Aromatic components of two ferric enterobactin binding sites in Escherichia coli FepA

Citation
Zh. Cao et al., Aromatic components of two ferric enterobactin binding sites in Escherichia coli FepA, MOL MICROB, 37(6), 2000, pp. 1306-1317
Citations number
31
Categorie Soggetti
Microbiology
Journal title
MOLECULAR MICROBIOLOGY
ISSN journal
0950382X → ACNP
Volume
37
Issue
6
Year of publication
2000
Pages
1306 - 1317
Database
ISI
SICI code
0950-382X(200009)37:6<1306:ACOTFE>2.0.ZU;2-Z
Abstract
Ferric enterobactin is a catecholate siderophore that binds with high affin ity (K-d approximate to 10(-10) M) to the Escherichia coli outer membrane p rotein FepA. We studied the involvement of aromatic amino acids in its upta ke 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 struc ture 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 expr ession and localization. Among single mutants, Y260A and F329A were most de trimental, reducing the affinity between FepA and ferric enterobactin 100- and 10-fold respectively. Double substitutions involving Y260, Y272 and F32 9 impaired (100- to 2500-fold) adsorption of the iron chelate more strongly . For Y280A and Y272A, the drop in adsorption affinity caused commensurate decreases in transport efficiency, suggesting that the target residues prim arily act in ligand binding. F329A, like R316A, showed greater impairment o f 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 o f dual ligand binding sites in the FepA vestibule, and measurements of the rate of ferric enterobactin adsorption to fluoresceinated FepA mutant prote ins confirmed this conclusion. The initial, outermost site contains aromati c residues and probably functions through hydrophobic interactions, whereas the secondary site exists deeper in the vestibule, contains both charged a nd aromatic residues and probably acts through hydrophobic and electrostati c bonds.