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David S. Cafiso - One of the best experts on this subject based on the ideXlab platform.
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A Dynamic Protein–Protein Coupling between the TonB-Dependent Transporter FhuA and TonB
Biochemistry, 2018Co-Authors: Jessica L. Sarver, Michael Zhang, Lishan Liu, David A. Nyenhuis, David S. CafisoAbstract:Bacterial outer membrane TonB-dependent transporters function by executing cycles of binding and unbinding to the inner membrane Protein TonB. In the vitamin B12 transporter BtuB and the ferric citrate transporter FecA, substrate binding increases the periplasmic exposure of the Ton box, an energy-coupling segment. This increased exposure appears to enhance the affinity of the transporter for TonB. Here, continuous wave and pulse EPR spectroscopy were used to examine the state of the Ton box in the Escherichia coli ferrichrome transporter FhuA. In its apo state, the Ton box of FhuA samples a broad range of positions and multiple conformational substates. When bound to ferrichrome, the Ton box does not extend further into the periplasm, although the structural states sampled by the FhuA Ton box are altered. When bound to a soluble fragment of TonB, the TonB-FhuA complex remains heterogeneous and dynamic, indicating that TonB does not make strong, specific contacts with either the FhuA barrel or the core region of the transporter. This result differs from that seen in the crystal structure of the TonB-FhuA complex. These data indicate that unlike BtuB and FecA, the periplasmic exposure of the Ton box in FhuA does not change significantly in the presence of substrate and that allosteric control of transporter-TonB interactions functions by a different mechanism than that seen in either BtuB or FecA. Moreover, the data indicate that models involving a rotation of TonB relative to the transporter are unlikely to underlie the mechanism that drives TonB-dependent transport.
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a dynamic Protein Protein coupling between the TonB dependent transporter fhua and TonB
Biochemistry, 2018Co-Authors: Jessica L. Sarver, Michael Zhang, Lishan Liu, David A. Nyenhuis, David S. CafisoAbstract:Bacterial outer membrane TonB-dependent transporters function by executing cycles of binding and unbinding to the inner membrane Protein TonB. In the vitamin B12 transporter BtuB and the ferric citrate transporter FecA, substrate binding increases the periplasmic exposure of the Ton box, an energy-coupling segment. This increased exposure appears to enhance the affinity of the transporter for TonB. Here, continuous wave and pulse EPR spectroscopy were used to examine the state of the Ton box in the Escherichia coli ferrichrome transporter FhuA. In its apo state, the Ton box of FhuA samples a broad range of positions and multiple conformational substates. When bound to ferrichrome, the Ton box does not extend further into the periplasm, although the structural states sampled by the FhuA Ton box are altered. When bound to a soluble fragment of TonB, the TonB-FhuA complex remains heterogeneous and dynamic, indicating that TonB does not make strong, specific contacts with either the FhuA barrel or the core region of the transporter. This result differs from that seen in the crystal structure of the TonB-FhuA complex. These data indicate that unlike BtuB and FecA, the periplasmic exposure of the Ton box in FhuA does not change significantly in the presence of substrate and that allosteric control of transporter-TonB interactions functions by a different mechanism than that seen in either BtuB or FecA. Moreover, the data indicate that models involving a rotation of TonB relative to the transporter are unlikely to underlie the mechanism that drives TonB-dependent transport.
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A Dynamic Protein–Protein Coupling between the TonB-Dependent Transporter FhuA and TonB
2018Co-Authors: Jessica L. Sarver, Lishan Liu, Michael Zhang, David A. Nyenhuis, David S. CafisoAbstract:Bacterial outer membrane TonB-dependent transporters function by executing cycles of binding and unbinding to the inner membrane Protein TonB. In the vitamin B12 transporter BtuB and the ferric citrate transporter FecA, substrate binding increases the periplasmic exposure of the Ton box, an energy-coupling segment. This increased exposure appears to enhance the affinity of the transporter for TonB. Here, continuous wave and pulse EPR spectroscopy were used to examine the state of the Ton box in the Escherichia coli ferrichrome transporter FhuA. In its apo state, the Ton box of FhuA samples a broad range of positions and multiple conformational substates. When bound to ferrichrome, the Ton box does not extend further into the periplasm, although the structural states sampled by the FhuA Ton box are altered. When bound to a soluble fragment of TonB, the TonB-FhuA complex remains heterogeneous and dynamic, indicating that TonB does not make strong, specific contacts with either the FhuA barrel or the core region of the transporter. This result differs from that seen in the crystal structure of the TonB–FhuA complex. These data indicate that unlike BtuB and FecA, the periplasmic exposure of the Ton box in FhuA does not change significantly in the presence of substrate and that allosteric control of transporter–TonB interactions functions by a different mechanism than that seen in either BtuB or FecA. Moreover, the data indicate that models involving a rotation of TonB relative to the transporter are unlikely to underlie the mechanism that drives TonB-dependent transport
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Allosteric Signaling Is Bidirectional in an Outer-Membrane Transport Protein.
Biophysical journal, 2016Co-Authors: Arthur Sikora, Benesh Joseph, Morgan Matson, Jacob Staley, David S. CafisoAbstract:In BtuB, the Escherichia coli TonB-dependent transporter for vitamin B12, substrate binding to the extracellular surface unfolds a conserved energy coupling motif termed the Ton box into the periplasm. This transmembrane signaling event facilitates an interaction between BtuB and the inner-membrane Protein TonB. In this study, continuous-wave and pulse electron paramagnetic resonance in a native outer-membrane preparation demonstrate that signaling also occurs from the periplasmic to the extracellular surface in BtuB. The binding of a TonB fragment to the periplasmic interface alters the configuration of the second extracellular loop and partially dissociates a spin-labeled substrate analog. Moreover, mutants in the periplasmic Ton box that are transport-defective alter the binding site for vitamin B12 in BtuB. This work demonstrates that the Ton box and the extracellular substrate binding site are allosterically coupled in BtuB, and that TonB binding may initiate a partial round of transport.
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TonB Binding Produces Allosteric Changes in the Outer Loops and Substrate Binding Site of the TBDT BtuB
Biophysical Journal, 2016Co-Authors: Arthur Sikora, David S. CafisoAbstract:Outer-membrane TonB-dependent transporters function in the uptake of essential nutrients, and are important for the success of many bacterial pathogens. During transport, these Proteins undergo a cycle of binding and unbinding to the inner membrane Protein TonB, through an interaction that is mediated by the Ton box, an energy-coupling segment at the periplasmic interface. Previous work has shown that in BtuB, the vitamin B12 transporter in Escherichia coli, substrate binding unfolds the Ton box, an event which regulates the interaction between BtuB and TonB.In previous work (Joseph et al. (2015) Angew Chem Int Ed Engl. 54:6196) we demonstrated that electron-electron resonance (DEER) data could be obtained on BtuB in native outer membrane preparations and whole cells, and that distances were comparable in native and reconstituted systems. In the present study DEER was used in outer membranes to examine structural heterogeneity in the loops and the effect of ligands on loop configuration. The data show that the loops sample a large conformational space, but that the co-ligand calcium selects for one conformational substrate in the 2nd external loop. The ordering of this loop is correlated with a dramatic increase in the affinity of BtuB for B12. Interestingly, the binding of a C-terminal fragment of TonB to the periplasmic interface of BtuB produces significant changes in the exterior loops to generate a more open substrate binding pocket. Moreover, spectra from the spin-labeled cobalamin analog indicate that TonB weakens the affinity of the substrate and can displace vitamin B12 from BtuB. These data indicate that signal transduction moves in both directions in BtuB and that reduced B12 binding by TonB is a step in the transport process.This work was supported by NIGMS, GM035215.
Kathleen Postle - One of the best experts on this subject based on the ideXlab platform.
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Going Outside the TonB Box: Identification of Novel FepA-TonB Interactions In Vivo.
Journal of bacteriology, 2017Co-Authors: Michael G. Gresock, Kathleen PostleAbstract: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 vivoIMPORTANCE 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.
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From Homodimer to Heterodimer and Back: Elucidating the TonB Energy Transduction Cycle
Journal of bacteriology, 2015Co-Authors: Michael G. Gresock, Kyle A. Kastead, Kathleen PostleAbstract:ABSTRACT The 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). In Escherichia 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. IMPORTANCE Canonical 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.
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His20 Provides the Sole Functionally Significant Side Chain in the Essential TonB Transmembrane Domain
Journal of bacteriology, 2007Co-Authors: Ray A. Larsen, Gail E. Deckert, Kyle A. Kastead, Surendranathan Devanathan, Kimberly L. Keller, Kathleen PostleAbstract: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.
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Conserved Residues Ser16 and His20 and Their Relative Positioning Are Essential for TonB Activity, Cross-linking of TonB with ExbB, and the Ability of TonB to Respond to Proton Motive Force
The Journal of biological chemistry, 2000Co-Authors: Ray A. Larsen, Kathleen PostleAbstract:The cytoplasmic membrane Protein TonB couples the proton electrochemical potential of the cytoplasmic membrane to transport events at the outer membrane of Gram-negative bacteria. The amino-terminal signal anchor of TonB and its interaction with the cytoplasmic membrane Protein ExbB are essential to this process. The TonB signal anchor is predicted to form an alpha-helix, with a conserved face comprised of residues Ser(16), His(20), Leu(27), and Ser(31). Deletion of either Ser(16) or His(20) or of individual intervening but not flanking residues rendered TonB inactive and unable to assume a proton motive force-dependent conformation. In vivo formaldehyde cross-linking experiments revealed that the ability of this subset of mutants to form a characteristic heterodimer with ExbB was greatly diminished. Replacement of residues 17-19 by three consecutive alanines produced a wild type TonB allele, indicating that the intervening residues (Val, Cys, and Ile) contributed only to spacing. These data indicated that the spatial relationship of Ser(16) to His(20) was essential to function and suggested that the motif HXXXS defines the minimal requirement for the coupling of TonB to the cytoplasmic membrane electrochemical gradient. Deletion of Trp(11) resulted in a TonB that remained active yet was unable to cross-link with ExbB. Because Trp(11) was demonstrably not involved in the actual cross-linking, these results suggest that the TonB/ExbB interaction detected by cross-linking occurred at a step in the energy transduction cycle distinct from the coupling of TonB to the electrochemical gradient.
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Cell Envelope Signaling in Escherichia coli LIGAND BINDING TO THE FERRICHROME-IRON RECEPTOR FhuA PROMOTES INTERACTION WITH THE ENERGY-TRANSDUCING Protein TonB
The Journal of biological chemistry, 1997Co-Authors: Gregory S. Moeck, James W Coulton, Kathleen PostleAbstract:Abstract The ferrichrome-iron receptor ofEscherichia coli is FhuA, an outer membrane Protein that is dependent upon the energy-coupling Protein TonB to enable active transport of specific hydroxamate siderophores, infection by certain phages, and cell killing by the Protein antibiotics colicin M and microcin 25. In vivo cross-linking studies were performed to establish at the biochemical level the interaction between FhuA and TonB. In an E. coli strain in which both Proteins were expressed from the chromosome, a high molecular mass complex was detected when the ferrichrome homologue ferricrocin was added immediately prior to addition of cross-linker. The complex included both Proteins; it was absent from strains of E. coli that were devoid of either FhuA or TonB, and it was detected with anti-FhuA and anti-TonB monoclonal antibodies. These results indicate that,in vivo, the binding of ferricrocin to FhuA enhances complex formation between the receptor and TonB. An in vitro system was established with which to examine the FhuA-TonB interaction. Incubation of TonB with histidine-tagged FhuA followed by addition of Ni2+-nitrilotriacetate-agarose led to the specific recovery of both TonB and FhuA. Addition of ferricrocin or colicin M to FhuA in this system greatly increased the coupling between FhuA and TonB. Conversely, a monoclonal antibody that binds near the N terminus of FhuA reduced the retention of TonB by histidine-tagged FhuA. These studies demonstrate the significance of ligand binding at the external surface of the cell to mediate signal transduction across the outer membrane.
Robert J. Kadner - One of the best experts on this subject based on the ideXlab platform.
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The Escherichia coli outer membrane cobalamin transporter BtuB: structural analysis of calcium and substrate binding, and identification of orthologous transporters by sequence/structure conservation.
Journal of molecular biology, 2003Co-Authors: David P. Chimento, Robert J. Kadner, Michael C WienerAbstract:Gram-negative bacteria possess specialized active transport systems that function to transport organometallic cofactors or carriers, such as cobalamins, siderophores, and porphyrins, across their outer membranes. The primary components of each transport system are an outer membrane transporter and the energy-coupling Protein TonB. In Escherichia coli, the TonB-dependent outer membrane transporter BtuB carries out active transport of cobalamin (Cbl) substrates across its outer membrane. Cobalamins bind to BtuB with nanomolar affinity. Previous studies implicated calcium in high-affinity binding of cyanocobalamin (CN-Cbl) to BtuB. We previously solved four structures of BtuB or BtuB complexes: an apo-structure of a methionine-substitution mutant (used to obtain experimental phases by selenomethionine single-wavelength anomalous diffraction studies); an apo-structure of wild-type BtuB; a binary complex of calcium and wild-type BtuB; and a ternary complex of calcium, CN-Cbl and wild-type BtuB. We present an analysis of the binding of calcium in the binary and ternary complexes, and show that calcium coordination changes upon substrate binding. High-affinity CN-Cbl binding and calcium coordination are coupled. We also analyze the binding mode of CN-Cbl to BtuB, and compare and contrast this binding to that observed in other Proteins that bind Cbl. BtuB binds CN-Cbl in a manner very different from Cbl-utilizing enzymes and the periplasmic Cbl binding Protein BtuF. Homology searches of bacterial genomes, structural annotation based on the presence of conserved Cbl-binding residues identified by analysis of our BtuB structure, and detection of homologs of the periplasmic Cbl-binding binding Protein BtuF enable identification of putative BtuB orthologs in enteric and non-enteric bacterial species.
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Competing ligands stabilize alternate conformations of the energy coupling motif of a TonB-dependent outer membrane transporter
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Gail E. Fanucci, Robert J. Kadner, Nathalie Cadieux, David S. CafisoAbstract:BtuB is a TonB-dependent outer-membrane transporter for vitamin B12 (or cyanocobalamin, CN-Cbl) in Escherichia coli. The binding of CN-Cbl is believed to promote an unfolding or undocking of the Ton box, the conserved N-terminal energy coupling motif at the periplasmic surface of the transporter. This structural change may facilitate the interaction of BtuB with the inner membrane Protein TonB. In this work, the effect of the receptor binding fragment of colicin E3 (E3R) on the conformation of the Ton box was examined with site-directed spin labeling. Addition of E3R reverses the undocking of the Ton box that is promoted by CN-Cbl, consistent with a competitive binding between the substrate and the colicin fragment. EPR spectroscopy indicates that the Ton box is in a two-state equilibrium between docked and undocked conformations. In the absence of substrate, the docked conformation is the predominant state; however, the equilibrium can be shifted to the undocked state by the addition of detergents or site-specific proline substitutions. Even when the undocking is induced by detergents or by certain proline mutations, E3R binding shifts the equilibrium to the docked conformation. Thus, two competitive extracellular ligands, CN-Cbl and ER3, transduce opposite conformations of the N-terminal Ton box. Substrate binding stabilizes an undocked conformation, whereas E3R binding stabilizes a docked conformation of the Ton box.
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Transport-defective mutations alter the conformation of the energy-coupling motif of an outer membrane transporter.
Biochemistry, 2001Co-Authors: Kelly A. Coggshall, Robert J. Kadner, Nathalie Cadieux, Christie A. Piedmont, David S. CafisoAbstract:The bacterial outer membrane transporter for vitamin B(12), BtuB, derives its energy for transport by interacting with the trans-periplasmic membrane Protein TonB. This interaction with TonB occurs in part through an N-terminal segment in the BtuB sequence called the Ton box. In the present study, site-directed spin labeling of intact outer membrane preparations was used to investigate the conformation of the Ton box in wild-type BtuB and in two transport-defective mutants, L8P and V10P. In the wild-type Protein, the Ton box is folded into the barrel of the transporter. The conformation of this segment is dramatically different in the transport-defective mutants L8P and V10P, where the Ton box is found to be flexible, and undocked from the transporter barrel with a greater exposure to the periplasm. In the wild-type Protein, vitamin B(12) induces an undocking of the Ton box, but its addition to these transport defective mutants produces little or no change in the conformation of the Ton box. Proline substitutions at positions that do not alter transport do not alter the wild-type conformation of the Ton box; thus, the effect of substituting proline at positions 8 and 10 on the docked state of the Ton box appears to be unique. The failure of these mutants to execute the B(12) transport cycle may be a result of the altered conformation of the Ton box.
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Sequence Changes in the Ton Box Region of BtuB Affect Its Transport Activities and Interaction with TonB Protein
Journal of bacteriology, 2000Co-Authors: Nathalie Cadieux, Clive Bradbeer, Robert J. KadnerAbstract:Uptake of cobalamins by the transporter Protein BtuB in the outer membrane of Escherichia coli requires the proton motive force and the transperiplasmic Protein TonB. The Ton box sequence near the amino terminus of BtuB is conserved among all TonB-dependent transporters and is the only known site of mutations that confer a transport-defective phenotype which can be suppressed by certain substitutions at residue 160 in TonB. The crystallographic structures of the TonB-dependent transporter FhuA revealed that the region near the Ton box, which itself was not resolved, is exposed to the periplasmic space and undergoes an extensive shift in position upon binding of substrate. Site-directed disulfide bonding in intact cells has been used to show that the Ton box of BtuB and residues around position 160 of TonB approach each other in a highly oriented and specific manner to form BtuB-TonB heterodimers that are stimulated by the presence of transport substrate. Here, replacement of Ton box residues with proline or cysteine revealed that residue side chain recognition is not important for function, although replacement with proline at four of the seven Ton box positions impaired cobalamin transport. The defect in cobalamin utilization resulting from the L8P substitution was suppressed by cysteine substitutions in adjacent residues in BtuB or in TonB. This suppression did not restore active transport of cobalamins but may allow each transporter to function at most once. The uncoupled proline substitutions in BtuB markedly affected the pattern of disulfide bonding to TonB, both increasing the extent of cross-linking and shifting the pairs of residues that can be joined. Cross-linking of BtuB and TonB in the presence of the BtuB V10P substitution became independent of the presence of substrate, indicating an additional distortion of the exposure of the Ton box in the periplasmic space. TonB action thus requires a specific orientation for functional contact with the Ton box, and changes in the conformation of this region block transport by preventing substrate release and repeated transport cycles.
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Substrate-induced exposure of an energy-coupling motif of a membrane transporter
Nature Structural Biology, 2000Co-Authors: Helen J. Merianos, Robert J. Kadner, Nathalie Cadieux, Cindy H. Lin, David S. CafisoAbstract:BtuB is an outer membrane Protein responsible for the uptake of vitamin B_12 by Escherichia coli. It belongs to a family of bacterial transport Proteins that derive energy for transport by coupling to the trans-periplasmic energy-coupling Protein TonB. Using site-directed spin labeling and EPR we investigated the structure and substrate-induced changes in the TonB box, a highly conserved region in all TonB dependent transporters that may couple to TonB. In the absence of substrate, the line widths and collision parameters from EPR are consistent with this domain existing in a structured helical conformation that contacts the barrel of the transporter. Addition of substrate converts this segment into an extended structure that is highly dynamic, disordered and probably extended into the periplasm. This structural change demonstrates that the TonB box cycles between sequestered and accessible states in a substrate-dependent fashion. In a transport defective mutant of BtuB, this conformational cycle is disrupted and the TonB box appears to be extended even in the absence of substrate. These data suggest that the TonB box extends into the periplasm and interacts with TonB only in the presence of substrate.
Jessica L. Sarver - One of the best experts on this subject based on the ideXlab platform.
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a dynamic Protein Protein coupling between the TonB dependent transporter fhua and TonB
Biochemistry, 2018Co-Authors: Jessica L. Sarver, Michael Zhang, Lishan Liu, David A. Nyenhuis, David S. CafisoAbstract:Bacterial outer membrane TonB-dependent transporters function by executing cycles of binding and unbinding to the inner membrane Protein TonB. In the vitamin B12 transporter BtuB and the ferric citrate transporter FecA, substrate binding increases the periplasmic exposure of the Ton box, an energy-coupling segment. This increased exposure appears to enhance the affinity of the transporter for TonB. Here, continuous wave and pulse EPR spectroscopy were used to examine the state of the Ton box in the Escherichia coli ferrichrome transporter FhuA. In its apo state, the Ton box of FhuA samples a broad range of positions and multiple conformational substates. When bound to ferrichrome, the Ton box does not extend further into the periplasm, although the structural states sampled by the FhuA Ton box are altered. When bound to a soluble fragment of TonB, the TonB-FhuA complex remains heterogeneous and dynamic, indicating that TonB does not make strong, specific contacts with either the FhuA barrel or the core region of the transporter. This result differs from that seen in the crystal structure of the TonB-FhuA complex. These data indicate that unlike BtuB and FecA, the periplasmic exposure of the Ton box in FhuA does not change significantly in the presence of substrate and that allosteric control of transporter-TonB interactions functions by a different mechanism than that seen in either BtuB or FecA. Moreover, the data indicate that models involving a rotation of TonB relative to the transporter are unlikely to underlie the mechanism that drives TonB-dependent transport.
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A Dynamic Protein–Protein Coupling between the TonB-Dependent Transporter FhuA and TonB
Biochemistry, 2018Co-Authors: Jessica L. Sarver, Michael Zhang, Lishan Liu, David A. Nyenhuis, David S. CafisoAbstract:Bacterial outer membrane TonB-dependent transporters function by executing cycles of binding and unbinding to the inner membrane Protein TonB. In the vitamin B12 transporter BtuB and the ferric citrate transporter FecA, substrate binding increases the periplasmic exposure of the Ton box, an energy-coupling segment. This increased exposure appears to enhance the affinity of the transporter for TonB. Here, continuous wave and pulse EPR spectroscopy were used to examine the state of the Ton box in the Escherichia coli ferrichrome transporter FhuA. In its apo state, the Ton box of FhuA samples a broad range of positions and multiple conformational substates. When bound to ferrichrome, the Ton box does not extend further into the periplasm, although the structural states sampled by the FhuA Ton box are altered. When bound to a soluble fragment of TonB, the TonB-FhuA complex remains heterogeneous and dynamic, indicating that TonB does not make strong, specific contacts with either the FhuA barrel or the core region of the transporter. This result differs from that seen in the crystal structure of the TonB-FhuA complex. These data indicate that unlike BtuB and FecA, the periplasmic exposure of the Ton box in FhuA does not change significantly in the presence of substrate and that allosteric control of transporter-TonB interactions functions by a different mechanism than that seen in either BtuB or FecA. Moreover, the data indicate that models involving a rotation of TonB relative to the transporter are unlikely to underlie the mechanism that drives TonB-dependent transport.
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Site-Directed Spin Labeling Reveals Multiple Modes for Regulating Protein-Protein Interactions in Bacterial Outer Membrane Transport
Biophysical Journal, 2013Co-Authors: Jessica L. Sarver, Audrey Mokdad, David S. CafisoAbstract:Structural transitions in three outer-membrane bacterial transport Proteins have been investigated using site-directed spin labeling and EPR spectroscopy. In the vitamin B12 transporter BtuB and the ferric citrate transporter FecA, EPR spectra reveal the existence of a substrate-dependent order-to-disorder transition in the energy coupling motif (Ton box), which is localized at the periplasmic surface of the transporter. In BtuB, the Ton box unfolds into the periplasm, and in FecA, an N-terminal transcriptional domain disengages from the Ton box. Both these events expose the Ton box and initiate interactions with the inner membrane Protein TonB, which drives transport. However, this disorder transition is not observed in all transporters, and in the ferrichrome transporter FhuA the EPR spectra indicate that the Ton box is constitutively disordered. Distance measurements using double electron-electron resonance (DEER) indicate how the FhuA Ton box is regulated, and each transporter regulates interactions with the inner membrane Protein TonB using a distinct molecular mechanism. This work indicates that a variety of mechanisms may used to regulate Protein-Protein interactions in a single transporter family.
Audrey Mokdad - One of the best experts on this subject based on the ideXlab platform.
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Monomeric TonB and the Ton box are required for the formation of a high-affinity transporter-TonB complex.
Biochemistry, 2013Co-Authors: Daniel M. Freed, Audrey Mokdad, Arthur Sikora, Stephen M. Lukasik, David S. CafisoAbstract:The energy-dependent uptake of trace nutrients by Gram-negative bacteria involves the coupling of an outer membrane transport Protein to the transperiplasmic Protein TonB. In this study, a soluble construct of Escherichia coli TonB (residues 33-239) was used to determine the affinity of TonB for outer membrane transporters BtuB, FecA, and FhuA. Using fluorescence anisotropy, TonB(33-239) was found to bind with high affinity (tens of nanomolar) to both BtuB and FhuA; however, no high-affinity binding to FecA was observed. In BtuB, the high-affinity binding of TonB(33-239) was eliminated by mutations in the Ton box, which yield transport-defective Protein, or by the addition of a Colicin E3 fragment, which stabilizes the Ton box in a folded state. These results indicate that transport requires a high-affinity transporter-TonB interaction that is mediated by the Ton box. Characterization of TonB(33-239) using double electron-electron resonance (DEER) demonstrates that a significant population of TonB(33-239) exists as a dimer; moreover, interspin distances are in approximate agreement with interlocked dimers observed previously by crystallography for shorter TonB fragments. When the TonB(33-239) dimer is bound to the outer membrane transporter, DEER shows that the TonB(33-239) dimer is converted to a monomeric form, suggesting that a dimer-monomer conversion takes place at the outer membrane during the TonB-dependent transport cycle.
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Site-Directed Spin Labeling Reveals Multiple Modes for Regulating Protein-Protein Interactions in Bacterial Outer Membrane Transport
Biophysical Journal, 2013Co-Authors: Jessica L. Sarver, Audrey Mokdad, David S. CafisoAbstract:Structural transitions in three outer-membrane bacterial transport Proteins have been investigated using site-directed spin labeling and EPR spectroscopy. In the vitamin B12 transporter BtuB and the ferric citrate transporter FecA, EPR spectra reveal the existence of a substrate-dependent order-to-disorder transition in the energy coupling motif (Ton box), which is localized at the periplasmic surface of the transporter. In BtuB, the Ton box unfolds into the periplasm, and in FecA, an N-terminal transcriptional domain disengages from the Ton box. Both these events expose the Ton box and initiate interactions with the inner membrane Protein TonB, which drives transport. However, this disorder transition is not observed in all transporters, and in the ferrichrome transporter FhuA the EPR spectra indicate that the Ton box is constitutively disordered. Distance measurements using double electron-electron resonance (DEER) indicate how the FhuA Ton box is regulated, and each transporter regulates interactions with the inner membrane Protein TonB using a distinct molecular mechanism. This work indicates that a variety of mechanisms may used to regulate Protein-Protein interactions in a single transporter family.
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Ligand-induced structural changes in the Escherichia coli ferric citrate transporter reveal modes for regulating Protein-Protein interactions.
Journal of molecular biology, 2012Co-Authors: Audrey Mokdad, Miyeon Kim, Dawn Z. Herrick, Ali K. Kahn, Emily Andrews, David S. CafisoAbstract:Outer-membrane TonB-dependent transporters, such as the Escherichia coli ferric citrate transporter FecA, interact with the inner-membrane Protein TonB through an energy-coupling segment termed the Ton box. In FecA, which regulates its own transcription, the Ton box is preceded by an N-terminal extension that interacts with the inner‐membrane Protein FecR. Here, site-directed spin labeling was used to examine the structural basis for transcriptional signaling and Ton box regulation in FecA. EPR spectroscopy indicates that regions of the N-terminal domain are in conformational exchange, consistent with its role as a Protein binding element; however, the local fold and dynamics of the domain are not altered by substrate or TonB. Distance restraints derived from pulse EPR were used to generate models for the position of the extension in the apo, substrate-, and TonB-bound states. In the apo state, this domain is positioned at the periplasmic surface of FecA, where it interacts with the Ton box and blocks access of the Ton box to the periplasm. Substrate addition rotates the transcriptional domain and exposes the Ton box, leading to a disorder transition in the Ton box that may facilitate interactions with TonB. When a soluble fragment of TonB is bound to FecA, the transcriptional domain is displaced to one edge of the barrel, consistent with a proposed β-strand exchange mechanism. However, neither substrate nor TonB displaces the N-terminus further into the periplasm. This result suggests that the intact TonB system mediates both signaling and transport by unfolding portions of the transporter.
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Pathway for Transmembrane Signaling in the Bacterial Ferric Citrate Transporter FecA
Biophysical Journal, 2011Co-Authors: Audrey Mokdad, Ali K. Khan, David S. CafisoAbstract:FecA is an outer-membrane TonB-dependent transporter, which binds and transports ferric citrate into the periplasm. FecA is also one of a number of TonB-dependent transporters which regulate its own transcription. FecA contains a Ton box, which interacts with the inner membrane Protein TonB, as well as an N-terminal signaling motif, which interacts with the inner membrane Protein FecR and up-regulates FecA expression. Site-directed spin labeling has been used to study the signal-transduction pathway through FecA. We have shown that mutants that interfere with transcriptional signaling also interfere with a substrate-induced unfolding of the Ton box, indicating that the two processes share a common pathway. We have also used double electron-electron resonance (DEER) to measure distances and determine the position of the N-terminal signaling motif of FecA in the periplasmic space using simulated annealing. The position of this motif has been determined in the presence and absence of substrate, and provides an indication of the molecular mechanisms that may underlie transcriptional signaling.
