The Intrinsically Disordered Region of ExbD is Required for Signal Transduction

Abstract

ABSTRACTThe TonB system actively transports vital nutrients across the unenergized outer membranes of the majority of Gram-negative bacteria. In this system, integral membrane proteins ExbB, ExbD, and TonB work together to transduce the protonmotive force (PMF) of the inner membrane to customized active transporters in the outer membrane by direct and cyclic binding of TonB to the transporters. A PMF-dependent TonB-ExbD interaction is prevented by 10-residue deletions within a periplasmic disordered domain of ExbD adjacent to the cytoplasmic membrane. Here we explored the function of the ExbD disordered domain in more detail.In vivophoto-cross-linking through sequential pBpa substitutions in the ExbD disordered domain captured five different ExbD complexes, some of which had been previously detected using in vivo formaldehyde crosslinking, a technique that lacks the residue-specific information that can be achieved through photo-cross-linking: 2 ExbB-ExbD heterodimers (one of which had not been detected previously), previously detected ExbD homodimers, previously detected PMF-dependent ExbD-TonB heterodimers, and for the first time, a predicted, ExbD-TonB PMF-independent interaction. The fact that multiple complexes were captured by the same pBpa substitution indicated the dynamic nature of ExbD interactions as the energy transduction cycle proceeded in vivo. In this study, we also discovered that a conserved motif, (V45, V47, L49, P50), within the disordered domain was required for signal transduction to TonB and to the C-terminal domain of ExbD and was the source of its essentiality.ImportanceThe TonB system is a virulence factor for many Gram-negative pathogens including E-S-K-A-P-E pathogenic speciesKlebsiella pneumoniae,Acinetobacter baumannii,and Pseudomonas aeruginosa. Because the majority of protein-protein interactions in the TonB system occur in the periplasm, it is an appealing target for novel antibiotics. Understanding the molecular mechanism of the TonB system will provide valuable information for design of potential inhibitors targeting the system.