Structure-guided mutation of the conserved G3-box glycine in Rheb generates a constitutively activated regulator of mammalian target of rapamycin (mTOR).

ABSTRACT: 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. Ras Q61L) 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 ultraconserved glycine residue in the G3-box motif (DXXG). Substitution of Rheb Gly-63 with alanine impaired both intrinsic and TSC2 GTPase-activating protein (GAP)-mediated GTP hydrolysis by displacing the hydrolytic water molecule, whereas introducti

SUBMITTER: Mazhab-Jafari MT 

PROVIDER: S-EPMC4007420 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature