Docking of the periplasmic FecB binding protein to the FecCD transmembrane proteins in the ferric citrate transport system of Escherichia coli

Abstract

Citrate-mediated iron transport across the cytoplasmic membrane is catalyzed by an ABC transporter that consists of the periplasmic binding protein FecB, the transmembrane proteins FecC and FecD, and the ATPase FecE. Salt bridges between glutamate residues of the binding protein and arginine residues of the transmembrane proteins are predicted to mediate the positioning of the substrate-loaded binding protein on the transmembrane protein, based on the crystal structures of the ABC transporter for vitamin B(12), consisting of the BtuF binding protein and the BtuCD transmembrane proteins (E. L. Borths et al., Proc. Natl. Acad. Sci. USA 99:16642-16647, 2002). Here, we examined the role of the residues predicted to be involved in salt-bridge formation between FecB and FecCD by substituting these residues with alanine, cysteine, arginine, and glutamate and by analyzing the citrate-mediated iron transport of the mutants. Replacement of E93 in FecB with alanine [FecB(E93A)], cysteine, or arginine nearly abolished citrate-mediated iron transport. Mutation FecB(E222R) nearly eliminated transport, and FecB(E222A) and FecB(E222C) strongly reduced transport. FecD(R54C) and FecD(R51E) abolished transport, whereas other R-to-C mutations in putative interaction sites between FecCD and FecB substantially reduced transport. The introduced cysteine residues in FecB and FecCD also served to examine the formation of disulfide bridges in place of salt bridges between the binding protein and the transmembrane proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results suggest cross-linking of FecB(E93C) to FecD(R54C) and FecB(E222C) to FecC(R60C). The data are consistent with the proposal that FecB(E93) is contained in the region that binds to FecD and FecB(E222) in the region that binds to FecC.

FIG. 1.

Crystal structures of the BtuF periplasmic binding protein of vitamin B₁₂ transport (2), the BtuC transmembrane dimer, and the BtuD ATPase (15), as assembled by Borths et al. (2), modified by labeling the amino acids proposed to form salt bridges between BtuF and BtuC.

FIG. 2.

Sequence alignment of the proposed interacting regions (2) of the periplasmic binding proteins BtuF, FecB, and FhuD (A) and the transmembrane proteins BtuC, FecCD, and FhuB (B) of E. coli. Two BtuC molecules form the transmembrane channel. FhuB is twice the size of the other transmembrane proteins, and its two halves are homologous. The amino acid residues predicted to form salt bridges between the periplasmic binding proteins and the transmembrane proteins are shown in boldface. The numbers indicate the positions in the mature proteins.

FIG. 3.

Citrate-mediated ⁵⁵Fe³⁺ transport into cells of E. coli AA93 transformed with plasmids encoding the wild-type FecIRABCDE proteins (⧫) and encoding these proteins with the following mutations: FecC(R60C) (•), FecB(E93C) FecC(R60C) (▵), FecB(E222C) FecC (R60C) (*), and FecB(E93C E222C) FecC(R60C) (▴).