SITE-DIRECTED SPIN-LABELING STUDY OF LIGAND-INDUCED CONFORMATIONAL CHANGE IN THE FERRIC ENTEROBACTIN RECEPTOR, FEPA

The ferric enterobactin receptor, FepA, is a TonB-dependent gated pori n that transports the siderophore ferric enterobactin across the outer membrane of gram-negative bacteria. We have created two site-directed mutants of Escherichia coli FepA, in both cases introducing a cystein e residue into the putative Ligand-binding domain. The introduced cyst eines were then modified with nitroxide spin labels for structural and dynamic studies using electron spin resonance (ESR) spectroscopy. The mutants were fully functional, as indicated by their ability to grow under iron-limiting conditions, their uptake of [Fe-59]enterobactin, a nd their sensitivity to colicin B. Labeling of the mutant FepA recepto rs proceeded easily upon incubation with sulfhydryl-specific spin labe ls, e.g. MTSL, (1-oxy-2,2,5,5-tetramethylpyrrolidin-3-yl)methyl methan ethiosulfonate. In contrast, spin labeling of the two native cysteines (Cys486 and Cys493) within wild-type FepA occurred only after treatme nt with a thiol reducing agent and partial denaturation in urea, sugge sting that the native cysteines are disulfide-linked. ESR spectra show ed a high degree of motional restriction for all three sites. Continuo us wave (CW) saturation studies indicated that one of the mutationally introduced sites (Cys280) was surface-localized as evidenced by its e xposure to the aqueous paramagnetic relaxation agent chromium oxalate and its low accessibility to O-2. The other (Cys310) apparently occupi es a site near the membrane/aqueous interface. The native cysteines oc cupy a site tightly packed within the protein structure with low acces sibility to both CROX and O-2, A shift in both conventional and satura tion-transfer ESR spectra of MTSL-labeled E280C and E310C (but not MTS L-labeled wild type) FepA was observed upon addition of ferric enterob actin. The ESR spectral shift was dependent on ferric enterobactin con centration and did not occur with siderophores not recognized by FepA. Ferric enterobactin binding did not alter the CW saturation propertie s of MTSL bound to these sites, but did influence their accessibility to O-2. These results provide consistent evidence for a ligand specifi c conformational change in the surface peptides of FepA upon the bindi ng of ferric enterobactin.