TonB induces conformational changes in surface-exposed loops of FhuA, outer membrane receptor of Escherichia coli.
FhuA, outer membrane receptor of Escherichia coli, transports hydroxamate-type siderophores into the periplasm. Cytoplasmic membrane-anchored TonB transduces energy to FhuA to facilitate siderophore transport. Because the N-terminal cork domain of FhuA occludes the C-terminal beta-barrel lumen, conformational changes must occur to enable siderophore passage. To localize conformational changes at an early stage of the siderophore transport cycle, four anti-FhuA monoclonal antibodies (mAbs) were purified to homogeneity, and the epitopes that they recognize were determined by phage display. We mapped continuous and discontinuous epitopes to outer surface-exposed loops 3, 4, and 5 and to beta-barrel strand 14. To probe for conformational changes of FhuA, surface plasmon resonance measured mAb binding to FhuA in its apo- and siderophore-bound states. Changes in binding kinetics were observed for mAbs whose epitopes were mapped to outer surface-exposed loops. Further, we measured mAb binding in the absence and presence of TonB. After forming immobilized FhuA-TonB complexes, changes in kinetics of mAb binding to FhuA were even more pronounced compared with kinetics of binding in the absence of TonB. Measurement of extrinsic fluorescence of the dye MDCC conjugated to residue 336 in outer surface-exposed loop 4 revealed 33% fluorescence quenching upon ferricrocin binding and up to 56% quenching upon TonB binding. Binding of mAbs to apo- and ferricrocin-bound FhuA complemented by fluorescence spectroscopy studies showed that their cognate epitopes on loops 3, 4, and 5 undergo conformational changes upon siderophore binding. Further, our data demonstrate that TonB binding promotes conformational changes in outer surface-exposed loops of FhuA.
James
Karron J
KJ
Department of Microbiology and Immunology, McGill University, Montreal, Quebec H3A 2B4, Canada.
Hancock
Mark A
MA
Moreau
Violaine
V
Molina
Franck
F
Coulton
James W
JW
D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D018604:Epitope Mapping; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D005291:Ferrichrome / Q000031:analogs & derivatives / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D019151:Peptide Library; D011487:Protein Conformation
D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; C050900:FhuA protein, E coli; D008565:Membrane Proteins; D019151:Peptide Library; C489181:tonB protein, E coli; D005291:Ferrichrome; C021678:ferricrocin