Ligand-induced structural changes in the Escherichia coli ferric citrate transporter reveal modes for regulating protein-protein interactions.

Outer-membrane TonB-dependent transporters, such as the Escherichia coli ferric citrate transporter FecA, interact with the inner-membrane protein TonB through an energy-coupling segment termed the Ton box. In FecA, which regulates its own transcription, the Ton box is preceded by an N-terminal extension that interacts with the inner-membrane protein FecR. Here, site-directed spin labeling was used to examine the structural basis for transcriptional signaling and Ton box regulation in FecA. EPR spectroscopy indicates that regions of the N-terminal domain are in conformational exchange, consistent with its role as a protein binding element; however, the local fold and dynamics of the domain are not altered by substrate or TonB. Distance restraints derived from pulse EPR were used to generate models for the position of the extension in the apo, substrate-, and TonB-bound states. In the apo state, this domain is positioned at the periplasmic surface of FecA, where it interacts with the Ton box and blocks access of the Ton box to the periplasm. Substrate addition rotates the transcriptional domain and exposes the Ton box, leading to a disorder transition in the Ton box that may facilitate interactions with TonB. When a soluble fragment of TonB is bound to FecA, the transcriptional domain is displaced to one edge of the barrel, consistent with a proposed β-strand exchange mechanism. However, neither substrate nor TonB displaces the N-terminus further into the periplasm. This result suggests that the intact TonB system mediates both signaling and transport by unfolding portions of the transporter.