Iron is essential for cell function causing competition for it between pathogens and their hosts. In the gut and stomach, pathogens like Helicobacter, Salmonella and Campylobacter rely on the uptake of Fe(II). Although Fe(II)-uptake is critical for virulence, little is known about the mechanisms of its uptake. The goal of this project is to understand the function of the membrane protein FeoB which is essential for Fe(II)-uptake in bacteria. FeoB is both novel and unique. Notably, the amino acid sequence of FeoB predicts a GTP-binding domain that is connected to a bundle of several putative transmembrane alpha-helices. Based on this design, we hypothesize that FeoB may have served as primordial ancestor for G protein-coupled receptors and/or channels. We will employ biochemical, genetic and biophysical tools to test this hypothesis and to establish the role of FeoB for Fe(II) uptake. The results of our work are important for understanding iron homeostasis in pathogens and may enable us to identify new strategies for treating microbial infections.
The first aim i s to determine the function of FeoB in Fe(II) uptake. We show that the N-terminal domain of FeoB acts as a regulatory GTP alpha-binding protein. However, the function of the membrane embedded domain remains unknown. We will combine in vivo Fe(II) uptake experiments with in vitro measurements of FeoB's Fe(II)-binding and Fe(II)-transport properties to establish whether FeoB functions as transporter/channel or acts as a receptor protein.
The second aim i s to determine the function of FeoB's G protein in Fe(II)-uptake, and how the activity of the G protein is regulated. We will determine whether FeoB itself rather than a downstream target is regulated by the N-terminal domain and whether Fe(II) can modify the activity of the G protein.
The third aim i s to identify the molecular basis for a guanine-nucleotide-exchange-factor like activity that we discovered in FeoB's N-terminal domain. We will disable this activity by mutagenesis and determine the importance of nucleotide exchange for Fe(II) uptake in vivo.
The fourth aim i s to generate crystals of FeoB embedded in a lipid bilayer. Ultimately, this will allow visualization of FeoB, and reveal whether its structure is related to other G protein-coupled membrane proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM066145-05
Application #
7118798
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Chin, Jean
Project Start
2002-09-01
Project End
2008-08-31
Budget Start
2006-09-01
Budget End
2008-08-31
Support Year
5
Fiscal Year
2006
Total Cost
$247,182
Indirect Cost
Name
Yale University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
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