Site-Directed Spin Labeling Reveals Multiple Modes For Regulating Protein-Protein Interactions In Bacterial Outer Membrane Transport

Structural transitions in three outer-membrane bacterial transport proteins have been investigated using site-directed spin labeling and EPR spectroscopy. In the vitamin B12 transporter BtuB and the ferric citrate transporter FecA, EPR spectra reveal the existence of a substrate-dependent order-to-disorder transition in the energy coupling motif (Ton box), which is localized at the periplasmic surface of the transporter. In BtuB, the Ton box unfolds into the periplasm, and in FecA, an N-terminal transcriptional domain disengages from the Ton box. Both these events expose the Ton box and initiate interactions with the inner membrane protein TonB, which drives transport. However, this disorder transition is not observed in all transporters, and in the ferrichrome transporter FhuA the EPR spectra indicate that the Ton box is constitutively disordered. Distance measurements using double electron-electron resonance (DEER) indicate how the FhuA Ton box is regulated, and each transporter regulates interactions with the inner membrane protein TonB using a distinct molecular mechanism. This work indicates that a variety of mechanisms may used to regulate protein-protein interactions in a single transporter family.