Biphasic binding kinetics between FepA and its ligands.
The Escherichia coli FepA protein is an energy- and TonB-dependent, ligand-binding porin that functions as a receptor for the siderophore ferric enterobactin and colicins B and D. We characterized the kinetic and thermodynamic parameters associated with the initial, energy-independent steps in ligand binding to FepA. In vivo experiments produced Kd values of 24, 185, and 560 nM for ferric enterobactin, colicin B, and colicin D, respectively. The siderophore and colicin B bound to FepA with a 1:1 stoichiometry, but colicin D bound to a maximum level that was 3-fold lower. Preincubation with ferric enterobactin prevented colicin B binding, and preincubation with colicin B prevented ferric enterobactin binding. Colicin B release from FepA was unexpectedly slow in vivo, about 10-fold slower than ferric enterobactin release. This slow dissociation of the colicin B.FepA complex facilitated the affinity purification of FepA and FepA mutants with colicin B-Sepharose. Analysis of a fluorescent FepA derivative showed that ferric enterobactin and colicin B adsorbed with biphasic kinetics, suggesting that both ligands bind in at least two distinct steps, an initial rapid stage and a subsequent slower step, that presumably establishes a transport-competent complex.
Payne
M A
MA
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019, USA.
Igo
J D
JD
Cao
Z
Z
Foster
S B
SB
Newton
S M
SM
Klebba
P E
PE
D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000378:metabolism*; D002846:Chromatography, Affinity; D003087:Colicins / Q000378:metabolism; D003902:Detergents; D004758:Enterobactin / Q000378:metabolism; D007473:Ion Channels / Q000378:metabolism*; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D017830:Octoxynol; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D003087:Colicins; D003902:Detergents; D007473:Ion Channels; D008024:Ligands; D011956:Receptors, Cell Surface; C029489:enterobactin receptor; D004758:Enterobactin; D017830:Octoxynol; D007501:Iron