Article | Published:

Cryo-EM structure of human mTOR complex 2

Cell Researchvolume 28pages518528 (2018) | Download Citation


Mechanistic target of rapamycin (mTOR) complex 2 (mTORC2) plays an essential role in regulating cell proliferation through phosphorylating AGC protein kinase family members, including AKT, PKC and SGK1. The functional core complex consists of mTOR, mLST8, and two mTORC2-specific components, Rictor and mSin1. Here we investigated the intermolecular interactions within mTORC2 complex and determined its cryo-electron microscopy structure at 4.9 Å resolution. The structure reveals a hollow rhombohedral fold with a 2-fold symmetry. The dimerized mTOR serves as a scaffold for the complex assembly. The N-terminal half of Rictor is composed of helical repeat clusters and binds to mTOR through multiple contacts. mSin1 is located close to the FRB domain and catalytic cavity of mTOR. Rictor and mSin1 together generate steric hindrance to inhibit binding of FKBP12-rapamycin to mTOR, revealing the mechanism for rapamycin insensitivity of mTORC2. The mTOR dimer in mTORC2 shows more compact conformation than that of mTORC1 (rapamycin sensitive), which might result from the interaction between mTOR and Rictor-mSin1. Structural comparison shows that binding of Rictor and Raptor (mTORC1-specific component) to mTOR is mutually exclusive. Our study provides a basis for understanding the assembly of mTORC2 and a framework to further characterize the regulatory mechanism of mTORC2 pathway.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    Heitman, J., Movva, N. R. & Hall, M. N. Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science 253, 905–909 (1991).

  2. 2.

    Sabatini, D. M., Erdjument-Bromage, H., Lui, M., Tempst, P. & Snyder, S. H. RAFT1: a mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs. Cell 78, 35–43 (1994).

  3. 3.

    Loewith, R. et al. Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. Mol. Cell 10, 457–468 (2002).

  4. 4.

    Wedaman, K. P. et al. Tor kinases are in distinct membrane-associated protein complexes in Saccharomyces cerevisiae. Mol. Biol. Cell 14, 1204–1220 (2003).

  5. 5.

    Saxton, R. A. & Sabatini, D. M. mTOR signaling in growth, metabolism, and disease. Cell 168, 960–976 (2017).

  6. 6.

    Wullschleger, S., Loewith, R. & Hall, M. N. TOR signaling in growth and metabolism. Cell 124, 471–484 (2006).

  7. 7.

    Sarbassov, D. D., Guertin, D. A., Ali, S. M. & Sabatini, D. M. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307, 1098–1101 (2005).

  8. 8.

    Sarbassov, D. D. et al. Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr. Biol. 14, 1296–1302 (2004).

  9. 9.

    Garcia-Martinez, J. M. & Alessi, D. R. mTOR complex 2 (mTORC2) controls hydrophobic motif phosphorylation and activation of serum- and glucocorticoid-induced protein kinase 1 (SGK1). Biochem. J. 416, 375–385 (2008).

  10. 10.

    Sparks, C. A. & Guertin, D. A. Targeting mTOR: prospects for mTOR complex 2 inhibitors in cancer therapy. Oncogene 29, 3733–3744 (2010).

  11. 11.

    Kim, D. H. et al. GbetaL, a positive regulator of the rapamycin-sensitive pathway required for the nutrient-sensitive interaction between raptor and mTOR. Mol. Cell 11, 895–904 (2003).

  12. 12.

    Kim, D. H. et al. mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 110, 163–175 (2002).

  13. 13.

    Hara, K. et al. Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action. Cell 110, 177–189 (2002).

  14. 14.

    Jacinto, E. et al. Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat. Cell Biol. 6, 1122–1128 (2004).

  15. 15.

    Frias, M. A. et al. mSin1 is necessary for Akt/PKB phosphorylation, and its isoforms define three distinct mTORC2s. Curr. Biol. 16, 1865–1870 (2006).

  16. 16.

    Jacinto, E. et al. SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity. Cell 127, 125–137 (2006).

  17. 17.

    Baretic, D. & Williams, R. L. PIKKs — the solenoid nest where partners and kinases meet. Curr. Opin. Struct. Biol. 29, 134–142 (2014).

  18. 18.

    Keith, C. T. & Schreiber, S. L. PIK-related kinases: DNA repair, recombination, and cell cycle checkpoints. Science 270, 50–51 (1995).

  19. 19.

    Yang, H. et al. mTOR kinase structure, mechanism and regulation. Nature 497, 217–223 (2013).

  20. 20.

    Liu, P. et al. PtdIns(3,4,5)P3-dependent activation of the mTORC2 kinase complex. Cancer Discov. 5, 1194–1209 (2015).

  21. 21.

    Aylett, C. H. et al. Architecture of human mTOR complex 1. Science 351, 48–52 (2016).

  22. 22.

    Baretic, D., Berndt, A., Ohashi, Y., Johnson, C. M. & Williams, R. L. Tor forms a dimer through an N-terminal helical solenoid with a complex topology. Nat. Commun. 7, 11016 (2016).

  23. 23.

    Yang, H. et al. 4.4 A Resolution cryo-EM structure of human mTOR complex 1. Protein Cell 7, 878–887 (2016).

  24. 24.

    Yang, H. et al. Mechanisms of mTORC1 activation by RHEB and inhibition by PRAS40. Nature 552, 368–373 (2017).

  25. 25.

    Gaubitz, C. et al. Molecular basis of the rapamycin insensitivity of target of rapamycin complex 2. Mol. Cell 58, 977–988 (2015).

  26. 26.

    Karuppasamy, M. et al. Cryo-EM structure of Saccharomyces cerevisiae target of rapamycin complex 2. Nat. Commun. 8, 1729 (2017).

  27. 27.

    Gaubitz, C., Prouteau, M., Kusmider, B. & Loewith, R. TORC2 structure and function. Trends Biochem. Sci. 41, 532–545 (2016).

  28. 28.

    Zhou, P., Zhang, N., Nussinov, R. & Ma, B. Defining the domain arrangement of the mammalian target of rapamycin complex component Rictor protein. J. Comput. Biol. 22, 876–886 (2015).

  29. 29.

    Choi, J., Chen, J., Schreiber, S. L. & Clardy, J. Structure of the FKBP12-rapamycin complex interacting with the binding domain of human FRAP. Science 273, 239–242 (1996).

  30. 30.

    Tatebe, H. et al. Substrate specificity of TOR complex 2 is determined by a ubiquitin-fold domain of the Sin1 subunit. Elife 6, (2017).

  31. 31.

    Furuita, K. et al. Utilization of paramagnetic relaxation enhancements for high-resolution NMR structure determination of a soluble loop-rich protein with sparse NOE distance restraints. J. Biomol. NMR 61, 55–64 (2015).

  32. 32.

    Cameron, A. J., Linch, M. D., Saurin, A. T., Escribano, C. & Parker, P. J. mTORC2 targets AGC kinases through Sin1-dependent recruitment. Biochem. J. 439, 287–297 (2011).

  33. 33.

    Kastner, B. et al. GraFix: sample preparation for single-particle electron cryomicroscopy. Nat. Methods 5, 53–55 (2008).

  34. 34.

    Yang, B. et al. Identification of cross-linked peptides from complex samples. Nat. Methods 9, 904–906 (2012).

  35. 35.

    Combe, C. W., Fischer, L. & Rappsilber, J. xiNET: cross-link network maps with residue resolution. Mol. Cell Proteom. 14, 1137–1147 (2015).

  36. 36.

    Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

  37. 37.

    Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

  38. 38.

    Rohou, A. & Grigorieff, N. CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

  39. 39.

    Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

  40. 40.

    Pettersen, E. F. et al. UCSF Chimera — a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

  41. 41.

    Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014).

  42. 42.

    Rossmann, M. G., Bernal, R. & Pletnev, S. V. Combining electron microscopic with X-ray crystallographic structures. J. Struct. Biol. 136, 190–200 (2001).

  43. 43.

    Emsley, P., Lohkamp, B., Scott, W. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

  44. 44.

    Adams, P. D. et al PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

  45. 45.

    Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr 26, 283–291 (1993).

  46. 46.

    Vriend, G. WHAT IF: a molecular modeling and drug design program. J. Mol. Graph. 8, 52–56 (1990).

Download references


We thank Center of Cryo-Electron Microscopy, Zhejiang University School of Medicine, Center for Biological Imaging of Institute of Biophysics (IBP) of Chinese Academy of Sciences (CAS), and National Center for Protein Science Shanghai (NCPSS) for the support on cryo-EM data collection and analyses. We thank the staff members at Biomedical Core Facility, Fudan University and NCPSS for their help with mass spectrometry analyses. This work was supported by the Ministry of Science and Technology of China (2016YFA0500700, 2016YFA0501100), the National Natural Science Foundation of China (31770781, U1432242, 31425008, 91419301), the National Program for support of Top-Notch Young Professionals (Y.X.), and the Strategic Priority Research Program of CAS (XDB08000000).

Author information

Author notes

  1. These authors contributed equally: Xizi Chen, Mengjie Liu In PDF, please change running title to "Cryo-EM structure of human mTORC2"


  1. Fudan University Shanghai Cancer Center, Institutes of Biomedical Sciences, Shanghai Medical College of Fudan University, Shanghai, 200032, China

    • Xizi Chen
    • , Mengjie Liu
    • , Yuan Tian
    • , Jiabei Li
    • , Yilun Qi
    • , Dan Zhao
    • , Zihan Wu
    • , Huirong Yang
    •  & Yanhui Xu
  2. Key Laboratory of Molecular Medicine, Ministry of Education, Department of Systems Biology for Medicine, School of Basic Medical Sciences, Shanghai Medical College of Fudan University, Shanghai, 200032, China

    • Xizi Chen
    • , Mengjie Liu
    • , Yuan Tian
    • , Jiabei Li
    • , Yilun Qi
    • , Dan Zhao
    • , Zihan Wu
    • , Huirong Yang
    •  & Yanhui Xu
  3. State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, School of Life Sciences, Fudan University, Shanghai, 200433, China

    • Xizi Chen
    • , Mengjie Liu
    • , Yuan Tian
    • , Jiabei Li
    • , Yilun Qi
    • , Dan Zhao
    • , Zihan Wu
    • , Huirong Yang
    •  & Yanhui Xu
  4. National Center for Protein Science Shanghai, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 201210, China

    • Min Huang
    •  & Catherine C. L. Wong
  5. Center for Precision Medicine Multi-Omics Research (CPMMOR), Peking University Health Science Center, Beijing, 100871, China

    • Catherine C. L. Wong
  6. State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Science, Peking University, Beijing, 100871, China

    • Catherine C. L. Wong
  7. Ministry of Education Key Laboratory of Protein Sciences, Tsinghua-Peking Joint Center for Life Sciences, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, 100084, China

    • Hong-Wei Wang
    •  & Jiawei Wang
  8. CAS Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, 200031, China

    • Yanhui Xu


  1. Search for Xizi Chen in:

  2. Search for Mengjie Liu in:

  3. Search for Yuan Tian in:

  4. Search for Jiabei Li in:

  5. Search for Yilun Qi in:

  6. Search for Dan Zhao in:

  7. Search for Zihan Wu in:

  8. Search for Min Huang in:

  9. Search for Catherine C. L. Wong in:

  10. Search for Hong-Wei Wang in:

  11. Search for Jiawei Wang in:

  12. Search for Huirong Yang in:

  13. Search for Yanhui Xu in:


X.C., M.L., H.Y., and Y.X. designed the experiments. X.C., J.L., and D.Z. purified the proteins. M.L., Y.T., Y.Q., H-W.W., and Z.W. prepared the cryo-EM sample, collected the data and determined the structure. J.W. built the structural model. X.C. and H.Y. performed biochemical analyses. X.C., M.H., and C.L. performed cross-linking MS and analyzed the data. X.C., M.L., H.Y., and Y.X. analyzed the data and wrote the manuscript. Y.X. supervised the project.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Huirong Yang or Yanhui Xu.

Electronic supplementary material

About this article

Publication history





Issue Date


Further reading