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Publication Abstract from PubMed

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 A 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 alpha-helices and no beta-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 M930, 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 M930 and L1019 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.

Crystal structures of Human TBC1D1 and TBC1D4 (AS160) RabGAP domains reveal critical elements for GLUT4 translocation.,Park SY, Jin W, Woo JR, Shoelson SE J Biol Chem. 2011 Mar 23. PMID:21454505^[4]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.