DOUBLE MUTAGENESIS OF A POSITIVE CHARGE CLUSTER IN THE LIGAND-BINDINGSITE OF THE FERRIC ENTEROBACTIN RECEPTOR, FEPA

Siderophores and colicins enter bacterial cells through TonB-dependent outer membrane proteins, Using site-directed substitution mutagenesis , we studied ligand recognition by a prototypic Escherichia coli sider ophore receptor, FepA, that binds the iron chelate ferric enterobactin and colicins B and D, These genetic experiments identified a common b inding site for two of the three ligands, containing multiple positive charges, within cell surface residues of FepA. Elimination of single residues in this region did mat impair the adsorption or transport of ferric enterobactin, but double mutagenesis in the charge cluster iden tified amino acids (Arg-286 and Arg-316) that participate in sideropho re binding and function in FepA-mediated killing by colicins B and D. Ferric enterobactin binding, furthermore, prevented covalent modificat ion of FepA within this domain by either a fluorescent probe or an arg inine-specific reagent, corroborating the involvement of this site in ligand recognition, These results identify, for the first time, residu es in a TonB-dependent outer membrane protein that participate in liga nd binding, They also explain the competition between ferric enterobac tin and the colicins on the bacterial cell surface: all three ligands interact with the same arginine residues within FepA during their pene tration through the outer membrane.