Abstract
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.
Highlights
Like other TonB-dependent outer membrane proteins, FepA serves as a receptor for a metal chelate (ferric enterobactin (FeEnt))1 and for noxious agents (colicin B (ColB) and colicin D (ColD))
Colicin binding experiments were incubated from 10 min to 3 h with no significant change in the parameters, indicating that the reactions were at or near equilibrium
Affinity purification of siderophore receptors by binding to their antagonistic ligands, bacteriocins, was reported by Oudega et al [21], who used cloacin DF13-Sepharose to purify an outer membrane (OM) protein later recognized as the ferric aerobactin receptor, IutA [33]
Summary
FeEnt, ferric enterobactin; ColB, colicin B; ColD, colicin D; DM, dodecyl maltoside; OM, outer membrane; PL5, fifth proposed surface loop; PAGE, polyacrylamide gel electrophoresis; MOPS, 4-morpholinepropanesulfonic acid. Colicins may traverse the OM through porin channels [14] This idea originates in part from experiments on a colicinresistant mutant of OmpF (G119D) with an occluded channel [15]. Colicins A and E1 bind to external loops of OmpF and BtuB, respectively, and contain N-terminal determinants that confer pore specificity [16], suggesting that after adsorption they interact with the underlying transmembrane channels. We examined the mechanism of ColB binding to FepA in vivo with an 125I-colicin binding assay and in vitro with a flourescent derivative of FepA. In both conditions the siderophore and the colicins showed complex binding kinetics. The high affinity binding equilibria between ColB and FepA facilitated purification of the receptor and its mutants, by affinity chromatography with immobilized ColB
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