Structure-guided mutation of the conserved G3-box glycine in Rheb generates a constitutively activated regulator of mTOR [Molecular Biophysics]

March 19th, 2014 by Mazhab-Jafari, M. T., Marshall, C. B., Ho, J., Ishiyama, N., Stambolic, V., Ikura, M.

Constitutively activated variants of small GTPases, which provide valuable functional probes of their role in cellular signaling pathways, can often be generated by mutating the canonical catalytic residue (e.g., RasQ61L) to impair GTP hydrolysis. However, this general approach is ineffective for a substantial fraction of the small GTPase family in which this residue is not conserved (e.g., Rap) or not catalytic (e.g., Rheb). Using a novel engineering approach, we have manipulated nucleotide binding through structure-guided substitutions of an ultra-conserved glycine residue in the G3-box motif (DxxG). Substitution of Rheb Gly63 with alanine impaired both intrinsic and TSC2 GTPase activating protein (GAP)-mediated GTP hydrolysis by displacing the hydrolytic water molecule, while introduction of a bulkier valine sidechain selectively blocked GTP binding by steric occlusion of the γ-phosphate. Rheb G63A stimulated phosphorylation of the mTORC1 substrate p70S6 kinase more strongly than wild-type, thus offering a new tool for mTOR signaling.
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