Studies on theEscherichia coliExbD Transmembrane Domain, Residue L132, and an Inhibitory Cyclic Peptide

Abstract

The TonB system of Escherichia coli uses the cytoplasmic membrane protonmotive force (PMF) to energize active transport of nutrients across the otherwise unenergized outer membrane. Because it overcomes limitations for nutrient diffusion through outer membrane size-limiting porins, it provides a growth advantage and is widespread among Gram-negative bacteria. It consists of three known cytoplasmic membrane proteins, TonB, ExbB and ExbD that energize a variety of customized TonB-dependent transporters in the outer membrane. The sole ExbD transmembrane domain is proposed to consist of residues 23-43 (Kampfenkel and Braun, 1992, J. Bacteriol. 174:5485-7). Here we showed that the charge and location of residue Asp25 were essential for activity of the TonB system, thus identifying it as the only PMF-responsive element in the TonB system. The proposed boundaries of the transmembrane domain α-helix were revised to consist of residues 23-39, with residues 40-43 initiating the subsequent disordered region required for signal transduction (Kopp and Postle, 2020, J. Bacteriol. 202, e00687-19). Trapping of disulfide-linked ExbD homodimers through T42C or V43C prevented TonB system activity that was restored by addition of the reducing agent dithiothreitol, indicating a requirement for motion. In vivo photo-cross-linking experiments suggested that motion was rotation of ExbD transmembrane domains. Inactivity of ExbD L132Q, the first ExbD mutant identified, was likely due to steric hindrance. A conserved and defined site of in vivo ExbD interaction with TonB was identified. Exogenous addition of a cyclic peptide based on that site inhibited ExbD-TonB interaction while concomitantly decreasing iron transport efficiency. This suggested that a novel antimicrobial strategy against ESKAPE and other Gram-negative pathogens could be developed by targeting ExbD protein-protein interactions.

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

“…In the solved structures, ExbB is a pentamer with homodimerized transmembrane domains of ExbD occupying the entirety of its lumen (77,78). Like the solved structures, the in vivo studies also show that ExbD homodimerizes through its transmembrane domain (45); unlike the solved structures, ExbB forms a dimer of dimers, a tetramer, in vivo (38). The in vivo studies also show that ExbB formaldehyde cross-links to both TonB and ExbD in vivo, likely through their transmembrane domains [ (19,57); Fig.…”

“…In vivo and in vitro views of ExbB, ExbD, and TonB. There are similarities and differences between solved structures of ExbB5/ExbD2 and in vivo data (42,45,57). In the solved structures, ExbB is a pentamer with homodimerized transmembrane domains of ExbD occupying the entirety of its lumen (77,78).…”

“…To test this idea, we examined the effect of DCCD on a form of ExbD that is stalled at Stage II in the energy transduction cycle, ExbD (D25N) [Fig. 2; (45,47)]. Treatment of ExbD (D25N) with DCCD caused the higher mass smear to collapse into a single higher mass form as well as an increased amount of monomer, suggesting that, at Stage II, only a single DCCD-modifiable conformation was available (Fig.…”

“…TonB homodimerized near its transmembrane domain (green line) remains active throughout the cycle, suggesting it functions as a homodimer during energy transduction (42). The ExbD transmembrane domain also homodimerizes [green line; (45)], but it is not known if the (35,47,51).…”

“…Conservative replacement with Glu at ExbD residue 25 decreases TonB system activity to 5-10% of the wild-type value, whilst replacements with all other residues inactivates ExbD. Likewise, the Asp 25 residue cannot be relocated to another position in the transmembrane domain and support activity (45). ExbD appears to use cytoplasmic membrane PMF to correctly configure the TonB periplasmic carboxy terminus for transmission of energy to FepA (19,46,47).…”