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

Huirong Yang,Dan Tan,Catherine C L Wong,Mengjie Liu,Yanhui Xu,Jiawei Wang,Hong-Wei Wang,Jia Wang,Min Huang,Meng-Qiu Dong,Xizi Chen

doi:10.1007/s13238-016-0346-6

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-016-0346-6) contains supplementary material, which is available to authorized users.

Highlights

Mechanistic target of rapamycin is a Ser/Thr kinase that belongs to the family of phosphoinositide-3-kinase-related kinases (PIKK) and is structurally and functionally conserved from yeast to mammals. mTOR exists in two distinct protein complexes: mTOR complex 1 and mTOR complex 2, which share two core components, the mTOR protein and the mammalian lethal with SEC13 protein 8. mTORC1 contains a unique subunit, regulatory-associated protein of mTOR (Raptor), whereas mTORC2 is defined by rapamycin insensitive companion of mTOR (Rictor)
The mTORC1 was transiently expressed with myc-mTOR, Flag-Raptor, Flag-mLST8 co-transfected into HEK293F cells in suspension culture
The purified mTORC1 consists of mTOR, Raptor, and mLST8 in stoichiometry and exhibits kinase activity on S6 Kinase 1 (S6K1) and 4E (eIF4E) binding protein 1 (4EBP1), which can be FRB + Kinase domain

Summary

Introduction

Mechanistic target of rapamycin (mTOR) is a Ser/Thr kinase that belongs to the family of phosphoinositide-3-kinase-related kinases (PIKK) and is structurally and functionally conserved from yeast to mammals. mTOR exists in two distinct protein complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), which share two core components, the mTOR protein and the mammalian lethal with SEC13 protein 8 (mLST8, known as GβL). mTORC1 contains a unique subunit, regulatory-associated protein of mTOR (Raptor), whereas mTORC2 is defined by rapamycin insensitive companion of mTOR (Rictor). Mechanistic target of rapamycin (mTOR) is a Ser/Thr kinase that belongs to the family of phosphoinositide-3-kinase-related kinases (PIKK) and is structurally and functionally conserved from yeast to mammals. MTORC1 contains a unique subunit, regulatory-associated protein of mTOR (Raptor), whereas mTORC2 is defined by rapamycin insensitive companion of mTOR (Rictor). Rapamycin inhibits mTORC1 by forming a complex with immunophilin FKBP12 (12 kDa FK506-binding protein) (Loewith et al, 2002; Sarbassov et al, 2004). In response to multiple growth factors, energy status, and stress pathways, Tuberous Sclerosis Complex 1/2 (TSC1/2). 4.4 Å Resolution Cryo-EM structure of human mTORC1.

Methods

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Conclusion