Aromatic components of two ferric enterobactin binding sites in Escherichia coli FepA.
Ferric enterobactin is a catecholate siderophore that binds with high affinity (Kd approximately 10-10 M) to the Escherichia coli outer membrane protein FepA. We studied the involvement of aromatic amino acids in its uptake by determining the binding affinities, kinetics and transport properties of site-directed mutants. We replaced seven aromatic residues (Y260, Y272, Y285, Y289, W297, Y309 and F329) in the central part of FepA primary structure with alanine, individually and in double combinations, and determined the ability of the mutant proteins to interact with ferric enterobactin and the protein toxins colicins B and D. All the constructs showed normal expression and localization. Among single mutants, Y260A and F329A were most detrimental, reducing the affinity between FepA and ferric enterobactin 100- and 10-fold respectively. Double substitutions involving Y260, Y272 and F329 impaired (100- to 2500-fold) adsorption of the iron chelate more strongly. For Y260A and Y272A, the drop in adsorption affinity caused commensurate decreases in transport efficiency, suggesting that the target residues primarily act in ligand binding. F329A, like R316A, showed greater impairment of transport than binding, intimating mechanistic involvement during ligand internalization. Furthermore, immunochemical studies localized F329 in the FepA ligand binding site. The mutagenesis results suggested the existence of dual ligand binding sites in the FepA vestibule, and measurements of the rate of ferric enterobactin adsorption to fluoresceinated FepA mutant proteins confirmed this conclusion. The initial, outermost site contains aromatic residues and probably functions through hydrophobic interactions, whereas the secondary site exists deeper in the vestibule, contains both charged and aromatic residues and probably acts through hydrophobic and electrostatic bonds.
Cao
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Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019, USA.
Qi
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Sprencel
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Newton
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SM
Klebba
P E
PE
D000409:Alanine; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000598:Amino Acids, Cyclic / Q000737:chemistry / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003087:Colicins / Q000494:pharmacology; D004758:Enterobactin / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D021381:Protein Transport; D011956:Receptors, Cell Surface; D017386:Sequence Homology, Amino Acid
D000598:Amino Acids, Cyclic; D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D003087:Colicins; D011956:Receptors, Cell Surface; C029489:enterobactin receptor; D004758:Enterobactin; D007501:Iron; D000409:Alanine