Characterization of interactions of TonB and Colicin M with FhuA reconstituted into Nanodiscs

TonB-dependent transporters are β-barrel outer membrane proteins that depend on interactions with the inner membrane protein TonB to drive import of scarce nutrients. Upon becoming ligand-loaded, TonB-dependent transporters bind TonB through a β-strand exchange. FhuA is the TonB-dependent transporter that transports hydroxamate iron siderophores, such as ferrichrome and ferricrocin, into the periplasm and also acts as the receptor and transporter of the antimicrobial protein colicin M. The interactions of FhuA with TonB have previously been investigated in detergents, which can affect the conformations of TonB-dependent transporters and alter their interaction with TonB. To exclude the potential negative effects of detergent, FhuA was reconstituted into Nanodiscs that reconstitute a membrane-like environment suitable for biochemical analysis. Binding of TonB to FhuA was found to be strongly dependent on the ligand-loaded state of FhuA. The binding affinity was relatively high (~200 nM) and enthalpy driven, suggesting a disorder-to-order interaction occurring during the β-strand exchange. Colicin M also bound Nanodisc reconstituted FhuA with a high affinity (~3.5 nM) through an entropy driven interaction that may reflect a hydrophobic interaction. The ligand ferricrocin inhibited the binding of colicin M to FhuA. While TonB is required for transport of colicin M into cells, colicin M was not observed to cause the recruitment of TonB to FhuA. Finally, the conformation of FhuA was investigated in the presence of ferricrocin and colicin M by partial proteolysis.