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

Abstract

Ferric siderophores, vitamin B 12 , and group B colicins are taken up through the outer membranes of Escherichia coli cells by an energy-coupled process. Energy from the cytoplasmic membrane is transferred to the outer membrane with the aid of the Ton system, consisting of the proteins TonB, ExbB, and ExbD. In this paper we describe two point mutations which inactivate ExbD. One mutation close to the N-terminal end of ExbD is located in the cytoplasmic membrane, and the other mutation close 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, the only charged amino acid in the predicted membrane-spanning region of ExbD, was replaced by asparagine, failed to restore the Ton activities of strain CHO3 and negatively complemented ExbD ؉ strains, indicating an interaction of this mutated ExbD with wild-type ExbD or with another component. This component was shown to be ExbB. ExbB that was labeled with 6 histidine residues at its C-terminal end and that bound to 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 complex in which the energized conformation of TonB opens the channels in the outer membrane receptor proteins.

TonB protein appears to transduce energy by shuttling between the cytoplasmic membrane and the outer membrane in Escherichia coli

“…TonB has been demonstrated to interact directly with ExbB (Larsen et al, 1994;Skare et al, 1993) and ExbB has been demonstrated to interact directly with ExbD (Braun et al, 1996). In this study, T7-ExbB and T7-ExbD localized with the cytoplasmic membrane, under conditions where chromosomally encoded TonB in the same gradients localized in both cytoplasmic membrane and outer membrane fractions.…”

“…ExbB and ExbD localize with the cytoplasmic membrane TonB and the cytoplasmic membrane proteins ExbB and ExbD have been demonstrated to interact closely (Braun et al, 1996;Skare et al, 1993). If TonB existed in specialized regions of the cytoplasmic membrane that have a density equal to that of the outer membrane, ExbB and ExbD should also be found in these specialized high-density regions of the cytoplasmic membrane.…”

“…However, ironsiderophore complexes and vitamin B12 are too large, too scarce, and too important for their transport across the outer membrane to be left to simple diffusion. These compounds are instead actively transported across the outer membrane into the periplasmic space (most recently reviewed by Braun et al, 1996).…”

The proline‐rich domain of TonB possesses an extended polyproline II‐like conformation of sufficient length to span the periplasm of Gram‐negative bacteria

“…ExbB and ExbD, which exhibit distant homology with components of the flagellar motor, 9 appear to form a complex with TonB. 10,11 Suppressor mutation analysis has suggested that ExbB interacts with TonB through the transmembrane domain 12 while an NMR study suggested that ExbD interacts weakly with the periplasmic domain. 13 The crystal structures of several TonB-dependent receptors show a common architecture.…”

Analysis of Haptoglobin and Hemoglobin-Haptoglobin Interactions with theNeisseria meningitidisTonB-Dependent Receptor HpuAB by Flow Cytometry

“…The conserved interaction of a family of high-affinity outer membrane (OM) transporters with the TonB complex suggests that many, if not all, gram-negative bacteria have adopted similar strategies to transport Fe across the OM. TonB is anchored in the inner membrane in association with ExbBD and spans the periplasm, presumably to transduce the energy of the proton motive force (PMF) to OM receptors (4,34,38,40). Although TonB physically interacts with OM receptors (1,8,32,45,83), the way in which TonB might convey PMF energy to these receptors to drive solute uptake remains unclear (10,46).…”