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 K-d 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 coli cin 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.