Epitope Insertions Define Functional and Topological Features of the Escherichia coli Ferric Enterobactin Receptor (∗)

• Published in: The Journal of biological chemistry 1995-02, Vol.270 (6), p.2483-2488
• Main Authors: Armstrong, Sandra K., McIntosh, Mark A.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacterial Outer Membrane Proteins - chemistry; Bacterial Outer Membrane Proteins - metabolism; Base Sequence; Carrier Proteins - chemistry; Carrier Proteins - metabolism; DNA, Bacterial; Enterobactin - metabolism; Epitopes - metabolism; Escherichia coli; Escherichia coli - metabolism; In Situ Hybridization; Molecular Sequence Data; Protein Conformation; Receptors, Cell Surface; Recombinant Fusion Proteins - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.6.2483

The outer membrane protein FepA of Escherichia coli is the receptor for the ferric enterobactin siderophore complex and colicins B and D. A foreign antigenic determinant inserted into selected FepA sites allowed mutational analysis of receptor function and in situ immunological tracking of specific protein domains with respect to the bacterial cell compartment. Immunoblot analysis of bacterial proteins using an epitope-specific antibody detected the peptide determinant in the receptor fusions. The impact of the insertions on FepA function was examined by ferric enterobactin-mediated iron uptake experiments and colicin sensitivity tests. In all cases, FepA retained biological activity despite introduction of the foreign sequence. To further develop the topological model of FepA, the peptide-specific antibody was used to localize epitope-carrying FepA domains in intact bacterial cells and their isolated membranes. One epitope resided in a region on the exterior of the cell, at the surface of the FepA protein, while other epitopes appeared to be localized to the periplasm or within the outer membrane.