Double mutagenesis of a positive charge cluster in the ligand-binding site 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 siderophore receptor, FepA, that binds the iron chelate ferric enterobactin and colicins B and D. These genetic experiments identified a common binding 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 not impair the adsorption or transport of ferric enterobactin, but double mutagenesis in the charge cluster identified amino acids (Arg-286 and Arg-316) that participate in siderophore binding and function in FepA-mediated killing by colicins B and D. Ferric enterobactin binding, furthermore, prevented covalent modification of FepA within this domain by either a fluorescent probe or an arginine-specific reagent, corroborating the involvement of this site in ligand recognition. These results identify, for the first time, residues in a TonB-dependent outer membrane protein that participate in ligand binding. They also explain the competition between ferric enterobactin and the colicins on the bacterial cell surface: all three ligands interact with the same arginine residues within FepA during their penetration through the outer membrane.
Newton
S M
SM
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019, USA.
Allen
J S
JS
Cao
Z
Z
Qi
Z
Z
Jiang
X
X
Sprencel
C
C
Igo
J D
JD
Foster
S B
SB
Payne
M A
MA
Klebba
P E
PE
D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003087:Colicins / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004758:Enterobactin / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D017262:Siderophores / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D003087:Colicins; D011956:Receptors, Cell Surface; D017262:Siderophores; C029489:enterobactin receptor; D004758:Enterobactin; D001120:Arginine