All organisms require metals as co-factors, and these essential metals must be obtained from external sources. Gram- negative bacteria possess specialized pathways that enable binding and transport across three cellular compartments: the outer membrane, the periplasm and the inner membrane. The outer membrane contains transporters specific for different metals or metal-containing compounds; in pathogenic bacteria, these transporters are implicated as virulence factors. As the outer membrane does not maintain an electrochemical gradient, energy- dependent steps in transport across the outer membrane are mediated through proteins that couple the inner membrane gradient to the outer membrane transporter. These bacterial systems provide a tractable model for a molecular dissection of active transport. We seek to understand the mechanism of these transport processes through crystallographic and structure/function studies of bacterial cobalamin transport. We have obtained three-dimensional crystals of the Escherichia coli vitamin cobalamin transporter BtuB that diffract to beyond 2.0 Angstrom units resolution (2.1 Angstrom units dataset collected). Datasets (2.6 Angstrom units resolution) of crystals soaked in cyanocobalamin (vitamin B12) have also been collected. BtuB mutants with altered cobalamin binding will be made and characterized, and these mutant structures will be solved. BtuB couples to the inner membrane via the TonB protein. We have expressed a domain of the TonB protein that interacts with BtuB, and will undertake structural studies of this domain alone and in complex with BtuB (also in the presence or absence of vitamin B12). Also, although the natural ligands for BtuB are cobalamins, BtuB is also the receptor to which bacterial toxins (colicins) and bacteriophage bind. The nature of these interactions will be investigated by making, crystallizing and solving complexes of receptor-binding domains of E colicins and BtuB.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK059999-01
Application #
6364683
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Sechi, Salvatore
Project Start
2001-08-01
Project End
2006-07-31
Budget Start
2001-08-01
Budget End
2002-07-31
Support Year
1
Fiscal Year
2001
Total Cost
$291,277
Indirect Cost
Name
University of Virginia
Department
Physiology
Type
Schools of Medicine
DUNS #
001910777
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Gumbart, James; Wiener, Michael C; Tajkhorshid, Emad (2007) Mechanics of force propagation in TonB-dependent outer membrane transport. Biophys J 93:496-504
Shultis, David D; Purdy, Michael D; Banchs, Christian N et al. (2006) Outer membrane active transport: structure of the BtuB:TonB complex. Science 312:1396-9
Shultis, David D; Purdy, Michael D; Banchs, Christian N et al. (2006) Crystallization and preliminary X-ray crystallographic analysis of the Escherichia coli outer membrane cobalamin transporter BtuB in complex with the carboxy-terminal domain of TonB. Acta Crystallogr Sect F Struct Biol Cryst Commun 62:638-41
Wiener, Michael C (2005) TonB-dependent outer membrane transport: going for Baroque? Curr Opin Struct Biol 15:394-400
Chimento, David P; Kadner, Robert J; Wiener, Michael C (2005) Comparative structural analysis of TonB-dependent outer membrane transporters: implications for the transport cycle. Proteins 59:240-51
Chimento, David P; Mohanty, Arun K; Kadner, Robert J et al. (2003) Crystallization and initial X-ray diffraction of BtuB, the integral membrane cobalamin transporter of Escherichia coli. Acta Crystallogr D Biol Crystallogr 59:509-11
Chimento, David P; Kadner, Robert J; Wiener, Michael C (2003) The Escherichia coli outer membrane cobalamin transporter BtuB: structural analysis of calcium and substrate binding, and identification of orthologous transporters by sequence/structure conservation. J Mol Biol 332:999-1014
Mohanty, Arun K; Bishop, Christopher M; Bishop, Thomas C et al. (2003) Enzymatic E-colicins bind to their target receptor BtuB by presentation of a small binding epitope on a coiled-coil scaffold. J Biol Chem 278:40953-8
Cadieux, Nathalie; Bradbeer, Clive; Reeger-Schneider, Eva et al. (2002) Identification of the periplasmic cobalamin-binding protein BtuF of Escherichia coli. J Bacteriol 184:706-17