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Evaluation of the Binding Kinetics of RHEB with mTORC1 by In-Cell and In Vitro Assays

1
Emergent Bioengineering Materials Research Team, RIKEN Center for Emergent Matter Science, RIKEN, Wako 351-0198, Saitama, Japan
2
Department of Life Science, Graduate School of Science and Engineering, Saitama University, Saitama City 338-8570, Saitama, Japan
3
Nano Medical Engineering Laboratory, RIKEN Cluster for Pioneering Research, RIKEN, Wako 351-0198, Saitama, Japan
*
Author to whom correspondence should be addressed.
Academic Editor: Sławomir Filipek
Int. J. Mol. Sci. 2021, 22(16), 8766; https://doi.org/10.3390/ijms22168766
Received: 10 June 2021 / Revised: 4 August 2021 / Accepted: 13 August 2021 / Published: 16 August 2021
(This article belongs to the Section Biochemistry)
The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) is activated by the small G-protein, Ras homolog enriched in brain (RHEB–GTPase). On lysosome, RHEB activates mTORC1 by binding the domains of N-heat, M-heat, and the focal adhesion targeting (FAT) domain, which allosterically regulates ATP binding in the active site for further phosphorylation. The crucial role of RHEB in regulating growth and survival through mTORC1 makes it a targetable site for anti-cancer therapeutics. However, the binding kinetics of RHEB to mTORC1 is still unknown at the molecular level. Therefore, we studied the kinetics by in vitro and in-cell protein–protein interaction (PPI) assays. To this end, we used the split-luciferase system (NanoBiT®) for in-cell studies and prepared proteins for the in vitro measurements. Consequently, we demonstrated that RHEB binds to the whole mTOR both in the presence or absence of GTPγS, with five-fold weaker affinity in the presence of GTPγS. In addition, RHEB bound to the truncated mTOR fragments of N-heat domain (∆N, aa 60–167) or M-heat domain (∆M, aa 967–1023) with the same affinity in the absence of GTP. The reconstructed binding site of RHEB, ∆N-FAT-M, however, bound to RHEB with the same affinity as ∆N-M, indicating that the FAT domain (∆FAT, aa 1240–1360) is dispensable for RHEB binding. Furthermore, RHEB bound to the truncated kinase domain (∆ATP, aa 2148–2300) with higher affinity than to ∆N-FAT-M. In conclusion, RHEB engages two different binding sites of mTOR, ∆N-FAT-M and ∆ATP, with higher affinity for ∆ATP, which likely regulates the kinase activity of mTOR through multiple different biding modes. View Full-Text
Keywords: mTORC1; RHEB; G-Protein; allosteric activation; kinase domain; binding kinetics mTORC1; RHEB; G-Protein; allosteric activation; kinase domain; binding kinetics
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MDPI and ACS Style

Shams, R.; Ito, Y.; Miyatake, H. Evaluation of the Binding Kinetics of RHEB with mTORC1 by In-Cell and In Vitro Assays. Int. J. Mol. Sci. 2021, 22, 8766. https://doi.org/10.3390/ijms22168766

AMA Style

Shams R, Ito Y, Miyatake H. Evaluation of the Binding Kinetics of RHEB with mTORC1 by In-Cell and In Vitro Assays. International Journal of Molecular Sciences. 2021; 22(16):8766. https://doi.org/10.3390/ijms22168766

Chicago/Turabian Style

Shams, Raef, Yoshihiro Ito, and Hideyuki Miyatake. 2021. "Evaluation of the Binding Kinetics of RHEB with mTORC1 by In-Cell and In Vitro Assays" International Journal of Molecular Sciences 22, no. 16: 8766. https://doi.org/10.3390/ijms22168766

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