Surface topology of the Escherichia coli K-12 ferric enterobactin receptor.
Monoclonal antibodies (MAb) were raised to the Escherichia coli K-12 ferric enterobactin receptor, FepA, and used to identify regions of the polypeptide that are involved in interaction with its ligands ferric enterobactin and colicins B and D. A total of 11 distinct FepA epitopes were identified. The locations of these epitopes within the primary sequence of FepA were mapped by screening MAb against a library of FepA::PhoA fusion proteins, a FepA deletion mutant, and proteolytically modified FepA. These experiments localized the 11 epitopes to seven different regions within the FepA polypeptide, including residues 2 to 24, 27 to 37, 100 to 178, 204 to 227, 258 to 290, 290 to 339, and 382 to 400 of the mature protein. Cell surface-exposed epitopes of FepA were identified and discriminated by cytofluorimetry and by the ability of MAb that recognize them to block the interaction of FepA with its ligands. Seven surface epitopes were defined, including one each in regions 27 to 37, 204 to 227, and 258 to 290 and two each in regions 290 to 339 and 382 to 400. One of these, within region 290 to 339, was recognized by MAb in bacteria containing intact (rfa+) lipopolysaccharide (LPS); all other surface epitopes were susceptible to MAb binding only in a strain containing a truncated (rfaD) LPS core, suggesting that they are physically shielded by E. coli K-12 LPS core sugars. Antibody binding to FepA surface epitopes within region 290 to 339 or 382 to 400 inhibited killing by colicin B or D and the uptake of ferric enterobactin. In addition to the FepA-specific MAb, antibodies that recognized other outer membrane components, including Cir, OmpA, TonA, and LPS, were identified. Immunochemical and biochemical characterization of the surface structures of FepA and analysis of its hydrophobicity and amphilicity were used to generate a model of the ferric enterobactin receptor's transmembrane strands, surface peptides, and ligand-binding domains.
Murphy
C K
CK
Department of Microbiology, Medical College of Wisconsin, Milwaukee 53226.
Kalve
V I
VI
Klebba
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PE
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D003001:Cloning, Molecular; D004758:Enterobactin / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005838:Genotype; D007106:Immune Sera; D007501:Iron / Q000378:metabolism*; D008969:Molecular Sequence Data; D010641:Phenotype; D010957:Plasmids; D011487:Protein Conformation; D011956:Receptors, Cell Surface
D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D007106:Immune Sera; D011956:Receptors, Cell Surface; C029489:enterobactin receptor; D004758:Enterobactin; D007501:Iron