title: 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 introduction of a bulkier valine side chain 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 mammalian target of rapamycin (mTOR) signaling.
authors.affiliation: From the Campbell Family Cancer Research Institute, Princess Margaret Cancer Centre and, Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 2M9, Canada.
authors.forename: Mohammad T
authors.initials: MT
authors.lastname: Mazhab-Jafari
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Marshall
authors.forename: Jason
authors.initials: J
authors.lastname: Ho
authors.forename: Noboru
authors.initials: N
authors.lastname: Ishiyama
authors.forename: Vuk
authors.initials: V
authors.lastname: Stambolic
authors.forename: Mitsuhiko
authors.initials: M
authors.lastname: Ikura
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D018360:Crystallography, X-Ray; D005998:Glycine / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D000076222:Mechanistic Target of Rapamycin Complex 1; D051379:Mice; D008958:Models, Molecular; D020559:Monomeric GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009479:Neuropeptides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D000076205:Ras Homolog Enriched in Brain Protein; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D058570:TOR Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism
chemical_list: D046912:Multiprotein Complexes; D009479:Neuropeptides; C490211:RHEB protein, human; D000076205:Ras Homolog Enriched in Brain Protein; D006160:Guanosine Triphosphate; C546842:MTOR protein, human; D000076222:Mechanistic Target of Rapamycin Complex 1; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D058570:TOR Serine-Threonine Kinases; D020559:Monomeric GTP-Binding Proteins; D000409:Alanine; D005998:Glycine
keywords: Constitutively Active; Crystal Structure; Enzyme Catalysis; G3-box; Protein Engineering; Rheb; Small GTPases; TSC2GAP; mTOR Complex (mTORC)