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 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 The Intrinsically Disordered Region of ExbD is Required for Signal Transduction

2019 ◽  
Author(s):  
Dale R. Kopp ◽  
Kathleen Postle
Keyword(s):  
Signal Transduction ◽  
Protein Interactions ◽  
Cross Linking ◽  
Protonmotive Force ◽  
Specific Information ◽  
Gram Negative ◽  
Intrinsically Disordered ◽  
Formaldehyde Crosslinking ◽  
Tonb System

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 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 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 species Klebsiella 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.

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 Interactions in the TonB-Dependent Energy Transduction Complex: ExbB and ExbD Form Homomultimers

1998 ◽  
Vol 180 (22) ◽  
pp. 6031-6038 ◽  
Author(s):  
Penelope I. Higgs ◽  
Paul S. Myers ◽  
Kathleen Postle
Keyword(s):  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Protein S ◽  
Energy Transduction ◽  
The Other ◽  
Receptor Protein ◽  
Cross Linking ◽  
Vitamin B ◽  
Outer Membrane Receptor

ABSTRACT The cytoplasmic membrane proteins ExbB and ExbD support TonB-dependent active transport of iron siderophores and vitamin B12 across the essentially unenergized outer membrane ofEscherichia coli. In this study, in vivo formaldehyde cross-linking analysis was used to investigate the interactions of T7 epitope-tagged ExbB or ExbD proteins. ExbB and ExbD each formed two unique cross-linked complexes which were not dependent on the presence of TonB, the outer membrane receptor protein FepA, or the other Exb protein. Cross-linking analysis of ExbB- and ExbD-derived size variants demonstrated instead that these ExbB and ExbD complexes were homodimers and homotrimers and suggested that ExbB also interacted with an unidentified protein(s). Cross-linking analysis of epitope-tagged ExbB and ExbD proteins with TonB antisera afforded detection of a previously unrecognized TonB-ExbD cross-linked complex and confirmed the composition of the TonB-ExbB cross-linked complex. The implications of these findings for the mechanism of TonB-dependent energy transduction are discussed.

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 TonB Interacts with Nonreceptor Proteins in the Outer Membrane of Escherichia coli

2002 ◽  
Vol 184 (6) ◽  
pp. 1640-1648 ◽  
Author(s):  
Penelope I. Higgs ◽  
Tracy E. Letain ◽  
Kelley K. Merriam ◽  
Neal S. Burke ◽  
HaJeung Park ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Outer Membrane Proteins ◽  
Membrane Receptors ◽  
Strong Interactions ◽  
Cross Linking ◽  
Outer Membrane Receptor ◽  
Outer Membranes

ABSTRACT The Escherichia coli TonB protein serves to couple the cytoplasmic membrane proton motive force to active transport of iron-siderophore complexes and vitamin B12 across the outer membrane. Consistent with this role, TonB has been demonstrated to participate in strong interactions with both the cytoplasmic and outer membranes. The cytoplasmic membrane determinants for that interaction have been previously characterized in some detail. Here we begin to examine the nature of TonB interactions with the outer membrane. Although the presence of the siderophore enterochelin (also known as enterobactin) greatly enhanced detectable cross-linking between TonB and the outer membrane receptor, FepA, the absence of enterochelin did not prevent the localization of TonB to the outer membrane. Furthermore, the absence of FepA or indeed of all the iron-responsive outer membrane receptors did not alter this association of TonB with the outer membrane. This suggested that TonB interactions with the outer membrane were not limited to the TonB-dependent outer membrane receptors. Hydrolysis of the murein layer with lysozyme did not alter the distribution of TonB, suggesting that peptidoglycan was not responsible for the outer membrane association of TonB. Conversely, the interaction of TonB with the outer membrane was disrupted by the addition of 4 M NaCl, suggesting that these interactions were proteinaceous. Subsequently, two additional contacts of TonB with the outer membrane proteins Lpp and, putatively, OmpA were identified by in vivo cross-linking. These contacts corresponded to the 43-kDa and part of the 77-kDa TonB-specific complexes described previously. Surprisingly, mutations in these proteins individually did not appear to affect TonB phenotypes. These results suggest that there may be multiple redundant sites where TonB can interact with the outer membrane prior to transducing energy to the outer membrane receptors.

scholarly journals Going Outside the TonB Box: Identification of Novel FepA-TonB Interactions In Vivo

2017 ◽  
Vol 199 (10) ◽  
Author(s):  
Michael G. Gresock ◽  
Kathleen Postle
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Current Model ◽  
Membrane Transporters ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Interaction Sites ◽  
Outer Membrane Transporters

ABSTRACT In Gram-negative bacteria, the cytoplasmic membrane protein TonB transmits energy derived from proton motive force to energize transport of important nutrients through TonB-dependent transporters in the outer membrane. Each transporter consists of a beta barrel domain and a lumen-occluding cork domain containing an essential sequence called the TonB box. To date, the only identified site of transporter-TonB interaction is between the TonB box and residues ∼158 to 162 of TonB. While the mechanism of ligand transport is a mystery, a current model based on site-directed spin labeling and molecular dynamics simulations is that, following ligand binding, the otherwise-sequestered TonB box extends into the periplasm for recognition by TonB, which mediates transport by pulling or twisting the cork. In this study, we tested that hypothesis with the outer membrane transporter FepA using in vivo photo-cross-linking to explore interactions of its TonB box and determine whether additional FepA-TonB interaction sites exist. We found numerous specific sites of FepA interaction with TonB on the periplasmic face of the FepA cork in addition to the TonB box. Two residues, T32 and A33, might constitute a ligand-sensitive conformational switch. The facts that some interactions were enhanced in the absence of ligand and that other interactions did not require the TonB box argued against the current model and suggested that the transport process is more complex than originally conceived, with subtleties that might provide a mechanism for discrimination among ligand-loaded transporters. These results constitute the first study on the dynamics of TonB-gated transporter interaction with TonB in vivo. IMPORTANCE The TonB system of Gram-negative bacteria has a noncanonical active transport mechanism involving signal transduction and proteins integral to both membranes. To achieve transport, the cytoplasmic membrane protein TonB physically contacts outer membrane transporters such as FepA. Only one contact between TonB and outer membrane transporters has been identified to date: the TonB box at the transporter amino terminus. The TonB box has low information content, raising the question of how TonB can discriminate among multiple different TonB-dependent transporters present in the bacterium if it is the only means of contact. Here we identified several additional sites through which FepA contacts TonB in vivo, including two neighboring residues that may explain how FepA signals to TonB that ligand has bound.

scholarly journals Coordination of peptidoglycan synthesis and outer membrane constriction during Escherichia coli cell division

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Andrew N Gray ◽  
Alexander JF Egan ◽  
Inge L van't Veer ◽  
Jolanda Verheul ◽  
Alexandre Colavin ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Cellular Structure ◽  
Cell Envelope ◽  
Energy State ◽  
Cross Linking ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Peptidoglycan Synthesis ◽  
Synthase Regulation

To maintain cellular structure and integrity during division, Gram-negative bacteria must carefully coordinate constriction of a tripartite cell envelope of inner membrane, peptidoglycan (PG), and outer membrane (OM). It has remained enigmatic how this is accomplished. Here, we show that envelope machines facilitating septal PG synthesis (PBP1B-LpoB complex) and OM constriction (Tol system) are physically and functionally coordinated via YbgF, renamed CpoB (Coordinator of PG synthesis and OM constriction, associated with PBP1B). CpoB localizes to the septum concurrent with PBP1B-LpoB and Tol at the onset of constriction, interacts with both complexes, and regulates PBP1B activity in response to Tol energy state. This coordination links PG synthesis with OM invagination and imparts a unique mode of bifunctional PG synthase regulation by selectively modulating PBP1B cross-linking activity. Coordination of the PBP1B and Tol machines by CpoB contributes to effective PBP1B function in vivo and maintenance of cell envelope integrity during division.

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