Evaluation Of The Binding Kinetics Of Rheb With Mtorc1 By In-Cell And In Vitro Assays

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(R)) 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 GTP gamma S, with five-fold weaker affinity in the presence of GTP gamma S. In addition, RHEB bound to the truncated mTOR fragments of N-heat domain ( increment N, aa 60-167) or M-heat domain ( increment M, aa 967-1023) with the same affinity in the absence of GTP. The reconstructed binding site of RHEB, increment N-FAT-M, however, bound to RHEB with the same affinity as increment N-M, indicating that the FAT domain ( increment FAT, aa 1240-1360) is dispensable for RHEB binding. Furthermore, RHEB bound to the truncated kinase domain ( increment ATP, aa 2148-2300) with higher affinity than to increment N-FAT-M. In conclusion, RHEB engages two different binding sites of mTOR, increment N-FAT-M and increment ATP, with higher affinity for increment ATP, which likely regulates the kinase activity of mTOR through multiple different biding modes.