Binding-protein-dependent transport systems in bacteria are members of the ATP-binding cassette (ABC) superfamily and genetic defects in human homologs are responsible for several serious human diseases including cystic fibrosis. The maltose transport system of Escherichia coli is well characterized and is therefore an ideal model for the study of ABC transporters. A periplasmic maltose binding-protein (MBP) directs maltose to a membrane- associated transport complex containing two transmembrane- spanning proteins, Ma1F and Ma1G and two copies of a peripheral ATP-binding-protein, Ma1K. ATP hydrolysis by the multisubunit complex drives maltose transport. Our long term objective is to understand how the structural and mechanistic features of the maltose transport system result in active transport. As this mechanism is complex, our approach is to break the overall reaction down into smaller partial reactions that can be studied in isolation. In this proposal, we will elucidate the molecular mechanism of maltose transport by developing assays for conformational changes within the multi-subunit transport complex that contribute to the overall translocation event. Given that conformational signals must pass between the hydrophobic domains and the ATP-binding domains, we will focus primarily on this interface. We have available highly purified preparations in which it is possible to monitor the dynamics of the transport complex using biochemical and biophysical techniques. These purified preparations will also be used in collaborative efforts to obtain a high resolution structure of the multi-subunit complex.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM049261-06A1
Application #
2852377
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1993-05-01
Project End
2003-04-30
Budget Start
1999-05-01
Budget End
2000-04-30
Support Year
6
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Baylor College of Medicine
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
074615394
City
Houston
State
TX
Country
United States
Zip Code
77030
Alvarez, Frances Joan D; Orelle, C├ędric; Huang, Yan et al. (2015) Full engagement of liganded maltose-binding protein stabilizes a semi-open ATP-binding cassette dimer in the maltose transporter. Mol Microbiol 98:878-94
Cui, Jinming; Qasim, Sabiha; Davidson, Amy L (2010) Uncoupling substrate transport from ATP hydrolysis in the Escherichia coli maltose transporter. J Biol Chem 285:39986-93
Alvarez, Frances Joan D; Orelle, Cedric; Davidson, Amy L (2010) Functional reconstitution of an ABC transporter in nanodiscs for use in electron paramagnetic resonance spectroscopy. J Am Chem Soc 132:9513-5
Orelle, Cedric; Alvarez, Frances Joan D; Oldham, Michael L et al. (2010) Dynamics of alpha-helical subdomain rotation in the intact maltose ATP-binding cassette transporter. Proc Natl Acad Sci U S A 107:20293-8
Davidson, Amy L; Alvarez, Frances Joan D (2010) Binding protein-dependent uptake of maltose into cells via an ATP-binding cassette transporter. EcoSal Plus 2010:
Khare, Dheeraj; Oldham, Michael L; Orelle, Cedric et al. (2009) Alternating access in maltose transporter mediated by rigid-body rotations. Mol Cell 33:528-36
Oldham, Michael L; Davidson, Amy L; Chen, Jue (2008) Structural insights into ABC transporter mechanism. Curr Opin Struct Biol 18:726-33
Lu, Gang; Westbrooks, James M; Davidson, Amy L et al. (2005) ATP hydrolysis is required to reset the ATP-binding cassette dimer into the resting-state conformation. Proc Natl Acad Sci U S A 102:17969-74
Davidson, Amy L; Chen, Jue (2004) ATP-binding cassette transporters in bacteria. Annu Rev Biochem 73:241-68
Chen, Jue; Lu, Gang; Lin, Jeffrey et al. (2003) A tweezers-like motion of the ATP-binding cassette dimer in an ABC transport cycle. Mol Cell 12:651-61