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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK053973-12
Application #
8009401
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Sechi, Salvatore
Project Start
1998-07-01
Project End
2014-12-31
Budget Start
2011-01-01
Budget End
2014-12-31
Support Year
12
Fiscal Year
2011
Total Cost
$333,749
Indirect Cost
Name
New York University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
121911077
City
New York
State
NY
Country
United States
Zip Code
10016
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
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
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
Mancusso, Romina; Karpowich, Nathan K; Czyzewski, Bryan K et al. (2011) Simple screening method for improving membrane protein thermostability. Methods 55:324-9
Waight, Andrew B; Love, James; Wang, Da-Neng (2010) Structure and mechanism of a pentameric formate channel. Nat Struct Mol Biol 17:31-7

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