Membrane transport, for example glucose uptake, Is essential to the cell. Such processes are often mediated by secondary membrane transporters. The largest secondary transporter family is the major facilitator superfamily (MFS), with more than 5,000 members identified to date, including the glucose transporter from muscle (Glut4) and the glucose-6-phosphate transporter (G6PT), both from human and both are involved in the pathogenesis of type 2 diabetes. We propose to study the transport mechanisms of a bacterial homolog of Glut4 and G6PT, the glycerol-3-phosphate transporter (GIpT) from E. coli, using structural biology, biochemistry and spectroscopic approaches. In the previous funding cycle, we determined the crystal structure of GIpT in a substrate-free form at 3.3 A resolution. The structure suggests a """"""""rocker-switch"""""""" mechanism for substrate transport. In the next funding cycle, to better understand GIpT's substrate specificity and substrate-induced conformational change, we plan to determine the crystal structures of the transporter in two substrate-bound forms. We will also test the rocker-switch mechanism and examine the role that key amino acid residues play using mutagenesis, transport assays and spectroscopic techniques. The structural and mechanistic information gained from GIpT can be used directly to improve our understanding of Glut4 and G6PT.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Biochemistry and Biophysics of Membranes Study Section (BBM)
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Sechi, Salvatore
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New York University
Anatomy/Cell Biology
Schools of Medicine
New York
United States
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Karpowich, Nathan K; Song, Jinmei; Wang, Da-Neng (2016) An Aromatic Cap Seals the Substrate Binding Site in an ECF-Type S Subunit for Riboflavin. J Mol Biol 428:3118-30
Sauer, David B; Karpowich, Nathan K; Song, Jin Mei et al. (2015) Rapid Bioinformatic Identification of Thermostabilizing Mutations. Biophys J 109:1420-8
Karpowich, Nathan K; Song, Jin Mei; Cocco, Nicolette et al. (2015) ATP binding drives substrate capture in an ECF transporter by a release-and-catch mechanism. Nat Struct Mol Biol 22:565-71
Mulligan, Christopher; Fitzgerald, Gabriel A; Wang, Da-Neng et al. (2014) Functional characterization of a Na+-dependent dicarboxylate transporter from Vibrio cholerae. J Gen Physiol 143:745-59
Karpowich, Nathan K; Wang, Da-Neng (2013) Assembly and mechanism of a group II ECF transporter. Proc Natl Acad Sci U S A 110:2534-9
Waight, Andrew B; Czyzewski, Bryan K; Wang, Da-Neng (2013) Ion selectivity and gating mechanisms of FNT channels. Curr Opin Struct Biol 23:499-506
Loew, Leslie M; Wang, Da-Neng (2013) Science communication: Quality at stake. Science 342:1169
Wang, Da-Neng; Stieglitz, Heather; Marden, Jennifer et al. (2013) Benjamin Franklin, Philadelphia's favorite son, was a membrane biophysicist. Biophys J 104:287-91
Czyzewski, Bryan K; Wang, Da-Neng (2012) Identification and characterization of a bacterial hydrosulphide ion channel. Nature 483:494-7
Mancusso, Romina; Gregorio, G Glenn; Liu, Qun et al. (2012) Structure and mechanism of a bacterial sodium-dependent dicarboxylate transporter. Nature 491:622-6

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