Ligand-induced conformational change in the ferric enterobactin receptor FepA as studied by site-directed spin labeling and time-domain ESR.
A mutant of the ferric enterobactin receptor, FepA, containing a valine to cysteine (V338C) substitution was made and the purified protein selectively modified with a sulfhydryl-specific nitroxide spin label. In reconstituted liposomes, interaction of the attached spin label with a combination of water-soluble and lipid-soluble relaxation agents indicated that the V338C site was located in the polar headgroup region of the membrane, approximately 1.5-4.5 A above the phosphate groups of the lipids. Binding of the ligand, ferric enterobactin (FeEnt), to the purified spin-labeled protein produced a significant decrease in both the rotational freedom and accessibility of the nitroxide, indicating the formation of new structural contacts between the spin label and either the protein or the bound ligand. Electron spin-echo (ESE) measurements of the nitroxide phase-memory relaxation rate in the presence and absence of bound ligand showed substantial dipolar coupling between the Fe3+ of FeEnt and the spin label and provided an iron-nitroxide distance estimate in the range of 20-30 A. We conclude that the ligand-induced changes in spin label motion and accessibility are due to new tertiary contacts with the protein and not to direct contact with the ligand. These studies suggest that V338C may occupy a hinge region connecting the ligand binding surface loop to the beta-barrel and provide the strongest evidence to date of an in vitro ligand-induced conformational change in FepA.
Klug
C S
CS
Biophysics Research Institute, Medical College of Wisconsin, Milwaukee 53226, USA.
Eaton
S S
SS
Eaton
G R
GR
Feix
J B
JB
D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004758:Enterobactin / Q000378:metabolism*; D008024:Ligands; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008081:Liposomes; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011956:Receptors, Cell Surface; D013113:Spin Labels; D014633:Valine / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D008024:Ligands; D008051:Lipid Bilayers; D008081:Liposomes; D011956:Receptors, Cell Surface; D013113:Spin Labels; C029489:enterobactin receptor; D004758:Enterobactin; D014633:Valine; D003545:Cysteine