Expression, purification, and structural characterization of CfrA, a putative iron transporter from Campylobacter jejuni

The gene for the Campylobacter ferric receptor (CfrA), a putative iron-siderophore transporter in the enteric food-borne pathogen Campylobacter jejuni, was cloned, and the membrane protein was expressed in Escherichia coli, affinity purified, and then reconstituted into model lipid membranes. Fourier transform infrared spectra recorded from the membrane-reconstituted CfrA are similar to spectra that have been recorded from other iron-siderophore transporters and are highly characteristic of a β-sheet protein (~44% β-sheet and ~10% α-helix). CfrA undergoes relatively extensive peptide hydrogen-deuterium exchange upon exposure to ²H₂O and yet is resistant to thermal denaturation at temperatures up to 95°C. The secondary structure, relatively high aqueous solvent exposure, and high thermal stability are all consistent with a transmembrane β-barrel structure containing a plug domain. Sequence alignments indicate that CfrA contains many of the structural motifs conserved in other iron-siderophore transporters, including the Ton box, PGV, IRG, RP, and LIDG motifs of the plug domain. Surprisingly, a homology model reveals that regions of CfrA that are expected to play a role in enterobactin binding exhibit sequences that differ substantially from the sequences of the corresponding regions that play an essential role in binding/transport by the E. coli enterobactin transporter, FepA. The sequence variations suggest that there are differences in the mechanisms used by CfrA and FepA to interact with bacterial siderophores. It may be possible to exploit these structural differences to develop CfrA-specific therapeutics.