains a conserved Ser2165 , which coordinates the ␤-phosphate of ATP. Like mTOR, PI3Ks and PKA have conserved ATP-interacting Ser residues in the P-loop, such as Ser675 (Vps34) and Ser53 (PKA). Following the P-loop, Lys2187 is another conserved mTOR residue, which interacts with ␣-and/or ␤-phosphate of ATP. Lys2187 is at the beginning of the loop connecting the last strand (k␤5) of the N-lobe ␤-sheet and the helix k␣3a (helix ␣C in PKA [42]). An equivalent Lys residue is found in PI3Ks and other PKs (e.g., Lys698 Drosophila melanogaster Vps34 and Lys72 of PKA). This loop emerges at the end of the central strand of the N-lobe ␤-sheet. The Lys at this position coordinates phosphates of the ATP in all of these enzymes, however, only in PKA does the equivalent residue also interact with the helix k␣3 (through residue Glu91 ). The absence of this salt link is characteristic of the inactive conformation of some PKs such as cyclin-dependent protein kinase 2 [43]. The importance of the Lys2187 equivalent residue is underscored in the observations that this residue is covalently modified by wortmannin in mTOR and the PI3Ks [44–46]. The equivalent of helix k␣3 serves as a basal element of the active site in the protein and lipid kinases (Fig. 4A). In mTOR, the amphipathic k␣3a helix exposes its conserved hydrophilic side toward the ATP-binding site residues (via Arg2193 Gln2194 Asp2195 , the RQD motif) and the FAT domain (Gln2200 [k␣3a]–Gln1941 [HRD]), whereas the face carrying the hydrophobic residues (Val2198 , Leu2201 , Phe2202 , Leu2204 , Val 2205 , Leu2208 and Leu2209 ) packs with helices k␣6, k␣8 and k␣9a (Fig. 4A). In the PI3Ks, the helix k␣3 makes equivalent interactions with the ATP-binding site, k␣6, k␣8 and k␣9 (Fig. 4A) and the helical domain (Vps34 Gln711 [k␣3]–Gln400 [helical domain]). In PKA, helix ␣C (k␣3) is hydrophilic, does not contain the RQD motif and makes close interactions with the active site and the ATP-binding site. Instead of interacting with the helical domain the solvent-exposed surface of the helix ␣C is reserved for the interaction with the regulatory subunit [47]. 4.2. The activation and the catalytic loop The C-lobe of the mTOR contains the majority of the active site. The mTOR crystal structure [22] reveals fully ordered structural elements crucial for the catalysis. They are known as the activation loop, the catalytic loop (both loops flanking bound ATP on the side opposite to the P-loop) and the FATC (Fig. 4A). The mTOR activation loop (26 residues) has two conserved motifs at the N- and the C-terminus, 2355 HIDFG2359 and 2371 P/YE(K/R)(V/I)PFRL2379 , respectively. The highly conserved DFG motif is present at the N-terminal end of the activation loop of both PI3Ks [48] and PKs [49], however, a similar motif at the C-terminal end of the activation loop, ERVPF(I/V)L, is found only in the class I PI3K isoforms. The shorter catalytic loop (9 residues) is extremely conserved among mTOR orthologues and it carries the characteristic sequence GLGD2338 RHPSN2343 . A DRHN motif is a typical signature sequence of the catalytic loop of PI3Ks (805 DRHN810 in D. melanogaster Vps34), whereas in the PKs it is 147 YRDN154 (PKA) or 127 HRDN134 (MAPK) [41] (Fig. 4A). From the structures of the PI3Ks and the PKs, the catalytic and the activation loops are locked in different conformations. In the crystal structure of Vps34, the Asp805 and the His807 residues of the DRHN motif are pointing toward the catalytic center (Fig. 4A), while in the structure of the other PI3K isoforms the equivalent residues are turned in the opposite direction, which may represent active and inactive states of the enzyme, respectively. In the recent crystal structure of the native mTOR complex, or the complex bound to the ATP␥S or the ADP-Mg-F, the conserved residues of both the DRHN and the DFG motifs are positioned toward the active site (Fig. 4A), which probably indicates

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Fig. 4. Comparison of mTOR with protein and lipid kinases. (A) Active site comparison of mTOR with protein and lipid kinases: PKA [PDB ID 1ATP], PI3K (Vps34) [PDB ID 2X6H] and mTOR [PDB ID 4JSP, mLST8 omitted]. (B) Catalytic and regulatory spines in PKA, PI3K (p110␤) [PDB ID 2Y3A] and mTOR are shown as dotted surfaces. Prepared in PyMOL.

that mTOR is an intrinsically active enzyme. The conformational “in/out” switching of the DFG activation loop residues exists as a mechanism of regulation of the PK activity [40]. However, no equivalent change in conformation has been observed for the PI3K family of enzymes. Furthermore, the PI3Ks are not regulated by activation loop phosphorylation.

4.3. Highly conserved C-terminus The FATC is composed of a pair of helices, k␣11 and a threesegmented k␣12 (a/b/c) at the end of the C-lobe (Fig. 4A). The k␣12 sequence is highly conserved among the mTOR orthologues (2542 YIGWCPFW2549 ), and it is thought to be involved

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in substrate recognition. The FATC is stabilized through interaction with the activation loop on one side and a predominantly hydrophobic interface with the LBE (helix-loop-helix motif, k␣4b and k␣4c) on the other side. The activation loop uses its Cterminal 2371 P/YE(K/R)(V/I)PFRL2379 motif to clamp the hinge of the FATC (between helices k␣11 and k␣12a) onto the LBE, where k␣12a also makes a single hydrogen-bond with mLST8 (Glu2536 [k␣12a]–Tyr222 [mLST8]). The activation loop then traverses along the FATC where it interacts with 2542 YWGWC2546 of helix k␣12b (Fig. 4B). In this way the activation loop ends up making a cage-like structure around the FATC hinge, stacking its Phe2371 perpendicular to the Tyr2542 and providing two more hydrogen bonds via residues Glu2373 and Arg2378 from two different sides. This helps to position an exposed hydrophobic patch of k␣12b toward the active site. Helix k␣12c anchors the base of the FATC by stacking against the LBE, where it makes a hydrophobic pocket. It seems there is no strong consensus motif at the phosphorylation sites of the mTOR physiological substrates. However, recent studies, including a positional scanning peptide array of mTOR substrates [50], revealed an mTOR preference for substrate with a Pro, hydrophobic or aromatic side chain at the position +1 to the phosphoacceptor site (Ser/Thr), and a lower selectivity for the substrate residues N-terminal to it (-4 and -5 positions). This is consistent with the idea that the k␣12b conserved hydrophobic patch (Tyr2542 Gly2544 Trp2545 Phe2371 ) could serve as a putative substrate binding site (Figs. 4B and 5A). The FATC helix k␣11 makes multiple interactions with the C-lobe via its two extremely conserved residues Gln2524 and Leu2528 . The Gln2524 makes a hydrogen bond network by contacting highly conserved Arg2408 [k␣8], Lys2507 [k␣10] and Asp2512 [k␣10] through an elbow between the FATC and helix k␣10. The Leu2528 fits into the hydrophobic interface created by the extremely conserved residues of the activation loop (Met2329 , Tyr2332 ) and the helix k␣6 (Pro2376 , Phe2377 ). Together with the helices k␣10 and k␣12, the k␣11 serves as a structural framework to the mTOR kinase domain encircling the active site elements, the activation and catalytic loops. 4.4. Regulatory arch The structural organization of the mTOR helices k␣10, k␣11 and k␣12 is reminiscent of the C-terminal regulatory arch composed of the equivalent helices in PI3Ks (k␣10, 11 and 12, which are arranged approximately in a plane) [32] (Fig. 4A), with a notable difference in the relative position and function of the helix k␣12. In contrast the conventional protein kinases, like PKA have no clear equivalent of these helices apart from the regulatory helix ␣F (mentioned in Section 4.5). In PI3Ks, helix k␣12 either sits on the surface of the C-lobe or protrudes from the kinase domain, while in mTOR, k␣12 is hidden behind the LBE (Fig. 4A). In the structures of the PI3K isoforms, k␣12 occupies different conformations that likely reflect an inactive-to-active state transition (Fig. 4A and B). For example, in p110␥ the capping k␣12 helix interacts with the 946 DRH948 motif (catalytic loop) through a hydrophobic contact with the conserved residue Trp1086 that packs against His948 . This interaction causes a change in the conformation of the catalytic loop, such that the His948 and Asp946 are pointing away from the active site and access to the lipid substrate appears to be restricted by the k␣12 so that the active site likely rests in a closed conformation [51]. In what could be the open conformation of Vps34, the equivalent helix k␣12 no longer protects the active site and is critical for interaction with the lipid membrane [52]. In the class IA PI3Ks, the arch of the p110 subunit appears to be important for interactions with the SH2 domains. In p110␤, the cSH2 domain of p85 clamps the k␣12 onto the p110 catalytic loop limiting access to the substrate [53]. In mTOR, k␣12 is locked between the activation loop, the LBE and mLST8, and it does

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not seem to be involved in an open-to-closed transition (Fig. 4A). Moreover, the mTOR structure and the in vitro kinase activity [22] suggest that the conformation of the kinase domain, with the k␣12 folded inside the C-lobe is inherently open and active in the absence of the regulatory subunits (RAPTOR and RICTOR/mSIN1, mentioned in Section 1). Together with the helices k␣10, 11 and 12, the C-terminal regulatory arch in mTOR contains a unique, fourth helix k␣9b (residues 2425–2436) that is conserved among mTOR orthologues and not present in PI3Ks (Fig. 4A). This helix packs against the activation loop, where it caps the catalytic site and overlaps with the region 2430–2450, known as the negative regulatory domain (RD). A major portion of the RD includes the unstructured k␣9b-k␣10 loop (residues 2437–2491) (Fig. 4A), which is variable in length and sequence across different species. Deletion of the RD causes an increase of the mTOR kinase activity [54–56], however, it is believed that this is due to k␣9b, since removal of the majority of the k␣9b-k␣10 loop (2443–2486) decreases mTOR activity [22]. One explanation might be that shortening the linker between helices k␣9b and k␣10 causes tighter packing of the two helices with the activation loop, further limiting accessibility to the active site. On the other hand, it has been proposed that a truncated k␣9b unplugs the end of the catalytic cleft widening the entry to the active site, causing mTOR activation [22]. As in p110␣, a number of the mTOR activating mutations (Fig. 3D) are found along the regulatory arch (helices k␣9b and k␣10). It is believed that RHEB activates mTOR via interaction with the kinase domain active site, mLST8 and RAPTOR [57]. Changes in the network of contacts within the regulatory arch could result in an allosteric regulation of mTOR. 4.5. Communicating substrate binding In the conventional PKs (PKA as an example) the central scaffold for the active protein kinase assembly is the helix ␣F (Fig. 4A), which may serve the functional role of the regulatory arch seen in mTOR and PI3Ks. Helix ␣F is in between the two conserved hydrophobic networks that span the kinase domain, which are present in active PKs (Fig. 4B). These structures are known as the regulatory (R) and the catalytic (C) spines, and they are disassembled in the inactive state of the PKs [58,59]. In canonical PKs, phosphorylation of the activation loop structures the R-spine (centered on the helix ␣C), whereas ATP binding results in formation of the complete C-spine (centered on the helix ␣F), bringing the kinase domain into a more compact global conformation. The fully operational R and C-spines prime the enzyme for catalysis because then the ATP is properly positioned and the substrate-binding surface is completely formed. The equivalent hydrophobic R and C-spines are conserved structures in the catalytic core of the PI3Ks and mTOR (Fig. 4B), although their dynamics might differ from the PKs. The PI3K crystal structures suggest that the R-spine is continuous in both active and inactive state of the kinase domain. The same is true for the R-spine of the intrinsically active conformation of the mTOR kinase domain. The beginning of the R-spine is crucial for positioning the DFG motif of the activation loop in all kinase families, the Phe185 (PKA), the Phe932 (p110␤) and the Phe2358 (mTOR) stack against the residues of the R-spine so that the Asp takes on an appropriate orientation for coordinating metal ion in the ATP-site (detailed in Section 4.6). The position of the R-spine appears to align with path that the activation loop traverses along the core of the kinase domain (Fig. 4B). In PI3Ks, the R-spine extends along the regulatory arch k␣10–k␣11 (p110␤), whereas in mTOR it ends before the helix k␣10. In mTOR, this ending is marked with the Phe2362 , which stacks against the R-spine formed between k␣6 and k␣10, just beneath the capping helix k␣9b. This interaction is probably important for stabilizing the putative substrate-binding site that forms between the Phe2371 and the C-spine (Fig. 4B).

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Fig. 5. The active site of the mTOR kinase. (A) The kinase active site is formed by three major loops: the P-loop (pink), the catalytic loop (orange, labeled CAT loop) and the activation loop (yellow, labeled ACT loop). The active site is surrounded by helix k␣9b and a disordered loop on top (dashed line), and the LBE on the front. A docked peptide is in dark blue with the phosphorylation site marked as 0 (purple). The C-terminal Pro is at position +1 and the N-terminal residues at positions −4 and −5. The region labeled as I marks the hydrophobic pocket formed by the activation loop and the FATC. This pocket appears to be crucial for substrate selection at position +1, and regions with less pronounced substrate specificity for residues at positions -4 and -5 are labeled as III and II. Model prepared in PyMOL (PDB ID: 4JSP). (B) Schematic view of critical elements for the catalytic mechanism.

In PI3Ks and mTOR the C-spine forms between the ATP-binding site and the hydrophobic center at the helix k␣12 (not conserved in PKA). The catalytic loop that stems from the R-spine anchors close to the k␣12 center of the C-spine (Leu2344 [mTOR] and Pro2341 [mTOR], and Ile919 [p110␤]) (Fig. 4B). The contacts that the catalytic loop makes with the C-spine are crucial for positioning the DRHN motif toward the ATP-site, both in PI3Ks and mTOR (as commented in Section 4.2). While it is likely that in PI3Ks the C-spine coordinates lipid membrane binding with changes in the ATP-pocket, the mTOR structure suggests that the C-spine is continuous so as to maintain the active status of the catalytic site. 4.6. Mechanism of catalysis The mTOR active site contacts are centered on the three crucial loops: (a) the activation loop, (b) the catalytic loop and (c) the P-loop (Fig. 4A). The fully ordered activation loop is part of the polypeptide-binding site, and it carries the DFG motif where the Asp2357 coordinates Mg2+ at the active site. In most PI3K structures, the activation loop is disordered. The activation loop of mTOR has a unique helical insertion, k˛AL, and this likely contributes to the stability of the activation loop. Order in the mTOR activation loop is probably also the result of a direct interaction of the loop with another part of the substrate-recognition mechanism, the FATC. The FATC contains a hydrophobic pocket essential for the substrate selectivity (discussed in Section 4.3). The catalytic loop carries three essential residues: Asp2338 that orientates the substrate and polarizes its phosphoacceptor hydroxyl group, His2340 postulated to stabilize the  -phosphate transition state and Asn2343 – the second metal ligand (Fig. 5A). The proposed mechanism for mTOR catalysis [22] is schematically presented in Fig. 5B. A number of known mTOR substrates do not have a strong consensus motif at their phosphorylation site, however, mTOR has higher affinity for peptide substrates with Ser over Thr at the phosphorylation site and Pro, hydrophobic or aromatic side chains C-terminal to it (+1 position) [16,22,50,55]. The conserved residues positioned at the entry to the catalytic cleft, His2247 [k␣4a], Asn2262 [k␣4b] and His2265 [k␣4b] might be responsible for a less selective substrate recognition at positions −4 and −5, although

there are several pockets around the catalytic site, which might contribute to this role (Fig. 5A). 5. Inhibiting mTOR kinase activity Before the structure of the kinase domain of mTOR was reported, inhibitors of mTOR catalysis were typically regarded as allosteric or active site inhibitors, with rapamycin being the classic example of an allosteric inhibitor. Rapamycin inhibits mTOR activity in a substrate and phosphorylation-site dependent manner [16,30,50]. A ternary complex consisting of rapamycin, FKBP12 and conserved residues of the mTOR FRB domain forms just in front of the catalytic cleft, constricting access to the active site (Fig. 6A) [60]. Although rapamycin can bind the FRB domain in the absence of its FKBP12 receptor, it shows 2000-fold higher affinity (Kd = 12 nM) for the FRB if in a complex with FKBP12 [61]. The proximity of the rapamycin binding site on the FRB, and its ability to inhibit mTORC1 and mTORC2 in the absence of FKBP12 (albeit at a much higher rapamycin concentration) prompted the proposal that rapamycin actually binds to a conserved secondary substrate binding site on the FRB, meaning that rapamycin is actually a competitive inhibitor for the protein substrate (but not ATP). This proposition is consistent with the ability of the isolated FRB to inhibit mTOR activity [22]. The organization of the ATP-binding site of mTOR resembles the ATP-binding site of PI3Ks [48]. Six conserved regions can be discerned in the ATP-binding pocket of mTOR: hydrophobic pocket I, the hinge (between N- and C-lobe, Trp2239 -Val2240 ), the specificity pocket, the P-loop, the DFG motif of the activation loop and the affinity pocket (also known as the hydrophobic pocket II) (Fig. 6B). Due to a pivotal role in the regulation of cell growth, mTOR has been a pharmacological target for treating various types of solid tumors and lymphomas. Thus, a number of potent mTOR ATP-competitive inhibitors have entered clinical trials as anti-cancer agents [62]. Specific inhibitors of mTOR with nanomolar or subnanomolar affinity such as PP242 [63], Torin2 [64] and the dual mTOR/PI3K inhibitor BEZ235 (Novartis) have been developed based on mTOR homology models, derived from PI3K structures. In a breakthrough effort, determinants of the potency and specificity of the mTOR

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Fig. 6. The mTOR kinase inhibitors. (A) A structural model of the quaternary complex consisting of mTOR, rapamycin, FKBP12 and mLST8. The structure of the ternary complex mFRB·rapamycin·FKBP12 [60] was superimposed onto the FRB of mTOR·ATP␥S·Mg2+ ·mLST8 kinase domain [22]. FKBP12 is shown as an orange surface. The mTOR kinase domain elements are colored as in Fig. 2 and labeled accordingly. The disordered loop between k␣9b and k␣10 is shown as a dotted line. (B) Close-up view of the ATP-binding pocket in mTOR in complex with the ATP␥S. (C) Torin2 interacting with mTOR (pale green = N-lobe, green = C-lobe). Orange dashed lines indicate ␲-stacking interactions, yellow curves hydrophobic interactions and pink dashed lines indicate a hydrogen bond. The compound does not make hydrogen bonds with Asp2195 , Asp2357 and Tyr2225 , as originally predicted based on the homology model [64]. (D) The PP242-mTOR structure depicted as in (C). The red arrow shows disposition of Tyr2225 that changes conformation on binding to the inhibitor. Both inhibitors make hydrogen bonds with the ‘hinge’ between Trp2239 (N-lobe) and Val2240 (C-lobe), which is typical for ATP-competitive inhibitors of the PI3Ks. Models prepared in PyMOL (PDB entry: 4JSP [mTOR·ATP␥S·Mg2+ ·mLST8], 4JSX [Torin2 complex] and 4JT5 [PP242 complex]).

inhibitors PP242 and Torin2 have been recently examined by Xray crystallography [22]. This study shows that a key interaction responsible for the 800-fold specificity of Torin2 for mTOR over PI3K [64] is a stacking of its tricyclic ring (benzonaphthyridinone) with Trp2239 in the ATP-binding site (although invariant among mTOR orthologues, the equivalent residue in the PI3Ks is always hydrophobic, but not Trp) as well as less extensive but significant interactions of the trifluoromethyl group with the specificity pocket (N-lobe) (Fig. 6C). Notably, the Torin2 complex revealed in the crystal structure [22] lacked several of the hydrogen bonding interactions that were proposed based on a homology model of mTOR kinase domain [64]. Unexpectedly, the crystal structures also showed that the inhibitor PP242 induces a conformational change in the ATP binding site where Tyr2225 swings out of the way of the hydroxyindole group of PP242 so as to deepen and expand the inner hydrophobic pocket where the inhibitor binds (Fig. 6D). When at low concentration, ATP-competitive inhibitors of mTOR in combination with rapamycin show a synergistic effect

in selectively blocking the activity of mTORC1, but not mTORC2, as evidenced in different cell lines [65]. This synergy is consistent with the combination blocking binding of both substrates (protein and ATP) in the active site. 6. Conclusion and perspective The mammalian target of rapamycin regulates eukaryotic cell growth and stress responses. Only recently, we have learned great details about the structural organization of its protein kinase domain. Although closely related to the catalytic domain of the PI3Ks, the bilobal mTOR kinase domain is about 200 residues larger, with unique and essential elements: the FRB acting as the gatekeeper and recruiting substrate; the LBE interacting with both FATC and mLST8; the 12-residue k␣9b protecting active site and participating in regulation; and the 35-residue FATC forming part of the substrate binding site. The 3D structures have already shown the basis of two distinct ATP-competitive inhibitors, led to a

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reinterpretation of the mode of inhibition by rapamycin and underscored the limitations of homology models in structure-based inhibitor design. Given that the mTOR signaling pathway is target of many clinical trials [62], these structures will be of great benefit in refining future designs. The additional domain that stabilizes the mTOR catalytic skeleton is the ␣-helical solenoid known as FAT that might also have an essential scaffolding function. Importantly, the accessory protein mLST8 was shown to be crucial for the mTOR kinase domain stability [7,22]. Unlike the PI3Ks and most classical protein kinases that transition between the catalytically active (open) and inactive (closed) stage, the mTOR kinase domain, surprisingly, appears to be inherently active (open). This is possible because the mTOR catalytic site is protected and the substrate is carefully selected. The mTOR activity is controlled by a bipartite substrate recruitment mechanism involving the FRB and the RAPTOR regulatory subunit [22]. In this way, the substrate is recognized via its FRB-binding motif on one side and the TOS motif on the other, before it is positioned for phosphorylation. Remarkably, the structure is consistent with the classical allosteric mTOR inhibitor, rapamycin, being a competitive inhibitor of substrate binding. The low resolution EM model of mTORC1 provides a hint about its overall architecture suggesting a dimer, which could form via interaction with RAPTOR and which disassembles in presence of rapamycin [12]. However, we do not know how RAPTOR talks to mTOR-mLST8, what is the physiological significance of dimerization of mTORC1 or how is the substrate recruitment and localization coupled to the activation by RHEB [57,66] or inhibition by DEPTOR [8]. Even more elusive is the nature of the interaction of RICTOR/mSIN1 subunits within the mTORC2, that are thought to be significantly more stable in presence of rapamycin, in both yeast and mammals [3,5]. The mTOR kinase is a member of a small PIKK family, whose representatives (DNA-PKcs, ATM, SMG-1, ATR and TRRAP) share many of the same structural features, especially the highly conserved kinase domain. Careful analysis by Yang and colleagues showed that the kinase domains of DNA-PKcs, SMG-1 and TRRAP all possess an FRB-domain. A very recent EM structure of the SMG-1, involved in the nonsense-mediated mRNA decay, indicates that the enzyme uses its FRB domain to recruit an appropriate factor (UPF2) to the vicinity of the kinase domain [21], similar to what was observed for mTOR. Although less conserved in length and sequence [18], the helical repeats are common among the PIKKs. This is well reflected in the crystal structure of DNA-PKcs (6.6 A˚ resolution) [20] and the EM structure of SMG-1 [21], where the long arrays of helical repeats position in front of the catalytic cleft ready to recruit specific binding partners and substrates. The same property of the helical solenoid might be expected for the mTOR kinase. The highresolution structural studies of the full-length mTOR in complex with the specific subunits will provide greater understating of the regulation of mTOR complexes, but will also well inform structural attempts in studying other members of the PIKK family. Acknowledgements DB was supported by an LMB Cambridge International Studentship, the Cambridge Overseas Trust and Trinity College Cambridge. This work was funded by UK MRC (MC U105184308 to RLW). References [1] Betz C, Hall MN. Where is mTOR and what is it doing there? J Cell Biol 2013;203:563–74. [2] Hara K, Maruki Y, Long X, Yoshino K, Oshiro N, Hidayat S, et al. Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action. Cell 2002;110:177–89.

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