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Abstract: PO1094

Structural Determinants of mTORC2 Substrate Specificity and SGK1 Phosphorylation Revealed by Cryogenic Electron Microscopy

Session Information

Category: Fluid, Electrolyte, and Acid-Base Disorders

  • 901 Fluid, Electrolyte, and Acid-Base Disorders: Basic

Authors

  • Chen, Junliang, SinoUnited Health, Shanghai, China
  • Yu, Zanlin, University of California San Francisco, San Francisco, California, United States
  • Wang, Feng, University of California San Francisco, San Francisco, California, United States
  • Liu, Xi, University of California San Francisco, San Francisco, California, United States
  • Takagi, Enzo, University of California San Francisco, San Francisco, California, United States
  • Joubert, Lydia-Marie, Stanford University, Stanford, California, United States
  • Jin, Mingliang, University of California San Francisco, San Francisco, California, United States
  • Nowotny, Carlos, University of California San Francisco, San Francisco, California, United States
  • Agard, David, University of California San Francisco, San Francisco, California, United States
  • Cheng, Yifan, University of California San Francisco, San Francisco, California, United States
  • Pearce, David, University of California San Francisco, San Francisco, California, United States
Background

mTORC2 is a multi-subunit kinase complex central to multiple essential signaling pathways. Notably, it responds to hormonal signals and local electrolyte concentrations to phosphorylate SGK1 and regulate K+ secretion in the renal tubules. Two core subunits, Rictor and mSin1 distinguish mTORC2 from its much better characterized relative, mTORC1. Two other subunits, mTOR itself and a small scaffold, mLST8, complete the core complex. Previous mTORC2 reconstructions have lacked key regions of the > 1 MDa complex, particularly determinants of specificity.

Methods

Core mTORC2 subunits were expressed in Expi293F cells and purified using new methods for on grid purification. cryo-EM was peformed using Krios at SLAC for high energy electrons for density maps of human mTORC2. Structures were solved for apo-complex at overall 3.23 Å resolution, and for co-complexes with substrates, SGK1 and Akt, at 3.38 and 3.44 Å, respectively.

Results

The apo-complex reveals architectural features of functionally important domains, including specific side chain positions and interactions, which are visualized for the first time. In particular Rictor/Ser-1624 and Ser-1625 were observed to engage in hydrogen bond interactions with mTOR/Thr-2098, in a manner that provides steric hindrance to binding of Rapamycin, and explains mTORC2 resistance to the effects of this clinically important mTORC1 inhibitor. In addition, mSin1, the other defining subunit of mTORC2, is seen to form extensive contacts with Rictor, including an extended strand, which makes multiple weak contacts with a Rictor helical cluster. Most notably for the role mTORC2 plays in renal electrolyte handling, in the co-complex structure with SGK1—but not the Akt co-complex, we see a marked change in the conformation of the mSin1 N-terminal extended strand in a manner consistent with previous functional data identifying this region as required for phosphorylation of SGK1, but not Akt, thus providing a structural basis for differential regulation.

Conclusion

These findings provide new structural insight into mTORC2 specificity and context-dependent activities, and foundation for further mechanistic studies. Further, these findings provide a potential avenue toward highly selective mTOR modulators with potential clinical utility.

Funding

  • NIDDK Support