The Experts below are selected from a list of 10881 Experts worldwide ranked by ideXlab platform
James W Coulton - One of the best experts on this subject based on the ideXlab platform.
-
tonb interacts with btuf the escherichia coli periplasmic binding protein for cyanocobalamin
Biochemistry, 2009Co-Authors: Karron J James, Mark A Hancock, Jeannicolas Gagnon, James W CoultonAbstract:By its direct contact with Outer Membrane Receptor BtuB, the cytoplasmic Membrane transducer TonB delivers energy that mediates cyanocobalamin uptake in Escherichia coli. This activity has been generally proposed to be the role of TonB in cyanocobalamin uptake. We now report the discovery and characterization of interactions between TonB and periplasmic binding protein BtuF. Phage display experiments predicted interaction between TonB and BtuF, identifying potential binding residues on each protein. Dynamic light scattering experiments measured a complex of 55 kDa, consistent with a TonB−BtuF heterodimer. The hydrodynamic radius of the complex was unchanged in the presence of cyanocobalamin. Surface plasmon resonance measured TonB−BtuF interaction kinetics that were independent of cyanocobalamin and that deviated from a simple binding model. Binding isotherms from intrinsic fluorescence suggested a multifaceted interaction that was independent of cyanocobalamin. In addition, the presence of TonB did not a...
-
tonb interacts with btuf the escherichia coli periplasmic binding protein for cyanocobalamin
Biochemistry, 2009Co-Authors: Karron J James, Mark A Hancock, Jeannicolas Gagnon, James W CoultonAbstract:By its direct contact with Outer Membrane Receptor BtuB, the cytoplasmic Membrane transducer TonB delivers energy that mediates cyanocobalamin uptake in Escherichia coli. This activity has been generally proposed to be the role of TonB in cyanocobalamin uptake. We now report the discovery and characterization of interactions between TonB and periplasmic binding protein BtuF. Phage display experiments predicted interaction between TonB and BtuF, identifying potential binding residues on each protein. Dynamic light scattering experiments measured a complex of 55 kDa, consistent with a TonB-BtuF heterodimer. The hydrodynamic radius of the complex was unchanged in the presence of cyanocobalamin. Surface plasmon resonance measured TonB-BtuF interaction kinetics that were independent of cyanocobalamin and that deviated from a simple binding model. Binding isotherms from intrinsic fluorescence suggested a multifaceted interaction that was independent of cyanocobalamin. In addition, the presence of TonB did not abrogate subsequent binding of cyanocobalamin by BtuF. Taken together, these data support a previously proposed model wherein TonB serves as a scaffold to optimally position BtuF for initial binding of cyanocobalamin and for its subsequent release. These results substantiate a diverse role for TonB with its multiple protein-protein interactions in bacterial nutrient uptake systems.
-
tonb induces conformational changes in surface exposed loops of fhua Outer Membrane Receptor of escherichia coli
Protein Science, 2008Co-Authors: Karron J James, Mark A Hancock, Violaine Moreau, Franck Molina, James W CoultonAbstract:Most bacteria require iron to grow, but this element exists predominantly in insoluble forms in the environment, making it difficult to absorb. To acquire iron, bacteria have developed effective transport mechanisms. Bacterial Outer Membrane (OM) Receptors bind and import small molecules called siderophores that chelate iron with high affinity (Ferguson and Deisenhofer 2004). FhuA, an OM Receptor protein of Escherichia coli, imports iron chelated by ferrichrome or by its structural analog ferricrocin, both hydroxamate-type siderophores. The iron transport cycle commences when FhuA binds a siderophore. Import of these hydroxamate siderophores through FhuA requires direct contact between FhuA and TonB, a protein anchored in the cytoplasmic Membrane (CM). At the CM, energy from the proton motive force is harnessed by TonB in complex with ExbB and ExbD and then transduced to FhuA by TonB. To better understand the transport mechanism, whereby E. coli acquires iron, molecular details of siderophore import need to be established. The structure of FhuA is characterized by a C-terminal transMembrane barrel domain composed of 22 antiparallel β-strands connected by long loops on the extracellular surface and short turns on the periplasmic side (Ferguson et al. 1998b; Locher et al. 1998). The interior of the barrel is occluded by an N-terminal globular cork domain. Residues 24–29 of FhuA adopt a helical conformation, referred to as the switch helix. N-terminal to the switch helix of FhuA is the Ton box (residues 7–13), a hepta-peptide motif DTITVTA that displays sequence conservation among OM Receptors. Similar structural features were identified in other E. coli TonB-dependent OM Receptors: FepA (Buchanan et al. 1999), Receptor for ferric enterobactin; FecA (Ferguson et al. 2002; Yue et al. 2003), Receptor for diferric dicitrate; BtuB (Chimento et al. 2003), Receptor for cyanocobalamin; Cir (Buchanan et al. 2007), Receptor for colicin Ia; as well as Pseudomonas aeruginosa TonB-dependent OM Receptors FptA (Cobessi et al. 2005b), Receptor for pyochelin; and FpvA (Cobessi et al. 2005a), Receptor for pyoverdine. Structural studies revealed conformational changes at the Outer surface of OM Receptors. The crystal structure of FecA plus diferric dicitrate shows a change in position of loops 7 and 8 by 11 A and 15 A, respectively, compared with their positions in apo-FecA (Ferguson et al. 2002; Yue et al. 2003). In the siderophore-bound conformation, the loops become oriented toward the barrel lumen. Their change in position may prevent siderophore escape into the environment at a later stage of the transport cycle when binding affinity for chelated iron must be reduced to promote its translocation. FepA cross-linking studies (Scott et al. 2002) document structural changes in loop 7 during transport of ferric enterobactin. Comparison of BtuB structures with and without bound cyanocobalamin and Ca2+ (Chimento et al. 2003) showed conformational changes in loop 19-20 and in an apical loop of the cork domain. In the cocrystal structure of cyanocobalamin-bound BtuB in complex with TonB (Shultis et al. 2006), a further shift in position of this apical loop was detected. Cocrystal structures of FhuA–TonB (Pawelek et al. 2006) and BtuB–TonB (Shultis et al. 2006) showed structural changes on the periplasmic side of the OM Receptors, where formation of an interstrand β-sheet between the Ton box of the Receptors and β3 of TonB was observed. Shared architecture of these TonB-dependent OM Receptors and their functional similarities suggest that they employ similar mechanisms for translocation of their cognate substrates. Examining crystal structures of apo- and ferrichrome-bound FhuA (Ferguson et al. 1998b; Locher et al. 1998) and our FhuA–TonB cocrystal structure (Pawelek et al. 2006) did not reveal changes in position of Outer surface-exposed loops. However, studies of whole cells indicated conformational changes in residues on loop 4 of FhuA upon binding of ferrichrome (Bos et al. 1998). Given this outcome and the structural changes in Outer surface-exposed loops observed in other TonB-dependent transporters, we propose that additional changes may occur at the Outer surface of FhuA that have not been revealed by previously adopted methods. In the present study, we report strategies to probe conformational changes in FhuA. By phage display, we mapped monoclonal antibody (mAb) epitopes to specific residues on FhuA. Using surface plasmon resonance (SPR), we measured binding of mAbs to residues on different Outer surface-exposed loops of FhuA in the absence and presence of both ferricrocin and TonB. By use of fluorescence spectroscopy, we measured quenching at residue 336 in the Outer surface-exposed loop 4 of FhuA upon ferricrocin binding and upon TonB binding. Our findings provide evidence that TonB induces conformational changes in Outer surface-exposed loops of FhuA.
-
structure of tonb in complex with fhua e coli Outer Membrane Receptor
Science, 2006Co-Authors: Peter D Pawelek, Christopher Ngthowhing, Natalia Moiseeva, Marc Allaire, Nathalie Croteau, Cezar M. Khursigara, James W CoultonAbstract:The cytoplasmic Membrane protein TonB spans the periplasm of the Gram-negative bacterial cell envelope, contacts cognate Outer Membrane Receptors, and facilitates siderophore transport. The Outer Membrane Receptor FhuA from Escherichia coli mediates TonB-dependent import of ferrichrome. We report the 3.3 angstrom resolution crystal structure of the TonB carboxyl-terminal domain in complex with FhuA. TonB contacts stabilize FhuA's amino-terminal residues, including those of the consensus Ton box sequence that form an interprotein β sheet with TonB through strand exchange. The highly conserved TonB residue arginine-166 is oriented to form multiple contacts with the FhuA cork, the globular domain enclosed by the β barrel.
-
Structure of TonB in complex with FhuA, E. coli Outer Membrane Receptor.
Science (New York N.Y.), 2006Co-Authors: Peter D Pawelek, Natalia Moiseeva, Marc Allaire, Nathalie Croteau, Cezar M. Khursigara, Christopher Ng-thow-hing, James W CoultonAbstract:The cytoplasmic Membrane protein TonB spans the periplasm of the Gram-negative bacterial cell envelope, contacts cognate Outer Membrane Receptors, and facilitates siderophore transport. The Outer Membrane Receptor FhuA from Escherichia coli mediates TonB-dependent import of ferrichrome. We report the 3.3 angstrom resolution crystal structure of the TonB carboxyl-terminal domain in complex with FhuA. TonB contacts stabilize FhuA's amino-terminal residues, including those of the consensus Ton box sequence that form an interprotein beta sheet with TonB through strand exchange. The highly conserved TonB residue arginine-166 is oriented to form multiple contacts with the FhuA cork, the globular domain enclosed by the beta barrel.
Franc Pattus - One of the best experts on this subject based on the ideXlab platform.
-
the metal dependence of pyoverdine interactions with its Outer Membrane Receptor fpva
Journal of Bacteriology, 2008Co-Authors: Jason Greenwald, Hervé Celia, Gabrielle Zederlutz, Agnes Hagege, Franc PattusAbstract:To acquire iron, Pseudomonas aeruginosa secretes the fluorescent siderophore pyoverdine (Pvd), which chelates iron and shuttles it into the cells via the specific Outer Membrane transporter FpvA. We studied the role of iron and other metals in the binding and transport of Pvd by FpvA and conclude that there is no significant affinity between FpvA and metal-free Pvd. We found that the fluorescent in vivo complex of iron-free FpvA-Pvd is in fact a complex with aluminum (FpvA-Pvd-Al) formed from trace aluminum in the growth medium. When Pseudomonas aeruginosa was cultured in a medium that had been treated with a metal affinity resin, the in vivo formation of the FpvA-Pvd complex and the recycling of Pvd on FpvA were nearly abolished. The accumulation of Pvd in the periplasm of Pseudomonas aeruginosa was also reduced in the treated growth medium, while the addition of 1 μM AlCl3 to the treated medium restored the effects of trace metals observed in standard growth medium. Using fluorescent resonance energy transfer and surface plasmon resonance techniques, the in vitro interactions between Pvd and detergent-solubilized FpvA were also shown to be metal dependent. We demonstrated that FpvA binds Pvd-Fe but not Pvd and that Pvd did not compete with Pvd-Fe for FpvA binding. In light of our finding that the Pvd-Al complex is transported across the Outer Membrane of Pseudomonas aeruginosa, a model for siderophore recognition based on a metal-induced conformation followed by redox selectivity for iron is discussed.
-
a β strand lock exchange for signal transduction in tonb dependent transducers on the basis of a common structural motif
Structure, 2007Co-Authors: Karl Brillet, Franc Pattus, Hervé Celia, Laure Journet, Laetitia Paulus, Aude Stahl, David CobessiAbstract:Summary Transport of molecules larger than 600 Da across the Outer Membrane involves TonB-dependent Receptors and TonB-ExbB-ExbD of the inner Membrane. The transport is energy consuming, and involves direct interactions between a short N-terminal sequence of Receptor, called the TonB box, and TonB. We solved the structure of the ferric pyoverdine (Pvd-Fe) Outer Membrane Receptor FpvA from Pseudomonas aeruginosa in its apo form. Structure analyses show that residues of the TonB box are in a β strand which interacts through a mixed four-stranded β sheet with the periplasmic signaling domain involved in interactions with an inner Membrane sigma regulator. In this conformation, the TonB box cannot form a four-stranded β sheet with TonB. The FhuA-TonB or BtuB-TonB structures show that the TonB-FpvA interactions require a conformational change which involves a β strand lock-exchange mechanism. This mechanism is compatible with movements of the periplasmic domain deduced from crystallographic analyses of FpvA, FpvA-Pvd, and FpvA-Pvd-Fe.
-
From the periplasmic signaling domain to the extracellular face of an Outer Membrane signal transducer of Pseudomonas aeruginosa: crystal structure of the ferric pyoverdine Outer Membrane Receptor.
Journal of Molecular Biology, 2007Co-Authors: Christophe Wirth, Franc Pattus, Wolfram Meyer-klaucke, David CobessiAbstract:The pyoverdine Outer Membrane Receptor, FpvA, from Pseudomonas aeruginosa translocates ferric pyoverdine across the Outer Membrane through an energy consuming mechanism using the proton motive force and the TonB-ExbB-ExbD energy transducing complex from the inner Membrane. We solved the crystal structure of the full-length FpvA bound to iron-pyoverdine at 2.7 A resolution. Signal transduction to an anti-sigma protein of the inner Membrane and to TonB-ExbB-ExbD involves the periplasmic domain, which displays a beta-alpha-beta fold composed of two alpha-helices sandwiched by two beta-sheets. One iron-pyoverdine conformer is bound at the extracellular face of FpvA, revealing the conformer selectivity of the binding site. The loop that contains the TonB box, involved in interactions with TonB, and connects the signaling domain to the plug domain of FpvA is not defined in the electron density following the binding of ferric pyoverdine. The high flexibility of this loop is probably necessary for signal transduction through the Outer Membrane.
-
Interaction of TonB with the Outer Membrane Receptor FpvA of Pseudomonas aeruginosa.
Journal of bacteriology, 2006Co-Authors: Hendrik Adams, Franc Pattus, Isabelle J. Schalk, Gabrielle Zeder-lutz, Hervé CeliaAbstract:Pseudomonas aeruginosa is a ubiquitous environmental gram-negative bacterium that has become one of the most prominent causative agents of opportunistic human infections (52). As it does for nearly all microorganisms, elemental iron plays an indispensable role in the growth and colonization of P. aeruginosa. In animal hosts, however, iron is usually bound to proteins such as transferrin and lactoferrin (53), and P. aeruginosa is therefore challenged with an environment that maintains a very small reservoir of free soluble iron. To overcome this problem, under these iron-limited conditions P. aeruginosa produces high-affinity iron chelators called siderophores (5). The major siderophore produced by P. aeruginosa is pyoverdine (Pvd), which sequesters iron from external sources and is concentrated as ferric Pvd on the Outer Membrane by the high-affinity transporter FpvA (38). Like all Outer Membrane siderophore Receptors (51), this transporter is composed of two domains, a C-terminal β-barrel made of 22 β-strands and an N-terminal, globular cork or plug domain residing inside the barrel. FpvA belongs to a subclass of iron transporters which has an additional N-terminal domain that is involved in the regulation of the transcription of the fpvA operon (29, 47). In vivo, the normal state of FpvA under iron limitation is the FpvA-Pvd complex (45, 46). The dissociation of Pvd from FpvA, before binding of extracellular Pvd-Fe and the subsequent iron transport into the periplasm, is dependent on the inner Membrane protein TonB and the proton motive force (PMF) (6, 45, 50). In the P. aeruginosa genome (49), three tonB genes have been identified, i.e., tonB1, tonB2, and tonB3. Disruption of tonB1 inhibits siderophore-mediated iron uptake and heme uptake (39, 54). Inactivation of tonB2 has no adverse effect on iron or heme acquisition, but tonB1-tonB2 double mutants are more compromised with respect to growth in iron-restricted medium than is a single tonB1 knockout mutant (55). Inactivation of tonB3 appears to result in a defective twitching motility (19), and the gene product is most likely not involved in iron uptake. Most knowledge about TonB function and structure has been obtained primarily from studies with Escherichia coli TonB (reviewed in reference 40). This protein comprises three domains: a hydrophobic amino-terminal helix that anchors the protein in the cytoplasmic Membrane, followed by a proline-rich region and a carboxyl-terminal globular domain that protrudes in the periplasm. Although P. aeruginosa TonB displays a high homology with E. coli TonB, the protein is distinguished by an N-terminal extension, which is important for the TonB activity in P. aeruginosa (54). Furthermore, full TonB function is dependent on the ExbB and ExbD proteins, which are located in the cytoplasmic Membrane (1, 14). The combined topologies of ExbB and ExbD mimic those of a signal transducer, with ExbD extending into the periplasmic space from its single transMembrane domain (21) and ExbB consisting of three transMembrane domains and a significant cytoplasmic domain (21, 23). Although P. aeruginosa homologues of ExbB and ExbD have been found, inactivation of these genes did not adversely affect the growth under iron limitation (55). Structural determinations of carboxyl-terminal domains of different lengths of TonB have been performed, either by X-ray diffraction (4, 27, 28) or by spectroscopy (37). These different models show that the protein forms structurally different dimers but may also exist as a monomer. Further work suggests that FhuA, the E. coli iron-ferrichrome transporter, is able to bind two TonB proteins in vitro and that the TonB protein dimerizes in vivo (15, 24, 25, 44). Whereas TonB is involved in the iron uptake by siderophore Receptors, the way it exerts its mode of action for the exchange of siderophore on FpvA and the internalization of ferric siderophore is still unclear. Evidence for a direct physical interaction between TonB and TonB-dependent transporters was gathered from a range of in vivo and in vitro experiments (3, 31, 34, 35, 48). Located at the N termini of all TonB-dependent Outer Membrane transporters is the TonB box, a short stretch of amino acids shared by all the transporters (2, 20, 40). The nature of the change in the TonB box conformation after substrate binding is not well defined. The TonB box is disordered in the structures of FhuA and FecA (ferrichrome and ferric-dicitrate transporters, respectively, in E. coli), but a short helix to which the TonB box is attached is unfolded in the ferric siderophore-bound structures and moves across the periplasm (10, 33). However, unfolding of a switch helix is not a general feature, because it is absent in FepA and BtuB, the ferric enterobactin and vitamin B12 transporters, respectively, in E. coli (11). Consistently, removal of the switch helix in FhuA resulted in a decrease in iron transport but still conferred colicin M and phage sensitivity (9). To date, two models for the activation of the Outer Membrane Receptor by the TonB machinery have been postulated. In the propeller model, a TonB dimer undergoes rotary motion, similar to the mechanism described for the bacterial flagellar motor that is powered by MotA and MotB, which are homologous to ExbB and ExbD (41). In such a system, the energy for iron transport is delivered by the torque of the TonB-ExbB-ExbD system. Alternatively, energized TonB shuttles from the inner to the Outer Membrane, thereby releasing its energy to the iron transporter (30, 41). The shuttle model is supported by in vivo labeling experiments that demonstrate periplasmic accessibility of the extreme N terminus of TonB to a specific Cys marker. In our laboratory we investigate the iron uptake mechanism of FpvA by in vivo and in vitro experiments (16, 45), and we recently solved the X-ray structure of FpvA (7). To gain more insight into the iron uptake mechanism in P. aeruginosa, we focused on the tonB1 gene product and its putative interaction with FpvA. In this study, we sought biochemical evidence for this interaction.
-
the crystal structure of the pyoverdine Outer Membrane Receptor fpva from pseudomonas aeruginosa at 3 6 a resolution
Journal of Molecular Biology, 2005Co-Authors: David Cobessi, Hervé Celia, Isabelle J. Schalk, Nicolas Folschweiller, Mohamed A Abdallah, Franc PattusAbstract:The pyoverdine Outer Membrane Receptor FpvA from Pseudomonas aeruginosa translocates ferric-pyoverdine across the Outer Membrane via an energy consuming mechanism that involves the inner Membrane energy transducing complex of TonB–ExbB–ExbD and the proton motive force. We solved the crystal structure of FpvA loaded with iron-free pyoverdine at 3.6 A resolution. The pyoverdine Receptor is folded in two domains: a transMembrane 22-stranded β-barrel domain occluded by an N-terminal domain containing a mixed four-stranded β-sheet (the plug). The β-strands of the barrel are connected by long extracellular loops and short periplasmic turns. The iron-free pyoverdine is bound at the surface of the Receptor in a pocket lined with aromatic residues while the extracellular loops do not completely cover the pyoverdine binding site. The TonB box, which is involved in intermolecular contacts with the TonB protein of the inner Membrane, is observed in an extended conformation. Comparison of this first reported structure of an iron-siderophore transporter from a bacterium other than Escherichia coli with the known structures of the E. coli TonB-dependent transporters reveals a high structural homology and suggests that a common sensing mechanism exists for the iron-loading status in all bacterial iron siderophore transporters.
Angela Wilks - One of the best experts on this subject based on the ideXlab platform.
-
contributions of the heme coordinating ligands of the pseudomonas aeruginosa Outer Membrane Receptor hasr to extracellular heme sensing and transport
Journal of Biological Chemistry, 2020Co-Authors: Alecia T Dent, Angela WilksAbstract:Pseudomonas aeruginosa exhibits a high requirement for iron, which it can acquire via several mechanisms, including the acquisition and utilization of heme. The P. aeruginosa genome encodes two heme uptake systems, the heme assimilation system (Has) and the Pseudomonas heme utilization (Phu) system. Extracellular heme is sensed via the Has system, which encodes an extracytoplasmic function (ECF) σ factor system. Previous studies have shown that the transfer of heme from the extracellular hemophore HasAp to the Outer Membrane Receptor HasR is required for activation of the σ factor HasI and upregulation of has operon expression. Here, employing site-directed mutagenesis, allelic exchange, quantitative PCR analyses, immunoblotting, and 13C-heme uptake experiments, we delineated the differential contributions of the extracellular FRAP/PNPNL loop residue His-624 in HasR and of His-221 in its N-terminal plug domain required for heme capture to heme transport and signaling, respectively. Specifically, we show that substitution of the N-terminal plug His-221 disrupts both signaling and transport, leading to dysregulation of both the Has and Phu uptake systems. Our results are consistent with a model wherein heme release from HasAp to the N-terminal plug of HasR is required to initiate signaling, whereas His-624 is required for simultaneously closing off the heme transport channel from the extracellular medium and triggering heme transport. Our results provide critical insight into heme release, signaling, and transport in P. aeruginosa and suggest a functional link between the ECF σ factor and Phu heme uptake system.
-
contributions of the heme coordinating ligands of the pseudomonas aeruginosa Outer Membrane Receptor hasr to extracellular heme sensing and transport
bioRxiv, 2020Co-Authors: Alecia T Dent, Angela WilksAbstract:ABSTRACT Pseudomonas aeruginosa exhibits a high requirement for iron which it can acquire via several mechanisms including the acquisition and utilization of heme. P. aeruginosa encodes two heme uptake systems, the heme assimilation system (Has) and the Pseudomonasheme utilization (Phu) system. Extracellular heme is sensed via the Has system that encodes an extra cytoplasmic function (ECF) σ factor system. Previous studies have shown release of heme from the extracellular hemophore HasAp to the Outer Membrane Receptor HasR is required for activation of the σ factor HasI. Herein, employing site-directed mutagenesis, allelic exchange, quantitative PCR analyses, immunoblotting and 13C-heme uptake studies, we characterize the differential contributions of the Outer Membrane Receptor HasR extracellular FRAP/PNPNL loop residue His-624 and the N-terminal plug residue His-221 to heme transport and signaling, respectively. Specifically, we show mutation of the N-terminal plug His-221 disrupts both signaling and transport. The data is consistent with a model where heme release from HasAp to the N-terminal plug of HasR is required to initiate signaling, whereas His624 is required for simultaneously closing off the heme transport channel from the extracellular medium and triggering heme transport. Furthermore, mutation of His-221 leads to dysregulation of both the Has and Phu uptake systems suggesting a possible functional link that is coordinated through the ECF σ factor system.
-
characterization of the Outer Membrane Receptor shua from the heme uptake system of shigella dysenteriae substrate specificity and identification of the heme protein ligands
Journal of Biological Chemistry, 2007Co-Authors: Kimberly Burkhard, Angela WilksAbstract:Abstract Shigella dysenteriae, like many bacterial pathogens, has evolved Outer Membrane Receptor-mediated pathways for the uptake and utilization of heme as an iron source. As a first step toward understanding the mechanism of heme uptake we have undertaken a site-directed mutagenesis, spectroscopic, and kinetic analysis of the Outer Membrane Receptor ShuA of S. dysenteriae. Purification of the Outer Membrane Receptor gave a single band of molecular mass 73 kDa on SDS-PAGE. Initial spectroscopic analysis of the protein in either detergent micelles or lipid bicelles revealed residual heme bound to the Receptor, with a Soret maximum at 413 nm. Titration of the protein with exogenous heme gave a Soret peak at 437 nm in detergent micelles, and 402 nm in lipid bicelles. However, transfer of heme from hemoglobin yields a Soret maximum at 413 nm identical to that of the isolated protein. Further spectroscopic and kinetic analysis revealed that hemoglobin in the oxidized state is the most likely physiological substrate for ShuA. In addition, mutation of the conserved histidines, H86A or H420A, resulted in a loss of the ability of the Receptor to efficiently extract heme from hemoglobin. In contrast the double mutant H86A/H420A was unable to extract heme from hemoglobin. These findings taken together confirm that both His-86 and His-420 are essential for substrate recognition, heme coordination, and transfer. Furthermore, the full-length TonB was shown to form a 1:1 complex with either apo-ShuA H86A/H420A or the wild-type ShuA. These observations provide a basis for future studies on the coordination and transport of heme by the TonB-dependent Outer Membrane Receptors.
-
characterization of the periplasmic heme binding protein shut from the heme uptake system of shigella dysenteriae
Biochemistry, 2005Co-Authors: Suntara Eakanunkul, Gudrun S Lukatrodgers, Suganya Sumithran, Arundhati Ghosh, Kenton R Rodgers, John H Dawson, Angela WilksAbstract:The heme uptake systems by which bacterial pathogens acquire and utilize heme have recently been described. Such systems may utilize heme directly from the host's hemeproteins or via a hemophore that sequesters and transports heme to an Outer Membrane Receptor and subsequently to the translocating proteins by which heme is further transported into the cell. However, little is known of the heme binding and release mechanisms that facilitate the uptake of heme into the pathogenic organism. As a first step toward elucidating the molecular level events that drive heme binding and release, we have undertaken a spectroscopic and mutational study of the first purified periplasmic heme-binding protein (PBP), ShuT from Shigella dysenteriae. On the basis of sequence identity, the ShuT protein is most closely related to the class of PBPs typified by the vitamin B12 (BtuF) and iron-hydroxamate (FhuD) PBPs and is a monomeric protein having a molecular mass of 28.5 kDa following proteolytic processing of the periplasmi...
Peter D Pawelek - One of the best experts on this subject based on the ideXlab platform.
-
structure of tonb in complex with fhua e coli Outer Membrane Receptor
Science, 2006Co-Authors: Peter D Pawelek, Christopher Ngthowhing, Natalia Moiseeva, Marc Allaire, Nathalie Croteau, Cezar M. Khursigara, James W CoultonAbstract:The cytoplasmic Membrane protein TonB spans the periplasm of the Gram-negative bacterial cell envelope, contacts cognate Outer Membrane Receptors, and facilitates siderophore transport. The Outer Membrane Receptor FhuA from Escherichia coli mediates TonB-dependent import of ferrichrome. We report the 3.3 angstrom resolution crystal structure of the TonB carboxyl-terminal domain in complex with FhuA. TonB contacts stabilize FhuA's amino-terminal residues, including those of the consensus Ton box sequence that form an interprotein β sheet with TonB through strand exchange. The highly conserved TonB residue arginine-166 is oriented to form multiple contacts with the FhuA cork, the globular domain enclosed by the β barrel.
-
Structure of TonB in complex with FhuA, E. coli Outer Membrane Receptor.
Science (New York N.Y.), 2006Co-Authors: Peter D Pawelek, Natalia Moiseeva, Marc Allaire, Nathalie Croteau, Cezar M. Khursigara, Christopher Ng-thow-hing, James W CoultonAbstract:The cytoplasmic Membrane protein TonB spans the periplasm of the Gram-negative bacterial cell envelope, contacts cognate Outer Membrane Receptors, and facilitates siderophore transport. The Outer Membrane Receptor FhuA from Escherichia coli mediates TonB-dependent import of ferrichrome. We report the 3.3 angstrom resolution crystal structure of the TonB carboxyl-terminal domain in complex with FhuA. TonB contacts stabilize FhuA's amino-terminal residues, including those of the consensus Ton box sequence that form an interprotein beta sheet with TonB through strand exchange. The highly conserved TonB residue arginine-166 is oriented to form multiple contacts with the FhuA cork, the globular domain enclosed by the beta barrel.
-
phage display reveals multiple contact sites between fhua an Outer Membrane Receptor of escherichia coli and tonb
Journal of Molecular Biology, 2006Co-Authors: David M Carter, Peter D Pawelek, Jeannicolas Gagnon, Moussab Damlaj, Suneeta Mandava, Lee Makowski, Diane J Rodi, James W CoultonAbstract:The ferric hydroxamate uptake Receptor FhuA from Escherichia coli transports siderophores across the Outer Membrane (OM). TonB-ExbB-ExbD transduces energy from the cytoplasmic Membrane to the OM by contacts between TonB and OM Receptors that contain the Ton box, a consensus sequence near the N terminus. Although the Ton box is a region of known contact between OM Receptors and TonB, our biophysical studies established that TonB binds to FhuA through multiple regions of interaction. Panning of phage-displayed random peptide libraries (Ph.D.-12, Ph.D.-C7C) against TonB identified peptide sequences that specifically interact with TonB. Analyses of these sequences using the Receptor Ligand Contacts (RELIC) suite of programs revealed clusters of multiply aligned peptides that mapped to FhuA. These clusters localized to a continuous periplasm-accessible surface: Ton box/switch helix; cork domain/beta1 strand; and periplasmic turn 8. Guided by such matches, synthetic oligonucleotides corresponding to DNA sequences identical to fhuA were fused to malE; peptides corresponding to the above regions were displayed at the N terminus of E.coli maltose-binding protein (MBP). Purified FhuA peptides fused to MBP bound specifically to TonB by ELISA. Furthermore, they competed with ligand-loaded FhuA for binding to TonB. RELIC also identified clusters of multiply aligned peptides corresponding to the Ton box regions in BtuB, FepA, and FecA; to periplasmic turn 8 in BtuB and FecA; and to periplasmic turns 1 and 2 in FepA. These experimental outcomes identify specific molecular contacts made between TonB and OM Receptors that extend beyond the well-characterized Ton box.
-
siderophore transport through escherichia coli Outer Membrane Receptor fhua with disulfide tethered cork and barrel domains
Journal of Biological Chemistry, 2005Co-Authors: Anne H Eisenhauer, Peter D Pawelek, Sofia Shames, James W CoultonAbstract:ThehydroxamatesiderophoreReceptorFhuAisaTonBdependent Outer Membrane protein of Escherichia coli composed of a C-terminal 22-stranded -barrel occluded by an N-terminal globular cork domain. During siderophore transport into the periplasm, the FhuA cork domain has been proposed to undergo conformational changes that allow transport through the barrel lumen; alternatively, the cork may be completely displaced from the barrel. To probe such changes, site-directed cysteine mutants in the cork domain (L109C and Q112C) and in the barrel domain (S356C and M383C) were created within the putative siderophore transport pathway. Molecular modeling predicted that the double cysteine mutants L109C/S356C and Q112C/M383C would form disulfide bonds, thereby tethering the cork and barrel domains. The double cysteine FhuA mutants were denatured under nonreducing conditions and fluorescently labeled with thiol-specific Oregon Green maleimide. Subsequent SDS-PAGE analysis revealed two distinct species: FhuA containing a disulfide bond and FhuA with free sulfhydryl groups. To address the role of the putative siderophore transport pathway and to evaluate possible rearrangements of the cork domain during ferricrocin transport, disulfide bond formation was enhanced by an oxidative catalyst. Cells containing double cysteine FhuA mutants that were subjected to oxidation during ferricrocin transport exhibited disulfide bond formation to near completion. After disulfide tethering of the cork to the barrel, ferricrocin transport was equivalent to transport by untreated cells. These results demonstrate that blocking the putative siderophore transport pathway does not abrogate ferricrocin uptake. We propose that, during siderophore transport through FhuA, the cork domain remains within the barrel rather than being displaced.
-
deletion of the proline rich region of tonb disrupts formation of a 2 1 complex with fhua an Outer Membrane Receptor of escherichia coli
Protein Science, 2005Co-Authors: Cezar M. Khursigara, Peter D Pawelek, Gregory De Crescenzo, James W CoultonAbstract:TonB protein of Escherichia coli couples the electrochemical potential of the cytoplasmic Membrane (CM) to active transport of iron-siderophores and vitamin B12 across the Outer Membrane (OM). TonB interacts with OM Receptors and transduces conformationally stored energy. Energy for transport is provided by the proton motive force through ExbB and ExbD, which form a ternary complex with TonB in the CM. TonB contains three distinct domains: an N-terminal signal/anchor sequence, a C-terminal domain, and a proline-rich region. The proline-rich region was proposed to extend TonB’s structure across the periplasm, allowing it to contact spatially distant OM Receptors. Having previously identified a 2:1 stoichiometry for the complex of full-length (FL) TonB and the OM Receptor FhuA, we now demonstrate that deletion of the proline-rich region of TonB (TonBΔ66-100) prevents formation of the 2:1 complex. Sedimentation velocity analytical ultracentrifugation of TonBΔ66-100 with FhuA revealed that a 1:1 TonB–FhuA complex is formed. Interactions between TonBΔ66-100 and FhuA were assessed by surface plasmon resonance, and their affinities were determined to be similar to those of TonB (FL)–FhuA. Presence of the FhuA-specific siderophore ferricrocin altered neither stoichiometry nor affinity of interaction, leading to our conclusion that the proline-rich region in TonB is important in forming a 2:1 high-affinity TonB–FhuA complex in vitro. Furthermore, TonBΔ66-100–FhuAΔ21-128 interactions demonstrated that the cork region of the OM Receptor was also important in forming a complex. Together, these results demonstrate a novel function of the proline-rich region of TonB in mediating TonB–TonB interactions within the TonB–FhuA complex.
David Cobessi - One of the best experts on this subject based on the ideXlab platform.
-
structure of the heme hemoglobin Outer Membrane Receptor shua from shigella dysenteriae heme binding by an induced fit mechanism
Proteins, 2010Co-Authors: David Cobessi, Ahmed Meksem, Karl BrilletAbstract:Shigella dysentriae and other Gram-negative human pathogens are able to use iron from heme bound to hemoglobin for growing. We solved at 2.6 A resolution the 3D structure of the TonB-dependent heme/hemoglobin Outer Membrane Receptor ShuA from S. dysenteriae. ShuA binds to hemoglobin and transports heme across the Outer Membrane. The structure consists of a C-terminal domain that folds into a 22-stranded transMembrane beta-barrel, which is filled by the N-terminal plug domain. One distal histidine ligand of heme is located at the apex of the plug, exposed to the solvent. His86 is situated 9.86 A apart from His420, the second histidine involved in the heme binding. His420 is in the extracellular loop L7. The heme coordination by His86 and His420 involves conformational changes. The comparisons with the hemophore Receptor HasR of Serratia marcescens bound to HasA-Heme suggest an extracellular induced fit mechanism for the heme binding. The loop L7 contains hydrophobic residues which could interact with the hydrophobic porphyring ring of heme. The energy required for the transport by ShuA is derived from the proton motive force after interactions between the periplasmic N-terminal TonB-box of ShuA and the inner Membrane protein, TonB. In ShuA, the TonB-box is buried and cannot interact with TonB. The structural comparisons with HasR suggest its conformational change upon the heme binding for interacting with TonB. The signaling of the heme binding could involve a hydrogen bond network going from His86 to the TonB-box.
-
a β strand lock exchange for signal transduction in tonb dependent transducers on the basis of a common structural motif
Structure, 2007Co-Authors: Karl Brillet, Franc Pattus, Hervé Celia, Laure Journet, Laetitia Paulus, Aude Stahl, David CobessiAbstract:Summary Transport of molecules larger than 600 Da across the Outer Membrane involves TonB-dependent Receptors and TonB-ExbB-ExbD of the inner Membrane. The transport is energy consuming, and involves direct interactions between a short N-terminal sequence of Receptor, called the TonB box, and TonB. We solved the structure of the ferric pyoverdine (Pvd-Fe) Outer Membrane Receptor FpvA from Pseudomonas aeruginosa in its apo form. Structure analyses show that residues of the TonB box are in a β strand which interacts through a mixed four-stranded β sheet with the periplasmic signaling domain involved in interactions with an inner Membrane sigma regulator. In this conformation, the TonB box cannot form a four-stranded β sheet with TonB. The FhuA-TonB or BtuB-TonB structures show that the TonB-FpvA interactions require a conformational change which involves a β strand lock-exchange mechanism. This mechanism is compatible with movements of the periplasmic domain deduced from crystallographic analyses of FpvA, FpvA-Pvd, and FpvA-Pvd-Fe.
-
From the periplasmic signaling domain to the extracellular face of an Outer Membrane signal transducer of Pseudomonas aeruginosa: crystal structure of the ferric pyoverdine Outer Membrane Receptor.
Journal of Molecular Biology, 2007Co-Authors: Christophe Wirth, Franc Pattus, Wolfram Meyer-klaucke, David CobessiAbstract:The pyoverdine Outer Membrane Receptor, FpvA, from Pseudomonas aeruginosa translocates ferric pyoverdine across the Outer Membrane through an energy consuming mechanism using the proton motive force and the TonB-ExbB-ExbD energy transducing complex from the inner Membrane. We solved the crystal structure of the full-length FpvA bound to iron-pyoverdine at 2.7 A resolution. Signal transduction to an anti-sigma protein of the inner Membrane and to TonB-ExbB-ExbD involves the periplasmic domain, which displays a beta-alpha-beta fold composed of two alpha-helices sandwiched by two beta-sheets. One iron-pyoverdine conformer is bound at the extracellular face of FpvA, revealing the conformer selectivity of the binding site. The loop that contains the TonB box, involved in interactions with TonB, and connects the signaling domain to the plug domain of FpvA is not defined in the electron density following the binding of ferric pyoverdine. The high flexibility of this loop is probably necessary for signal transduction through the Outer Membrane.
-
the crystal structure of the pyoverdine Outer Membrane Receptor fpva from pseudomonas aeruginosa at 3 6 a resolution
Journal of Molecular Biology, 2005Co-Authors: David Cobessi, Hervé Celia, Isabelle J. Schalk, Nicolas Folschweiller, Mohamed A Abdallah, Franc PattusAbstract:The pyoverdine Outer Membrane Receptor FpvA from Pseudomonas aeruginosa translocates ferric-pyoverdine across the Outer Membrane via an energy consuming mechanism that involves the inner Membrane energy transducing complex of TonB–ExbB–ExbD and the proton motive force. We solved the crystal structure of FpvA loaded with iron-free pyoverdine at 3.6 A resolution. The pyoverdine Receptor is folded in two domains: a transMembrane 22-stranded β-barrel domain occluded by an N-terminal domain containing a mixed four-stranded β-sheet (the plug). The β-strands of the barrel are connected by long extracellular loops and short periplasmic turns. The iron-free pyoverdine is bound at the surface of the Receptor in a pocket lined with aromatic residues while the extracellular loops do not completely cover the pyoverdine binding site. The TonB box, which is involved in intermolecular contacts with the TonB protein of the inner Membrane, is observed in an extended conformation. Comparison of this first reported structure of an iron-siderophore transporter from a bacterium other than Escherichia coli with the known structures of the E. coli TonB-dependent transporters reveals a high structural homology and suggests that a common sensing mechanism exists for the iron-loading status in all bacterial iron siderophore transporters.
-
The crystal structure of the pyoverdine Outer Membrane Receptor FpvA from Pseudomonas aeruginosa at 3.6 angstroms resolution
Journal of Molecular Biology, 2005Co-Authors: David Cobessi, Hervé Celia, Isabelle J. Schalk, Nicolas Folschweiller, M. A. Abdallah, Franc PattusAbstract:The pyoverdine Outer Membrane Receptor FpvA from Pseudomonas aeruginosa translocates ferric-pyoverdine across the Outer Membrane via an energy consuming mechanism that involves the inner Membrane energy transducing complex of TonB-ExbB-ExbD and the proton motive force. We solved the crystal structure of FpvA loaded with iron-free pyoverdine at 3.6 angstroms resolution. The pyoverdine Receptor is folded in two domains: a transMembrane 22-stranded beta-barrel domain occluded by an N-terminal domain containing a mixed four-stranded beta-sheet (the plug). The beta-strands of the barrel are connected by long extracellular loops and short periplasmic turns. The iron-free pyoverdine is bound at the surface of the Receptor in a pocket lined with aromatic residues while the extracellular loops do not completely cover the pyoverdine binding site. The TonB box, which is involved in intermolecular contacts with the TonB protein of the inner Membrane, is observed in an extended conformation. Comparison of this first reported structure of an iron-siderophore transporter from a bacterium other than Escherichia coli with the known structures of the E.coli TonB-dependent transporters reveals a high structural homology and suggests that a common sensing mechanism exists for the iron-loading status in all bacterial iron siderophore transporters.
