ENERGY-COUPLED TRANSPORT ACROSS THE OUTER-MEMBRANE OF ESCHERICHIA-COLI - EXBB BINDS EXBD AND TONB IN-VITRO, AND LEUCINE-132 IN THE PERIPLASMIC REGION AND ASPARTATE-25 IN THE TRANSMEMBRANE REGION ARE IMPORTANT FOR EXBD ACTIVITY

Ferric siderophores, vitamin B-12, and group B colicins are taken up t hrough the outer membranes of Escherichia coli cells by an energy-coup led process, Energy from the cytoplasmic membrane is transferred to th e outer membrane with the aid of the Ton system, consisting of the pro teins TonB, ExbB, and ExbD. In this paper we describe two point mutati ons which inactivate ExbD. One mutation close to the N-terminal end of ExbD is located in the cytoplasmic membrane, and the other mutation c lose to the C-terminal end is located in the periplasm. E. coli CHO3, carrying a chromosomal exbD mutation in which leucine at position 132 was replaced by glutamine, was devoid of all Ton-related activities. A plasmid-encoded ExbD derivative, in which aspartate at position 25, t he only charged amino acid in the predicted membrane-spanning region o f ExbD, was replaced by asparagine, failed to restore the Ton activiti es of strain CHO3 and negatively complemented ExbD(+) strains, indicat ing an interaction of this mutated ExbD with wild-type ExbD or with an other component. This component was shown to be ExbB. ExbB that was la beled with 6 histidine residues at its C-terminal end and that bound t o a nickel-nitrilotriacetic acid agarose column retained ExbD and TonB specifically; both were eluted with the ExbB labeled with 6 histidine residues, demonstrating interaction of ExbB with ExbD and TonB. These data further support the concept that TonB, ExbB, and ExbD form a com plex in which the energized conformation of TonB opens the channels in the outer membrane receptor proteins.