24In most Gram-negative bacteria, the TonB system is required to actively transport vital 25 nutrients across their unenergized outer-membrane barriers. The cytoplasmic membrane 26 proteins of the system ExbB, ExbD, and TonB work together to transduce the energy from the 27 protonmotive force (PMF) of the inner membrane to the ligand-specific, outer membrane TonB-28 dependent transporters. However, the precise coordination and interactions between ExbB, 29 ExbD, and TonB during energy transduction is unclear. Previously, deletions within a 30 periplasmic disordered domain of ExbD had been shown to be important for TonB system 31 activity and the essential ExbD-TonB PMF-dependent interaction. In this study, we have 32 discovered a conserved motif, ΨXΨXLP (Ψ = hydrophobic-branched residues; X = non-33 hydrophobic residues) specifically within this domain that was required for both TonB system 34 function and the ExbD-TonB PMF-dependent interaction. Alanine scanning mutagenesis found 35 that the only functionally important residues within the ExbD disordered domain were those 36 located in the motif. In addition, in vivo photo-cross-linking captured and identified five ExbD 37 complexes from pBpa substitutions within the ExbD disordered domain: 2-ExbB-ExbD 38 complexes, the ExbD homodimer, the PMF-dependent ExbD-TonB, and the first captured, 39 ExbD-TonB independent interaction. Interestingly, many of these complexes were captured 40 from single pBpa substitutions, which suggests that these residues are involved in multiple 41 interactions. The data presented in this study indicate that the ExbD disordered domain is a 42 highly dynamic region in vivo and a conserved motif within this domain is required for the TonB 43 system to respond to protonmotive force. 44 45 46 47Importance: 48 Within the context of pathogenicity, the TonB system is a virulence factor in many 49 Gram-negative pathogens including E-S-K-A-P-E pathogenic species Klebsiella pneumoniae, 50 Acinetobacter baumannii, and Pseudomonas aeruginosa. Because the TonB system is unique 51 to Gram-negative bacteria and is a periplasmically localized virulence factor, it is an appealing 52 target for novel antibiotics. However, there is no current antibiotic against this system. 53Understanding the mechanism by which the TonB system functions will provide valuable 54 information to design potential inhibitors targeting the system. 55 56 57 58 59 60 61 62 63 64 65 66 67 68 INTRODUCTION: 69The TonB system is required for most Gram-negative bacteria to acquire vital nutrients 70 such iron, vitamin B12, nickel, cobalt, copper, heme, maltodextrin, and sucrose across their 71 outer-membrane barriers (1), and is major virulence factor in many pathogenic Gram-negative 72 bacteria (2, 3). Under iron-limiting conditions, such as the human serum (3), Gram-negative 73 bacteria secrete iron-chelating compounds called siderophores that capture the iron with high 74 affinity. However, because iron-siderophore complexes are too large, too scare, and too 75...

The 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 proton motive 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 vivo photo-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 cross-linking, a technique that lacks the residue-specific information that can be achieved through photo-cross-linking: two 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, and 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 motif essentiality. IMPORTANCE The TonB system is a virulence factor for Gram-negative pathogens. The mechanism by which cytoplasmic membrane proteins of the TonB system transduce an electrochemical gradient into mechanical energy is a long-standing mystery. TonB, ExbB, and ExbD primary amino acid sequences are characterized by regions of predicted intrinsic disorder, consistent with a proposed multiplicity of protein-protein contacts as TonB proceeds through an energy transduction cycle, a complex process that has yet to be recapitulated in vitro. This study validates a region of intrinsic disorder near the ExbD transmembrane domain and identifies an essential conserved motif embedded within it that transduces signals to distal regions of ExbD suggested to configure TonB for productive interaction with outer membrane transporters.

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.

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