An ExbD Disordered Domain Peptide Inhibits TonB System Activity

Abstract

The TonB system energizes transport of essential nutrients, such as iron siderophores, across unenergized outer membranes of Gram-negative bacteria. The integral cytoplasmic membrane proteins of the TonB system--ExbB, ExbD, and TonB--transduce the protonmotive force of the cytoplasmic membrane to TonB-dependent outer membrane transporters for active transport. ExbD protein is anchored in the cytoplasmic membrane, with the majority of it occupying the periplasm. We previously identified a conserved motif within a periplasmic disordered domain that is essential for TonB system function. Here we demonstrated that export of a peptide derived from that motif into the periplasm prevented TonB system function and inhibited all known ExbD interactions in vivo. Formaldehyde crosslinking captured the ExbD peptide in multiple ExbD and TonB complexes. Furthermore, peptides with mutations in the conserved motif not only had significantly reduced ability to inhibit TonB system activity, but they also altered interactions with ExbD and TonB, indicating the specificity of the interaction. Conserved motif peptide interactions with ExbD and TonB mostly occurred between Stage II and Stage III of the TonB energy transduction cycle, a transition that is characterized by the use of protonmotive force. Taken together, the data suggest that the ExbD disordered domain motif has multiple interactions with TonB and ExbD during between Stage II and III of the TonB energization cycle. Because of the essentiality of the motif, it may be a potential template for design of novel antibiotics that target the TonB system.IMPORTANCEGram-negative bacteria are intrinsically antibiotic-resistant due to the diffusion barrier posed by their outer membranes. The TonB system allows them to circumvent this barrier for their own nutritional needs, including iron. The ability of bacteria to acquire iron is a virulence factor for many Gram-negative pathogens. However, no antibiotics currently target the TonB system. Because TonB and ExbD must interact productively in the periplasm for transport across the outer membrane, they constitute attractive targets for potential antibiotic development where chemical characteristics need not accommodate the need to cross the hydrophobic cytoplasmic membrane. Here we show that a small ExbD-derived peptide can interfere with the TonB-ExbD interaction to inhibit the TonB system in vivo.

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

“…We began to identify inhibitory regions of ExbD using endogenous secretion of promising small peptides into the periplasmic space. Secretion of a disordered region peptide (residues 44-63) inhibits TonB-dependent iron transport by inhibiting an essential ExbD-TonB interaction, thus establishing a proof of principle (63).…”

“…For example, ExbB formaldehyde cross-links as a dimer of homodimers in vivo (80). In another example, ExbD photo-cross-links to TonB in vivo through residues that boundary its transmembrane domain suggesting that the ExbD and TonB transmembrane domains are at some point adjacent (63), a result seemingly at odds with the structural data where the lumen of an ExbB pentamer cannot accommodate additional transmembrane domains beyond the two of ExbD.…”

“…It is included here for future researchers. Apparent chemical modification of full-length wild-type ExbD also occurs when an inhibitory ExbD peptide (residues 44-63) is expressed endogenously, resulting in the appearance of a higher mass ExbD species (63).…”

“…We were expecting to discover that a peptide that is inhibitory when supplied endogenously [residues 44-63; (63)] would also be inhibitory if supplied exogenously as a cyclic peptide (residues 45-51), but that was not the case. Possibly it had no effect because it was too short or possibly because an insufficient amount was able to cross the outer membrane and reach the periplasm.…”

“…To study the ability of the ExbD transmembrane domain to support homodimerization in the absence of its periplasmic domain, the DNA encoding ExbD residues V23-P63 was cloned into the TOXCAT vector, pccKan (58). The cloned region included the predicted ExbD transmembrane domain [(residues ; (20)] and a subsequent unstructured domain involved in signal transduction [(residues [44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62][63] (59,60)].…”