Identification of Functionally Important TonB-ExbD Periplasmic Domain Interactions In Vivo

Abstract

In Gram-negative bacteria, the cytoplasmic membrane proton-motive force energizes the active transport of TonB-dependent ligands through outer membrane TonB-gated transporters. In Escherichia coli , cytoplasmic membrane proteins ExbB and ExbD couple the proton-motive force to conformational changes in TonB, which are hypothesized to form the basis of energy transduction through direct contact with the transporters. While the role of ExbB is not well understood, contact between periplasmic domains of TonB and ExbD is required, with the conformational response of TonB to presence or absence of proton motive force being modulated through ExbD. A region (residues 92 to 121) within the ExbD periplasmic domain was previously identified as being important for TonB interaction. Here, the specific sites of periplasmic domain interactions between that region and the TonB carboxy terminus were identified by examining 270 combinations of 45 TonB and 6 ExbD individual cysteine substitutions for disulfide-linked heterodimer formation. ExbD residues A92C, K97C, and T109C interacted with multiple TonB substitutions in four regions of the TonB carboxy terminus. Two regions were on each side of the TonB residues known to interact with the TonB box of TonB-gated transporters, suggesting that ExbD positions TonB for correct interaction at that site. A third region contained a functionally important glycine residue, and the fourth region involved a highly conserved predicted amphipathic helix. Three ExbD substitutions, F103C, L115C, and T121C, were nonreactive with any TonB cysteine substitutions. ExbD D25, a candidate to be on a proton translocation pathway, was important to support efficient TonB-ExbD heterodimerization at these specific regions.

Membrane Protein Complex ExbB 4 -ExbD 1 -TonB 1 from Escherichia coli Demonstrates Conformational Plasticity

“…Whereas ExbD Ala92 was shown (15) to interact with TonB Ala150 (adjacent to our predicted region), it most likely contributes a different form of heterodimerization. A follow-up study by the same authors found that ExbD residues 92, 97, and 109 specifically and strongly cross-linked with TonB residues 150 to 212 (28). While at first these results may seem inconsistent with our phage display data, Ollis et al did not mutate the TonB residues identified here.…”

“…However, considering the nonphysiological conditions (pH 3 and low salt) and a propensity for protein aggregation, it is not surprising that this study did not reflect in vivo evidence of ExbD-TonB interactions (15,23,(25)(26)(27). Postle and coworkers identified periplasmic regions involved in ExbD and TonB interactions by disulfide crosslinking (26,28). In the absence of TonB, the same ExbD regions tend to promote their homodimerization (26).…”

“…Indeed, the TonB C-terminal domain in the cocrystal structures (30,31) accounts for approximately one-third of total residues (ϳ151 to 239), yet it occupies only ϳ2.5 nm of the ϳ21-nm periplasmic thickness (35,36). Overall, TonB's flexible and dynamic periplasmic domain (28,37) renders it an active protein in the periplasm. For example, certain regions of TonB have been demonstrated to mediate contacts with the periplasmic binding protein FhuD (38).…”

“…This in vivo evidence described an extended and dynamically flexible ExbD periplasmic domain. The biological relevance of the NMR structure (24) has therefore been questioned (26,28).…”

Going Outside the TonB Box: Identification of Novel FepA-TonB Interactions In Vivo

“…Previously, we engineered Cys substitutions in each of two TonB system proteins to identify sites of interaction via disulfide cross-linking (49)(50)(51)(52)(53)(54). Because the exact sites of interaction between the two proteins are unknown, many combinations of cysteine substitutions must be engineered in order to map the interaction interface.…”