In vivo tests of the E. coli TonB system working model—interaction of ExbB with unknown proteins, identification of TonB-ExbD transmembrane heterodimers and PMF-dependent ExbD structures

Le résumé fourni par la source

ABSTRACT The TonB system of Escherichia coli resolves 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 (ExbB 4 -TonB 2 ), and also engages dimeric ExbD (ExbB 4 -ExbD 2 ). 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 et al. , 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 as in vivo contacts for three heretofore undiscovered proteins, candidates to move ExbB complexes within the membrane. Support for the model also came from visualization of in vivo PMF-dependent conformational transitions in ExbD. Finally, we also show that TonB forms homodimers and heterodimers with ExbD through its transmembrane domain in vivo . This trio of in vivo observations suggest how and why solved in vitro structures of ExbB and ExbD differ significantly from the in vivo results and submit that future inclusion of the unknown ExbB-binding proteins may bring solved structures into congruence with proposed in vivo energy transduction cycle intermediates.

Ce résumé expose les affirmations des auteurs. BNTIC ne l’interprète pas comme une validation indépendante des résultats.