In vivotests of theE. coliTonB system working model—interaction of ExbB with unknown proteins, identification of TonB-ExbD transmembrane heterodimers and PMF-dependent ExbD structures

Abstract

ABSTRACTThe TonB system ofEscherichia coliresolves the dilemma posed by its outer membrane that protects it from a variety of external threats, but also constitutes a diffusion barrier to nutrient uptake. Our working model involves interactions among a set of cytoplasmic membrane-bound proteins: tetrameric ExbB that serves as a scaffold for a dimeric TonB complex (ExbB4-TonB2), and also engages dimeric ExbD (ExbB4-ExbD2). Through a set of synchronized conformational changes and movements these complexes are proposed to cyclically transduce cytoplasmic membrane protonmotive force to energize active transport of nutrients through TonB-dependent transporters in the outer membrane (described in Gresock etal., J. Bacteriol. 197:3433). In this work, we provide experimental validation of three important aspects of the model. The majority of ExbB is exposed to the cytoplasm, with an ∼90-residue cytoplasmic loop and an ∼50 residue carboxy terminal tail. Here we found for the first time, that the cytoplasmic regions of ExbB served asin vivocontacts for three heretofore undiscovered proteins, candidates to move ExbB complexes within the membrane. Support for the model also came from visualization ofin vivoPMF-dependent conformational transitions in ExbD. Finally, we also show that TonB forms homodimers and heterodimers with ExbD through its transmembrane domainin vivo. This trio ofin vivoobservations suggest how and why solvedin vitrostructures of ExbB and ExbD differ significantly from thein vivoresults and submit that future inclusion of the unknown ExbB-binding proteins may bring solved structures into congruence with proposedin vivoenergy transduction cycle intermediates.