Structual Characterization of the Na+/Glucose Cotransporter Family

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DESCRIPTION (provided by applicant): Membrane transport proteins govern energy transduction, modify ion concentrations, and actively import metabolites into the cell. Moreover, two of the most widely prescribed drugs internationally-fluoxetine and omeprazole-target membrane transport proteins. One important class of transporters is the Na+/Glucose cotransporter family (SGLT), which contains over 240 members and is present in all kingdoms of life. There are eleven human isoforms of SGLT, expressed in a variety of tissues. Mutations in at least three of these genes, encoding the Na+/sugar symporters SGLT1 and SGLT2, as well as the Na+/iodide cotransporter (NIS), are known to result in metabolic disorders. These proteins perform essential roles in physiology and are implicated in a number of diseases, most notably infectious diarrhea, diabetes and some cancers. Given the functional and pharmacological relevance of these proteins, a 3D crystal structure from this family of cotransporters, would greatly advance our understanding of how these cotransporters work and enable us to understand their role in health, disease and therapy. We have preliminary crystallographic data on the Na+/galactose cotransporter from Vibrio parahaemolyticus (vSGLT). These crystals are grown in the presence of Na+ and galactose and are currently diffracting to 4.5Angstroms, and optimization of these crystals is in progress. Using information from other members of the SGLT family and signature sequences for sugar- and Na+-binding sites, we have identified residues that should be influential in transport. We will construct mutants of vSGLT at these residues and analyze them using radioactive uptake and fluorescence assays. Using these methods we will be able to measure transport rates, apparent affinities for Na+ and galactose, and alterations in sugar specificity. Ultimately, this biochemical and biophysical data will be analyzed in conjunction with the atomic resolution structure of vSGLT. After obtaining the structure of vSGLT and identifying important mutants, we will use the backbone coordinates for molecular threading of SGLT1, SGLT2 and NIS symporters and refine the models by using available biochemical data. These models may be able to explain some key questions about these multifunctional proteins with a diverse range of substrates and possibly facilitate drug design.