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 beta-sheet protein (approximately 44% beta-sheet and approximately 10% alpha-helix). CfrA undergoes relatively extensive peptide hydrogen-deuterium exchange upon exposure to (2)H(2)O and yet is resistant to thermal denaturation at temperatures up to 95 degrees C. The secondary structure, relatively high aqueous solvent exposure, and high thermal stability are all consistent with a transmembrane beta-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.
Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 451 Smyth Rd., Ottawa, Ontario K1H 8M5, Canada.
Casey L
CL
Carswell
Marc D
MD
Rigden
John E
JE
Baenziger
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D016123:Campylobacter jejuni / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006358:Hot Temperature; D007501:Iron / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D017550:Spectroscopy, Fourier Transform Infrared
D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; D007501:Iron