J. Liu et al., SITE-DIRECTED SPIN-LABELING STUDY OF LIGAND-INDUCED CONFORMATIONAL CHANGE IN THE FERRIC ENTEROBACTIN RECEPTOR, FEPA, Biochemistry, 33(45), 1994, pp. 13274-13283
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.