scholarly journals From Homodimer to Heterodimer and Back: Elucidating the TonB Energy Transduction Cycle

2015 ◽  
Vol 197 (21) ◽  
pp. 3433-3445 ◽  
Author(s):  
Michael G. Gresock ◽  
Kyle A. Kastead ◽  
Kathleen Postle
Keyword(s):  
Outer Membrane ◽  
Active Transport ◽  
Cytoplasmic Membrane ◽  
Transmembrane Domain ◽  
Energy Transduction ◽  
Proton Gradient ◽  
Carboxy Terminus ◽  
Gram Negative ◽  
Amino Terminal ◽  
Tonb System

ABSTRACTThe TonB system actively transports large, scarce, and important nutrients through outer membrane (OM) transporters of Gram-negative bacteria using the proton gradient of the cytoplasmic membrane (CM). InEscherichia coli, the CM proteins ExbB and ExbD harness and transfer proton motive force energy to the CM protein TonB, which spans the periplasmic space and cyclically binds OM transporters. TonB has two activity domains: the amino-terminal transmembrane domain with residue H20 and the periplasmic carboxy terminus, through which it binds to OM transporters. TonB is inactivated by all substitutions at residue H20 except H20N. Here, we show that while TonB trapped as a homodimer through its amino-terminal domain retained full activity, trapping TonB through its carboxy terminus inactivated it by preventing conformational changes needed for interaction with OM transporters. Surprisingly, inactive TonB H20A had little effect on homodimerization through the amino terminus and instead decreased TonB carboxy-terminal homodimer formation prior to reinitiation of an energy transduction cycle. That result suggested that the TonB carboxy terminus ultimately interacts with OM transporters as a monomer. Our findings also suggested the existence of a separate equimolar pool of ExbD homodimers that are not in contact with TonB. A model is proposed where interaction of TonB homodimers with ExbD homodimers initiates the energy transduction cycle, and, ultimately, the ExbD carboxy terminus modulates interactions of a monomeric TonB carboxy terminus with OM transporters. After TonB exchanges its interaction with ExbD for interaction with a transporter, ExbD homodimers undergo a separate cycle needed to re-energize them.IMPORTANCECanonical mechanisms of active transport across cytoplasmic membranes employ ion gradients or hydrolysis of ATP for energy. Gram-negative bacterial outer membranes lack these resources. The TonB system embodies a novel means of active transport across the outer membrane for nutrients that are too large, too scarce, or too important for diffusion-limited transport. A proton gradient across the cytoplasmic membrane is converted by a multiprotein complex into mechanical energy that drives high-affinity active transport across the outer membrane. This system is also of interest since one of its uses in pathogenic bacteria is for competition with the host for the essential element iron. Understanding the mechanism of the TonB system will allow design of antibiotics targeting iron acquisition.

scholarly journals The Intrinsically Disordered Region of ExbD Is Required for Signal Transduction

2020 ◽  
Vol 202 (7) ◽  
Author(s):  
Dale R. Kopp ◽  
Kathleen Postle
Keyword(s):  
Signal Transduction ◽  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Intrinsic Disorder ◽  
Energy Transduction ◽  
Cross Linking ◽  
Gram Negative ◽  
Conserved Motif ◽  
Tonb System

ABSTRACT 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.

scholarly journals Characterization of Escherichia coli ExbD protein modification in vivo

2020 ◽  
Author(s):  
Aruna Kumar ◽  
Kathleen Postle
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Active Transport ◽  
Protein Modification ◽  
Cytoplasmic Membrane ◽  
Physical Contact ◽  
Protonmotive Force ◽  
Iron Starvation ◽  
Tonb System

ABSTRACTThe TonB system of Escherichia coli couples the protonmotive force of the cytoplasmic membrane to active transport of nutrients across the outer membrane. In the cytoplasmic membrane, this system consists of three known proteins, TonB, ExbB, and ExbD. ExbB and ExbD appear to harvest the protonmotive force and transmit it to TonB, which then makes direct physical contact with TonB-dependent active transport proteins in the outer membrane. Using two-dimensional gel electrophoresis, we found that ExbD exists as two different species with the same apparent molecular mass but with different pIs. The more basic ExbD species was consistently present, while the more acidic species arose when cells were starved for iron by the addition of iron chelators. The cause of the modification was, however, more complex than simple iron starvation. Absence of either TonB or ExbB protein also gave rise to modified ExbD under iron-replete conditions where the wild-type strain exhibited no ExbD modification. The effect of the tonB or exbB mutations were not entirely due to iron limitation since an equally iron-limited aroB mutation did not replicate the ExbD modification. This constitutes the first report of in vivo modification for any of the TonB system proteins.

scholarly journals The TonB Dimeric Crystal Structures Do Not Exist In Vivo

mBio ◽  
2010 ◽  
Vol 1 (5) ◽  
Author(s):  
Kathleen Postle ◽  
Kyle A. Kastead ◽  
Michael G. Gresock ◽  
Joydeep Ghosh ◽  
Cheryl D. Swayne
Keyword(s):  
Outer Membrane ◽  
Crystal Structures ◽  
Cytoplasmic Membrane ◽  
Proton Motive Force ◽  
Membrane Transporters ◽  
Motive Force ◽  
Carboxy Terminus ◽  
Tonb System ◽  
Outer Membrane Transporters

ABSTRACTThe TonB system energizes transport of nutrients across the outer membrane ofEscherichia coliusing cytoplasmic membrane proton motive force (PMF) for energy. Integral cytoplasmic membrane proteins ExbB and ExbD appear to harvest PMF and transduce it to TonB. The carboxy terminus of TonB then physically interacts with outer membrane transporters to allow translocation of ligands into the periplasmic space. The structure of the TonB carboxy terminus (residues ~150 to 239) has been solved several times with similar results. Our previous results hinted thatin vitrostructures might not mimic the dimeric conformations that characterize TonBin vivo. To test structural predictions and to identify irreplaceable residues, the entire carboxy terminus of TonB was scanned with Cys substitutions. TonB I232C and N233C, predicted to efficiently form disulfide-linked dimers in the crystal structures, did not do so. In contrast, Cys substitutions positioned at large distances from one another in the crystal structures efficiently formed dimers. Cys scanning identified seven functionally important residues. However, no single residue was irreplaceable. The phenotypes conferred by changes of the seven residues depended on both the specific assay used and the residue substituted. All seven residues were synergistic with one another. The buried nature of the residues in the structures was also inconsistent with these properties. Taken together, these results indicate that the solved dimeric crystal structures of TonB do not exist. The most likely explanation for the aberrant structures is that they were obtained in the absence of the TonB transmembrane domain, ExbB, ExbD, and/or the PMF.IMPORTANCEThe TonB system of Gram-negative bacteria is an attractive target for development of novel antibiotics because of its importance in iron acquisition and virulence. Logically, therefore, the structure of TonB must be accurately understood. TonB functions as a dimerin vivo, and two different but similar crystal structures of the dimeric carboxy-terminal ~90 amino acids gave rise to mechanistic models. Here we demonstrate that the crystal structures, and therefore the models based on them, are not biologically relevant. The idiosyncratic phenotypes conferred by substitutions at the only seven functionally important residues in the carboxy terminus suggest that similar to interaction of cytochromes P450 with numerous substrates, these residues allow TonB to differentially interact with different outer membrane transporters. Taken together, data suggest that TonB is maintained poised between order and disorder by ExbB, ExbD, and the proton motive force (PMF) before energy transduction to the outer membrane transporters.

scholarly journals His20 Provides the Sole Functionally Significant Side Chain in the Essential TonB Transmembrane Domain

2007 ◽  
Vol 189 (7) ◽  
pp. 2825-2833 ◽  
Author(s):  
Ray A. Larsen ◽  
Gail E. Deckert ◽  
Kyle A. Kastead ◽  
Surendranathan Devanathan ◽  
Kimberly L. Keller ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Cytoplasmic Membrane ◽  
Transmembrane Domain ◽  
Protonmotive Force ◽  
Wild Type ◽  
Carboxy Terminus ◽  
Amino Terminal ◽  
In The Wild ◽  
Mutant Phenotypes

ABSTRACT The cytoplasmic membrane protein TonB couples the protonmotive force of the cytoplasmic membrane to active transport across the outer membrane of Escherichia coli. The uncleaved amino-terminal signal anchor transmembrane domain (TMD; residues 12 to 32) of TonB and the integral cytoplasmic membrane proteins ExbB and ExbD are essential to this process, with important interactions occurring among the several TMDs of all three proteins. Here, we show that, of all the residues in the TonB TMD, only His20 is essential for TonB activity. When alanyl residues replaced all TMD residues except Ser16 and His20, the resultant “all-Ala Ser16 His20” TMD TonB retained 90% of wild-type iron transport activity. Ser16Ala in the context of a wild-type TonB TMD was fully active. In contrast, His20Ala in the wild-type TMD was entirely inactive. In more mechanistically informative assays, the all-Ala Ser16 His20 TMD TonB unexpectedly failed to support formation of disulfide-linked dimers by TonB derivatives bearing Cys substitutions for the aromatic residues in the carboxy terminus. We hypothesize that, because ExbB/D apparently cannot efficiently down-regulate conformational changes at the TonB carboxy terminus through the all-Ala Ser16 His20 TMD, the TonB carboxy terminus might fold so rapidly that disulfide-linked dimers cannot be efficiently trapped. In formaldehyde cross-linking experiments, the all-Ala Ser16 His20 TMD also supported large numbers of apparently nonspecific contacts with unknown proteins. The all-Ala Ser16 His20 TMD TonB retained its dependence on ExbB/D. Together, these results suggest that a role for ExbB/D might be to control rapid and nonspecific folding that the unregulated TonB carboxy terminus otherwise undergoes. Such a model helps to reconcile the crystal/nuclear magnetic resonance structures of the TonB carboxy terminus with conformational changes and mutant phenotypes observed at the TonB carboxy terminus in vivo.

scholarly journals Identification of New In Vivo TonB-FepA Rendezvous Sites

2021 ◽  
Author(s):  
Kathleen Postle ◽  
Kelvin Kho ◽  
Michael Gresock ◽  
Joydeep Ghosh ◽  
Ray Larsen
Keyword(s):  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Protonmotive Force ◽  
Amphipathic Helix ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Tonb System ◽  
Carboxy Terminal ◽  
Amino Terminal Domain

The TonB system of Gram-negative bacteria uses the protonmotive force of the cytoplasmic membrane to energize active transport of large or scarce nutrients across the outer membrane by means of customized beta-barrels known as TonB-dependent transporters (TBDTs). The lumen of each TBDT is occluded by an amino-terminal domain, called the cork, which must be displaced for transport of nutrients or translocation of the large protein toxins that parasitize the system. A complex of cytoplasmic membrane proteins consisting of TonB, ExbB and ExbD harnesses the protonmotive force that TonB transmits to the TBDT. The specifics of this energy transformation are a source of continuing interest. The amino terminal domain of a TBDT contains a region called the TonB box, that is essential for the reception of energy from TonB. This domain is the only identified site of in vivo interaction between the TBDT and TonB, occurring through a non-essential region centered on TonB residue Q160. Because TonB binds to TBDTs whether or not it is active or even intact, the mechanism and extent of cork movement in vivo has been challenging to discover. In this study, we used in vivo disulfide crosslinking between eight engineered Cys residues in Escherichia coli TonB and 42 Cys substitutions in the TBDT FepA, including the TonB box, to identify novel sites of interaction in vivo. The TonB Cys substitutions in the core of an essential carboxy terminal amphipathic helix (residues 199-216) were compared to TonB Q160C interactions. Functionality of the in vivo interactions was established when the presence of the inactive TonB H20A mutation inhibited them. A previously unknown functional interaction between the hydrophilic face of the amphipathic helix and the FepA TonB box was identified. Interaction of Q160C with the FepA TonB box appeared to be less functionally important. The two different parts of TonB also differed in their interactions with the FepA cork and barrel turns. While the TonB amphipathic helix Cys residues interacted only with Cys residues on the periplasmic face of the FepA cork, TonB Q160C interacted with buried Cys substitutions within the FepA cork, the first such interactions seen with any TBDT. Both sets of interactions required active TonB. Taken together, these data suggest a model where the amphipathic helix binds to the TonB box, causing the mechanically weak domain of the FepA cork to dip sufficiently into the periplasmic space for interaction with the TonB Q160 region, which is an interaction that does not occur if the TonB box is deleted. The TonB amphipathic helix also interacted with periplasmic turns between FepA β-strands in vivo supporting a surveillance mechanism where TonB searched for TBDTs on the periplasmic face of the outer membrane.

scholarly journals An ExbD Disordered Domain Peptide Inhibits TonB System Activity

2020 ◽  
Author(s):  
Dale R. Kopp ◽  
Kathleen Postle
Keyword(s):  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Protonmotive Force ◽  
Gram Negative Bacteria ◽  
System Function ◽  
Gram Negative ◽  
Conserved Motif ◽  
Outer Membranes ◽  
Tonb System

ABSTRACTThe 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.

scholarly journals Membrane fusion during phage lysis

2015 ◽  
Vol 112 (17) ◽  
pp. 5497-5502 ◽  
Author(s):  
Manoj Rajaure ◽  
Joel Berry ◽  
Rohit Kongari ◽  
Jesse Cahill ◽  
Ry Young
Keyword(s):  
Membrane Fusion ◽  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Bacterial Host ◽  
Inner Membrane ◽  
Gram Negative ◽  
Encoded Proteins ◽  
Coliphage Lambda ◽  
Necessary And Sufficient

In general, phages cause lysis of the bacterial host to effect release of the progeny virions. Until recently, it was thought that degradation of the peptidoglycan (PG) was necessary and sufficient for osmotic bursting of the cell. Recently, we have shown that in Gram-negative hosts, phage lysis also requires the disruption of the outer membrane (OM). This is accomplished by spanins, which are phage-encoded proteins that connect the cytoplasmic membrane (inner membrane, IM) and the OM. The mechanism by which the spanins destroy the OM is unknown. Here we show that the spanins of the paradigm coliphage lambda mediate efficient membrane fusion. This supports the notion that the last step of lysis is the fusion of the IM and OM. Moreover, data are provided indicating that spanin-mediated fusion is regulated by the meshwork of the PG, thus coupling fusion to murein degradation by the phage endolysin. Because endolysin function requires the formation of μm-scale holes by the phage holin, the lysis pathway is seen to require dramatic dynamics on the part of the OM and IM, as well as destruction of the PG.

scholarly journals A small-molecule inhibitor of BamA impervious to efflux and the outer membrane permeability barrier

2019 ◽  
Vol 116 (43) ◽  
pp. 21748-21757 ◽  
Author(s):  
Elizabeth M. Hart ◽  
Angela M. Mitchell ◽  
Anna Konovalova ◽  
Marcin Grabowicz ◽  
Jessica Sheng ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Small Molecule ◽  
Cytoplasmic Membrane ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Bam Complex ◽  
Induced Aggregation

The development of new antimicrobial drugs is a priority to combat the increasing spread of multidrug-resistant bacteria. This development is especially problematic in gram-negative bacteria due to the outer membrane (OM) permeability barrier and multidrug efflux pumps. Therefore, we screened for compounds that target essential, nonredundant, surface-exposed processes in gram-negative bacteria. We identified a compound, MRL-494, that inhibits assembly of OM proteins (OMPs) by the β-barrel assembly machine (BAM complex). The BAM complex contains one essential surface-exposed protein, BamA. We constructed a bamA mutagenesis library, screened for resistance to MRL-494, and identified the mutation bamAE470K. BamAE470K restores OMP biogenesis in the presence of MRL-494. The mutant protein has both altered conformation and activity, suggesting it could either inhibit MRL-494 binding or allow BamA to function in the presence of MRL-494. By cellular thermal shift assay (CETSA), we determined that MRL-494 stabilizes BamA and BamAE470K from thermally induced aggregation, indicating direct or proximal binding to both BamA and BamAE470K. Thus, it is the altered activity of BamAE470K responsible for resistance to MRL-494. Strikingly, MRL-494 possesses a second mechanism of action that kills gram-positive organisms. In microbes lacking an OM, MRL-494 lethally disrupts the cytoplasmic membrane. We suggest that the compound cannot disrupt the cytoplasmic membrane of gram-negative bacteria because it cannot penetrate the OM. Instead, MRL-494 inhibits OMP biogenesis from outside the OM by targeting BamA. The identification of a small molecule that inhibits OMP biogenesis at the cell surface represents a distinct class of antibacterial agents.

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