Structural and mechanistic studies of INDY proteins

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DESCRIPTION (provided by applicant): In liver and adipose cells, cytosolic citrate is a major precursor for the synthesis of fatty acids, triacylglycerols, cholesterol and low-density lipoprotein. The cytosolic citrate concentration partially depends on its direct import across the plasma membrane via the Na+-dependent citrate transporter, a member of the divalent anion/Na+ symporter (DASS) family. Mutations of the transporter gene in flies (INDY) result in reduced fat storage through calorie restriction. Knockout mice of the homologous gene are both slimmer and protected from obesity and insulin resistance. Thus, its central role in fatty acid biosynthesis makes NaCT a particularly attractive target of small-molecule therapeutic agents for obesity, diabetes and cardiovascular diseases. We have recently determined the 3.2 ¿ crystal structure of a bacterial INDY homolog in its inward-facing conformation. The crystal structure allows us to propose a detailed transport mechanism for the protein, including substrate specificity, ion specificity, ion- substrate coupling and conformational changes that the protein undergoes to accomplish substrate translocation across the membrane. In the current project, we will test this transport mechanism using mutagenesis and transport assays. We will further characterize the transport mechanism of the protein by determining the structure of the outward-facing conformation of the INDY protein from bacteria and mammals. Understanding of the transport mechanism of these transporters, particularly their substrate and ion specificity, will help in the design of drugs for obesity and diabetes.