Structural Studies of Sugar Transporters

New York University School Of Medicine, New York NY

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Abstract

DESCRIPTION (provided by applicant): Our goal is to understand the molecular mechanisms of solute transport by transmembrane proteins, in particular the sugar uptake in muscle and fat cells which, when impaired, causes type II diabetes. This transport process is mediated by Glut4, a transporter protein that spans the membrane 12 times. Human Glut4 is difficult to study due to its scarcity and heterogeneity. Therefore, we selected a related bacterial protein: the glycerol-3-phosphate (G3P) transporter (GlpT) from E. coli. Mutations in the human G3P transporter are thought to contribute to the susceptibility and severity of diabetes. Knowledge gained on E. coli GlpT will help understand the structure and mechanism of the human Glut4 protein. We will determine the structure of E. coli GlpT, a transmembrane protein, using X-ray crystallography of three-dimensional crystals. The structure will reveal the substrate-binding site and translocation pathway. Knowledge of the structure of GlpT mutants and GlpT in complex with inhibitors/substrates will reveal molecular mechanisms of transport. Mutagenesis and transport assays will provide information on the roles of key residues in substrate selectivity and translocation. The GlpT structure will serve as a paradigm for the mammalian glucose transporters, and other medically relevant proteins such as the dopamine transporter responsible for cocaine addiction. We will build a structural model for the human glucose transporters based on our GlpT structure, combining information from the conserved topology, and biochemical and mutagenesis data on mammalian sugar transporters. This model will be tested by mutagenesis in combination with activity assays on recombinant Glut4, and improved accordingly. Such a model will help us understand the cell's glucose uptake in muscle and fat tissues.