title: Crystal structures of human TBC1D1 and TBC1D4 (AS160) RabGTPase-activating protein (RabGAP) domains reveal critical elements for GLUT4 translocation.
abstract: We have solved the x-ray crystal structures of the RabGAP domains of human TBC1D1 and human TBC1D4 (AS160), at 2.2 and 3.5 Å resolution, respectively. Like the yeast Gyp1p RabGAP domain, whose structure was solved previously in complex with mouse Rab33B, the human TBC1D1 and TBC1D4 domains both have 16 α-helices and no β-sheet elements. We expected the yeast Gyp1p RabGAP/mouse Rab33B structure to predict the corresponding interfaces between cognate mammalian RabGAPs and Rabs, but found that residues were poorly conserved. We further tested the relevance of this model by Ala-scanning mutagenesis, but only one of five substitutions within the inferred binding site of the TBC1D1 RabGAP significantly perturbed catalytic efficiency. In addition, substitution of TBC1D1 residues with corresponding residues from Gyp1p did not enhance catalytic efficiency. We hypothesized that biologically relevant RabGAP/Rab partners utilize additional contacts not described in the yeast Gyp1p/mouse Rab33B structure, which we predicted using our two new human TBC1D1 and TBC1D4 structures. Ala substitution of TBC1D1 Met(930), corresponding to a residue outside of the Gyp1p/Rab33B contact, substantially reduced catalytic activity. GLUT4 translocation assays confirmed the biological relevance of our findings. Substitutions with lowest RabGAP activity, including catalytically dead RK and Met(930) and Leu(1019) predicted to perturb Rab binding, confirmed that biological activity requires contacts between cognate RabGAPs and Rabs beyond those in the yeast Gyp1p RabGAP/mouse Rab33B structure.
authors: Joslin Diabetes Center and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215, USA.
authors: Sang-Youn
authors: SY
authors: Park
authors: Wanzhu
authors: W
authors: Jin
authors: Ju Rang
authors: JR
authors: Woo
authors: Steven E
authors: SE
authors: Shoelson
mesh_terms: D000818:Animals; D018360:Crystallography, X-Ray; D020690:GTPase-Activating Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051275:Glucose Transporter Type 4; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D020691:rab GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D020690:GTPase-Activating Proteins; C416529:GYP1 protein, S cerevisiae; D051275:Glucose Transporter Type 4; C495692:SLC2A4 protein, human; D029701:Saccharomyces cerevisiae Proteins; C526441:TBC1D1 protein, human; C518767:TBC1D4 protein, human; C112838:Rab33B protein, mouse; D020691:rab GTP-Binding Proteins