Mutations in the Escherichia coli receptor FepA reveal residues involved in ligand binding and transport.
FepA is the Escherichia coli outer membrane receptor for ferric enterobactin, colicin D and colicin B. The transport processes through FepA are energy-dependent, relying on the periplasmic protein TonB to interact with FepA. Through this interaction, TonB tranduces energy derived from the cytoplasmic membrane across the periplasmic space to FepA. In this study, random mutagenesis strategies were used to define residues of FepA important for its function. Both polymerase chain reaction (PCR)-generated random mutations in the N-terminal 180 amino acids of FepA and spontaneous chromosomal fepA mutations were selected by resistance to colicin B. The PCR mutagenesis strategy targeted the N-terminus because it forms a plug inside the FepA barrel that is expected to be involved in ligand binding, ligand transport, and interaction with TonB. We report the characterization of 15 fepA missense mutations that were localized to three regions of the FepA receptor. The first region was a stretch of eight amino acids referred to as the TonB box. The second region included extracellular loops of both the barrel and the plug. A third region formed a cluster near the barrel wall around positions 75 and 126 of the plug. These mutations provide initial insight into the mechanisms of ligand binding and transport through the FepA receptor.
Barnard
T J
TJ
Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, Missouri 65212, USA.
Watson
M E
ME
McIntosh
M A
MA
D020816:Amino Acid Motifs; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003087:Colicins / Q000378:metabolism; D017124:Conserved Sequence; D004758:Enterobactin / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry; D008954:Models, Biological; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D011487:Protein Conformation; D021381:Protein Transport; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D003087:Colicins; D029968:Escherichia coli Proteins; D008024:Ligands; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; C029489:enterobactin receptor; C057616:tonB protein, Bacteria; C489181:tonB protein, E coli; D004758:Enterobactin