Structural Insights of Mtor Complex 1 Cell Research (2016) 26:267-268

Structural insights of mTOR complex 1 Cell Research (2016) 26:267-268. doi:10.1038/cr.2016.10; published online 22 January 2016

The mammalian target of ra- understanding of mTORC1 activation the reconstruction of mTORC1, the pamycin (mTOR), also known as and substrate selectivity are rather authors also resolved the structure of the mechanistic target of rapamycin, limited, chiefly due to the lack of three the fungus Chaetomium thermophilum is a central cell growth regulating dimensional structure of mTORC1. Raptor (CtRaptor), which exhibits 44% kinase that forms large molecular Understanding of mTORC1 molecular sequence identity to human Raptor. complexes in all eukaryotic cells. A architecture is also of high importance The overall shape of the reconstruc- paper recently published in Science for developing pharmacological drugs tion agrees with that observed by reports the architecture of mTOR to target this pathway. low-resolution cyro-EM [4]; however, complex 1 (mTORC1) and provides Cryo-electron microscopy (cryo- it appears that the handedness of the molecular insights into the regulation EM) studies have shown that mTOR previous reconstruction was not as- and substrate selectivity of mTORC1. forms an obligate dimer with an overall signed correctly. Generally, mTORC1 The mammalian target of rapamy- rhomboid shape and a central cav- adopts a cage-like, dimeric architecture cin (mTOR) exists in two different ity [4]. However, the reliability of the and appears in a hollow lozenge shape, complexes, mTORC1 and mTORC2, handedness of the reconstruction and in which Raptor and mLST8 contribute which are distinguished by unique the position of individual subunits was peripheral parts of the complex and accessory protein Raptor and Rictor, significantly compromised due to the make up the pinnacles of the longer and respectively [1]. Rapamycin is an low-resolution (26 Å) reconstruction. shorter axes of the lozenge, respectively. mTORC1-specific inhibitor, which A subsequent study presented the 3.2 Interestingly, the N-terminus of mTOR, complexes with the FK506-binding Å crystal structure of a complex of which was not resolved in previous 12 kDa protein (FKBP12) and inhibits N-terminally truncated human mTOR study [5], contains two α-helical so- Raptor-bound, but not Rictor-bound, and mLST8, which is a subunit com- lenoids. The larger section is a highly mTOR. Rapamycin analogs have been monly present in both mTORC1 and curved super-helix, which is named the used clinically to treat a number of hu- mTORC2. This crystal structure re- “horn”, while the smaller region adopts man diseases, including cancer [2]. A vealed more details of the structure a relatively linear arrangement and is wide range of both extra- and intracel- of mTOR kinase domain as well as its referred to as the “bridge” [6]. Both lular signals, including growth factors, inhibition by FKBP12-rapamycin com- sections are predominantly exposed to nutrient status and stress conditions, plex [5]. However, information on the the environment, indicating a potential have been shown to regulate mTORC1 subunit arrangement within mTORC1 role in binding mTOR regulators. In to control cell growth. Most notably, was missing because only a truncated addition, the horn and bridge HEAT mTORC1 is hyperactivated by onco- mTOR fragment was analyzed and the domains pack against one another, and genic PI3K-Akt signaling and promotes Raptor subunit, which plays a key role the first HEAT repeat of the horn region tumor growth [1]. in mTORC1 regulation and substrate interlocks with the adjacent mTOR FAT mTORC1 promotes cell growth selectivity, was lacking. domain, through which the two mTOR through phosphorylation of a large In a recent paper published in Science, subunits forms a dimer independent number of cellular proteins, including the architecture of human mTORC1 was of Raptor [6]. Another interesting ob- the ribosomal S6 kinase 1 (S6K1) and revealed by high-resolution cryo-EM servation is that the conformation of eIF-4E-binding protein 1 (4E-BP1) [3]. [6]. The authors purified mTORC1 the kinase domain appears unaffected Although mTORC2 shares the same complex (human mTOR together by dimerization, suggesting that the catalytic kinase subunit with mTORC1, with Raptor, and mLST8) bound to regulation of mTORC1 may be mainly it phosphorylates substrates very differ- FKBP12-rapamycin from insect cells. through controlling substrate access to ent from those of mTORC1 and thus ex- Single particle analysis of cryo-EM the active site. erts different cellular functions. Despite yielded a reconstruction with an overall The authors further investigated how the extensive studies, the mechanistic resolution of 5.9 Å. To complement Raptor contributes to the formation of npg 268 kinase domain of mTOR maintains a constitutively active conformation at all times. Association with Raptor lim- its substrate accessibility to the mTOR kinase active site as the Raptor RNC domain is positioned directly at the mTOR active site cleft, thereby explaining how Raptor modulates substrate selectivity of mTORC1. Furthermore, the new structure explains mTORC1 inhibition by FKBP12-rapamycin through block- Figure 1 Schematic model of mTORC1 substrate selectivity and delivery. (A) Sub- ing substrate accessibility to the mTOR strate recruitment is dependent on their TOR signaling (TOS) motif binding to Rap- tor TOS-binding site. mTOR FRB domain and mLST8 prevent phosphorylation to- kinase active site. It should be noted that wards non-cognate substrates by limiting the access to the ATP-binding cleft. Rap- the architecture of mTORC1 still needs tor binding further restricts the access to the active site, resulting in the enclosure further improvement as the current reso- of the active site cleft from all directions. Only when the TOS motif present in the lution (5.9 Å) is not sufficient to reveal substrate is recognized by Raptor, substrates can be delivered to the mTOR kinase active site and phosphorylated. (B) The FKBP12-rapamycin complex binding to the amino acid side chains of subunits and FRB domain further blocks the active site cleft and prevents the access of substrate critical sites for dimer formation and to kinase active site. activity control.

Hai-Xin Yuan1, Kun-Liang Guan2 mTORC1 complex. Raptor interacts ing access to the ATP-binding cleft [5]. with mTOR through an α-solenoid stack According to the current architecture, 1Molecular Cell Biology Laboratory, Institutes of formed between the horn and bridge Raptor binding further restricts the Biomedical Sciences, Shanghai Medical College, domains of mTOR via the Raptor ar- access to the active site, resulting in Fudan University, Shanghai 200032, China; 2De- madillo domain [6]. It is proposed that the enclosure of the active site cleft partment of Pharmacology and Moores Cancer Center, University of California, San Diego, La Raptor stabilizes the N-terminal region from all directions and reduction of its Jolla, CA 92093, USA of mTOR by providing roughly two- width to ~20 Å [6]. Moreover, binding Correspondence: Hai-Xin Yuana, thirds of the interaction surface with of FKBP12-rapamycin complex to the Kun-Liang Guanb a HEAT domains. As mentioned above, FRB domain of mTORC1 further re- E-mail: [email protected] bE-mail: [email protected] the formation of mTORC1 dimer is duces the active site cleft to ~10 Å [6]. dependent on interaction of mTOR Taken together, this model shows how References domains, but not Raptor, thus a model architectural subunits of mTORC1 and is suggested that Raptor binding may FKBP12-rapamycin limit access to the 1 Laplante M, Sabatini DM. Cell 2012; stabilize mTOR N-terminal conforma- recessed mTOR active site (Figure 1). 149:274-293. 2 Benjamin D, Colombi M, Moroni C, et al. tion without directly engaging in dimer It is notable that in contrary to previous Nat Rev Drug Discov 2011; 10:868-880. formation. findings [4], this study indicates that 3 Ma XM, Blenis J. Nat Rev Mol Cell Bio The structure of mTORC1 also FKBP12-rapamycin binding has no ef- 2009; 10:307-318. provides implications of mTORC1 sub- fect on mTORC1 stability. 4 Yip CK, Murata K, Walz T, et al. Mol Cell 2010; 38:768-774. strate selectivity and delivery. Previous In summary, the new structure 5 Yang H, Rudge DG, Koos JD, et al. Nature report has revealed that mTOR FRB reveals insights into the mTORC1 2013; 497:217-223. domain and mLST8 prevent activity architecture and important clues for 6 Aylett CH, Sauer E, Imseng S, et al. Science toward non-cognate substrates by limit- mTORC1 functional regulation. The 2016; 351:48-52.

Cell Research | Vol 26 No 3 | March 2016