Inactivation in vitro of the Escherichia coli outer membrane protein FhuA by a phage T5-encoded lipoprotein

Bacteriophage T5-encoded lipoprotein, synthesized by infected Escherichia coli cells, prevents superinfection of the host cell by this virus. The molecular basis of its ability to inactivate the receptor of phage T5, the FhuA protein, was investigated in vitro. Fully competent T5 lipoprotein, with a His tag attached to the C-terminus, was purified in detergent solution. Co-reconstitution with homogeneous FhuA protein into liposomes revealed that the lipoprotein inhibited the irreversible inactivation of phage T5 by FhuA protein. This phenomenon correlated with the inhibition of phage DNA ejection determined by fluorescence monitoring. Addition of detergent abolished the interaction between T5 lipoprotein and FhuA protein. When the signal sequence and N-terminal cysteinyl residue of the lipoprotein were removed by genetic truncation, the soluble polypeptide could be refolded and purified from inclusion bodies. The truncated lipoprotein interfered with infection of E. coli by phage T5, but only at very high concentrations. Circular dichroism spectra of both forms of T5 lipoprotein exhibited predominantly β-structure. T5 lipoprotein is sufficient for inactivation of the FhuA protein, presumably by inserting the N-terminal acyl chains into the membrane, thus increasing its local concentration. An in vitro stoichiometry of 10:1 has been calculated for the phage-encoded T5 lipoprotein to FhuA protein complex.     

Deletion of the proline-rich region of TonB disrupts formation of a 2:1 complex with FhuA, an outer membrane receptor of Escherichia coli

TonB protein of Escherichia coli couples the electrochemical potential of the cytoplasmic membrane (CM) to active transport of iron-siderophores and vitamin B(12) 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 (TonBDelta66-100) prevents formation of the 2:1 complex. Sedimentation velocity analytical ultracentrifugation of TonBDelta66-100 with FhuA revealed that a 1:1 TonB-FhuA complex is formed. Interactions between TonBDelta66-100 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, TonBDelta66-100-FhuADelta21-128 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.     

Crystal structure of the antibiotic albomycin in complex with the outer membrane transporter FhuA

One alternative method for drug delivery involves the use of siderophore-antibiotic conjugates. These compounds represent a specific means by which potent antimicrobial agents, covalently linked to iron-chelating siderophores, can be actively transported across the outer membrane of gram-negative bacteria. These "Trojan Horse" antibiotics may prove useful as an efficient means to combat multi-drug-resistant bacterial infections. Here we present the crystallographic structures of the natural siderophore-antibiotic conjugate albomycin and the siderophore phenylferricrocin, in complex with the active outer membrane transporter FhuA from Escherichia coli. To our knowledge, this represents the first structure of an antibiotic bound to its cognate transporter. Albomycins are broad-host range antibiotics that consist of a hydroxamate-type iron-chelating siderophore, and an antibiotically active, thioribosyl pyrimidine moiety. As observed with other hydroxamate-type siderophores, the three-dimensional structure of albomycin reveals an identical coordination geometry surrounding the ferric iron atom. Unexpectedly, this antibiotic assumes two conformational isomers in the binding site of FhuA, an extended and a compact form. The structural information derived from this study provides novel insights into the diverse array of antibiotic moieties that can be linked to the distal portion of iron-chelating siderophores and offers a structural platform for the rational design of hydroxamate-type siderophore-antibiotic conjugates.     

Characterization of outer membrane protein fractions of Bdellovibrionales

Bdellovibrio-and-like organisms (BALOs) are predatory bacteria that prey upon Gram-negative bacteria and are taxonomically subsumed in the order Bdellovibrionales. Despite their unique lifestyle, these bacteria show remarkable genotypic diversities. The outer membrane of the predators is likely to play an important role during the recognition and invasion stage, as well as in the intraperiplasmic growth phase. In this study, the outer membrane protein fractions of type strains of Bdellovibrio, Bacteriovorax and Peredibacter were investigated, revealing the presence of outer membrane proteins (Omps) similar to the major Omps of Bdellovibrio bacteriovorus. The primary structures of these Omps of Bdellovibrio sp. W, Bacteriovorax stolpii and Peredibacter starrii were elucidated by a combined mass spectrometric-reverse genetic approach. The similarity between the analyzed Omps of the investigated BALOs ranges from 32% to 89% showing conserved amino acid regions in their primary structure.     

Modulation by substrates of the interaction between the HasR outer membrane receptor and its specific TonB-like protein, HasB

TonB is a cytoplasmic membrane protein required for active transport of various essential substrates such as heme and iron siderophores through the outer membrane receptors of Gram-negative bacteria. This protein spans the periplasm, contacts outer membrane transporters by its C-terminal domain, and transduces energy from the protonmotive force to the transporters. The TonB box, a relatively conserved sequence localized on the periplasmic side of the transporters, has been shown to directly contact TonB.While Serratia marcescens TonB functions with various transporters, HasB, a TonB-like protein, is dedicated to the HasR transporter. HasR acquires heme either freely or via an extracellular heme carrier, the hemophore HasA, that binds to HasR and delivers heme to the transporter. Here, we study the interaction of HasR with a HasB C-terminal domain and compare it with that obtained with a TonB C-terminal fragment. Analysis of the thermodynamic parameters reveals that the interaction mode of HasR with HasB differs from that with TonB, the difference explaining the functional specificity of HasB for HasR. We also demonstrate that the presence of the substrate on the extracellular face of the transporter modifies, via enthalpy-entropy compensation, the interaction with HasB on the periplasmic face. The transmitted signal depends on the nature of the substrate. While the presence of heme on the transporter modifies only slightly the nature of interactions involved between HasR and HasB, hemophore binding on the transporter dramatically changes the interactions and seems to locally stabilize some structural motifs. In both cases, the HasR TonB box is the target for those modifications.     

Functional clues from the crystal structure of an orphan periplasmic ligand‐binding protein from Treponema pallidum

The spirochete Treponema pallidum is the causative agent of syphilis, a sexually transmitted infection of major global importance. Other closely related subspecies of Treponema also are the etiological agents of the endemic treponematoses, such as yaws, pinta, and bejel. The inability of T. pallidum and its close relatives to be cultured in vitro has prompted efforts to characterize T. pallidum's proteins structurally and biophysically, particularly those potentially relevant to treponemal membrane biology, with the goal of possibly revealing the functions of those proteins. This report describes the structure of the treponemal protein Tp0737; this polypeptide has a fold characteristic of a class of periplasmic ligand‐binding proteins associated with ABC‐type transporters. Although no ligand for the protein was observed in electron‐density maps, and thus the nature of the native ligand remains obscure, the structural data described herein provide a foundation for further efforts to elucidate the ligand and thus the function of this protein in T. pallidum.     

Biophysics of T5, IRA phages, Escherichia coli outer membrane protein FhuA and T5-FhuA interaction

In spite of the similarities in a structural organization of T5 and IRA phages their thermal and hydrodynamical peculiarities are completely different. One of the significant differences is observed in temperature value at which thermally induced DNA ejection starts. If in the case of physiological conditions this difference equals to 30°С, then it decreases as ionic strength of the solvent decreases. Also, from our experimental results follows that in the opening of phage tail channel for T5 phage (at pH7) significant role-play electrostatic forces. In spite of that both of these phages grow on the same Escherichia coli strain, we have shown that these phages need different receptors to penetrate into the bacterial cell precisely FhuA serves as receptor only for T5 phage. The higher FhuA concentration in T5 phage suspension is, the more intensive DNA ejection in environment is. The minimal FhuA/T5 ratio, which is 300/1, correspondingly, necessary for effective DNA ejection from the phage head was experimentally determined. For the first time the ejection of T5 phage DNA induced by FhuA was observed in an incessant regime. The deconvolution of calorimetric curve of FhuA’s denaturation has been shown that in a chosen condition there are four thermodynamically independent domains in the structure of FhuA.     

Overproduced and purified receptor binding protein pb5 of bacteriophage T5 binds to the T5 receptor protein FhuA

Abstract A promotor-less oad gene of bacteriophage T5, encoding the receptor binding protein pb5, was cloned into pT7-3 under the control of phage T7 promoter Φ10. Induction with IPTG resulted in enhanced production of pb5. Upon fractionation of the producing cells, most of the overproduced pb5 was found in the membrane fraction, which was most likely due to aggregation of the protein. The minor, soluble fraction of pb5 specifically inhibited adsorption of T5 to its FhuA receptor protein. Inhibition was also seen with trace amounts of pb5, and binding of pb5 to FhuA appeared to be almost irreversible. Purification of pb5 from the cytosolic fraction was performed by FPLC using a MonoQ column. pb5, which did not bind to the matrix of the column, was obtained in almost pure form. The purified protein also inhibited T5 adsorption.     

Characterization of a heat-stable fraction of human IgG

Heat aggregation of human IgG has been studied by photon correlation spectroscopy, ultracentrifugation, circular dichroism, and differential scanning calorimetry. It is found that pooled human IgG can be separated into two fractions of molecules, one that easily aggregates and one that is stable upon heating. In a buffer atpH=7.6 and 0.2 M NaCl it is found that about half of the original monomeric molecules do not aggregate even after heating at 62°C for 24 h. No differences in the antigen binding capacity of the heat-stable fraction and normal IgG are observed. Heat-stable molecules can partially be transformed to heat-aggregating molecules by a rapid acid denaturation followed by neutralization. Differential scanning calorimetry shows that the major heat denaturation, which is a two-phase process atpH=7.6, starts at about 63°C. Only minor differences between the heat-stable and the heat-labile fractions are observed in the thermograms. No differences are observed in the far-UV region of the CD spectra, indicating that the secondary structure of the heat-stable IgG does not differ from the native IgG molecule. While the aggregation of normal human IgG can be described by Smoluchowski kinetics, the heat-stable fraction follows another kinetics, which includes an activation step.     

Dimethyl sulfoxide at 2.5% (v/v) alters the structural cooperativity and unfolding mechanism of dimeric bacterial NAD+ synthetase

Dimethyl sulfoxide (DMSO) is commonly used as a cosolvent to improve the aqueous solubility of small organic compounds. Its use in a screen to identify novel inhibitors of the enzyme NAD(+) synthetase led to this investigation of its potential effects on the structure and stability of this 60-kD homodimeric enzyme. Although no effects are observed on the enzyme's catalytic activity, as low as 2.5% (v/v) DMSO led to demonstrable changes in the stability of the dimer and its unfolding mechanism. In the absence of DMSO, the dimer behaves hydrodynamically as a single ideal species, as determined by equilibrium analytical ultracentrifugation, and thermally unfolds according to a two-state dissociative mechanism, based on analysis by differential scanning calorimetry (DSC). In the presence of 2.5% (v/v) DMSO, an equilibrium between the dimer and monomer is now detectable with a measured dimer association constant, K(a), equal to 5.6 x 10(6)/M. DSC curve analysis is consistent with this finding. The data are best fit to a three-state sequential unfolding mechanism, most likely representing folded dimer <==> folded monomer <==> unfolded monomer. The unusually large change in the relative stabilities of dimer and monomer, e.g., the association equilibrium shifts from an infinitely large K(a) down to approximately 10(6)/M, in the presence of relatively low cosolvent concentration is surprising in view of the significant buried surface area at the dimer interface, roughly 20% of the surface area of each monomer is buried. A hypothetical structural mechanism to explain this effect is presented.     

Modeling and simulations of a bacterial outer membrane protein: OprF from Pseudomonas aeruginosa

OprF is a major outer membrane protein from Pseudomonas aeruginosa, a homolog of OmpA from Escherichia coli. The N-terminal domains of both proteins have been demonstrated to form low conductance channels in lipid bilayers. Homology models, consisting of an eight-stranded beta-barrel, of the N-terminal domain OprF have been constructed based on the crystal structure of the corresponding domain from E. coli OmpA. OprF homology models have been evaluated via a set (6 x 10 ns) of simulations of the beta-barrel embedded within a solvated dimyristoyl-phosphatidylcholine (DMPC) bilayer. The conformational stability of the models is similar to that of the crystal structure of OmpA in comparable simulations. There is a degree of water penetration into the pore-like center of the OprF barrel. The presence of an acidic/basic (E8/K121) side-chain interaction within the OprF barrel may form a "gate" able to close/open a central pore. Lipid-protein interactions within the simulations were analyzed and revealed that aromatic side-chains (Trp, Tyr) of OprF interact with lipid headgroups. Overall, the behavior of the OprF model in simulations supports the suggestion that this molecule is comparable to OmpA. The simulations help to explain the mechanism of formation of low conductance pores within the outer membrane.     

Monoclonal antibodies against the FhuA (TonA) protein of the Escherichia coli outer membrane

Abstract Outer membranes of Escherichia coli K-12 were used to isolate hybridoma cell lines that produce monoclonal antibodies against the FhuA (TonA) protein. Two monoclonal antibodies were obtained from independent immunization and fusion experiments. The antibodies belonged to the subclass IgG₁ and κ, and IgG_2b and κ, respectively. The latter antibody was purified by affinity chromatography on protein A-Sepharose. The culture supernatants of the hybridoma cell lines and the isolated antibody inhibited adsorption of the phages T5 and T1 to E. coli cells while binding of phage ø80, which also uses the FhuA protein as a receptor, remained unaffected. The specificity of the antibodies to the FhuA protein was supported by the prevention of killing of cells by colicin M and by the lack of inhibition of colicin B and of phage BG23. Transport of iron(III) as ferrichrome complex was not inhibited by the isolated antibody. However, partial competition with the adsorption of the phages T2, TuIb and T6 was observed which may indicate an organization of certain functional phage receptors into clusters.     

Transport across the outer membrane of Escherichia coli K12 via the FhuA receptor is regulated by the TonB protein of the cytoplasmic membrane

Summary Point mutations in the “TonB box” offhuA were suppressed by point mutations in thetonB gene, suggesting both a functional and physical interaction between the FhuA receptor protein in the outer membrane and the TonB protein in the cytoplasmic membrane ofEscherichia coli K12. Mutations influA were classified into four types according to the extent by which they impaired mutant cells in their growth on ferrichrome as sole iron source and in their sensitivity to the antibiotic albomycin and to colicin M. ThetonB mutation with a glutamine to leucine replacement at position 165 was less efficient in restoring the FhuA functions than the glutamine to lysine exchange at the same position. The better the coupling between FhuA and TonB the poorer was the inhibition of phage T1 binding to FhuA by ferrichrome. A working model is proposed in which the TonB protein assumes different conformations in response to the energized state of the cytoplasmic membrane and thereby allosterically regulates the activity of the FhuA receptor. This model implies an intermembrane coupling between two proteins in adjacent membranes.     

Characterization of in vitro interactions between a truncated TonB protein from Escherichia coli and the outer membrane receptors FhuA and FepA

High-affinity iron uptake in gram-negative bacteria depends upon TonB, a protein which couples the proton motive force in the cytoplasmic membrane to iron chelate receptors in the outer membrane. To advance studies on TonB structure and function, we expressed a recombinant form of Escherichia coli TonB lacking the N-terminal cytoplasmic membrane anchor. This protein (H(6)-'TonB; M(r), 24,880) was isolated in a soluble fraction of lysed cells and was purified by virtue of a hexahistidine tag located at its N terminus. Sedimentation experiments indicated that the H(6)-'TonB preparation was almost monodisperse and the protein was essentially monomeric. The value found for the Stokes radius (3.8 nm) is in good agreement with the value calculated by size exclusion chromatography. The frictional ratio (2.0) suggested that H(6)-'TonB adopts a highly asymmetrical form with an axial ratio of 15. H(6)-'TonB captured both the ferrichrome-iron receptor FhuA and the ferric enterobactin receptor FepA from detergent-solubilized outer membranes in vitro. Capture was enhanced by preincubation of the receptors with their cognate ligands. Cross-linking assays with the purified proteins in vitro demonstrated that there was preferential interaction between TonB and ligand-loaded FhuA. Purified H(6)-'TonB was found to be stable and thus shows promise for high-resolution structural studies.     

Molecular structure of fd (f1, M13) filamentous bacteriophage refined with respect to X-ray fibre diffraction and solid-state NMR data supports specific models of phage assembly at the bacterial membrane

Filamentous bacteriophage (Inovirus) is a simple and well-characterized model system. The phage particle, or virion, is about 60 angstroms in diameter and several thousand angstrom units long. The virions are assembled at the bacterial membrane as they extrude out of the host without killing it, an example of specific transport of nucleoprotein assemblages across membranes. The Ff group (fd, f1 and M13) has been especially widely studied. Models of virion assembly have been proposed based on a molecular model of the fd virion derived by X-ray fibre diffraction. A somewhat different model of the fd virion using solid-state NMR data has been proposed, not consistent with these models of assembly nor with the X-ray diffraction data. Here we show that reinterpreted NMR data are also consistent with the model derived from X-ray fibre diffraction studies, and discuss models of virion assembly.     

Inactivation of FhuA at the cell surface of Escherichia coli K-12 by a phage T5 lipoprotein at the periplasmic face of the outer membrane.

Inactivation of phage T5 by lysed cells after phage multiplication is prevented by a phage-encoded lipoprotein (Llp) that inactivates the FhuA outer membrane receptor protein (K. Decker, V. Krauel, A. Meesmann, and K. Heller, Mol. Microbiol. 12:321-332, 1994). Using FhuA derivatives carrying insertions of 4 and 16 amino acid residues and point mutations, we determined whether FhuA inactivation is caused by binding of Llp to FhuA and which regions of FhuA are important for inactivation by Llp. Cells expressing Llp were resistant not only to phage T5 but to all FhuA ligands tested, such as phage phi 80, colicin M, and albomycin, and they were strongly reduced in the uptake of ferrichrome. Most of the FhuA derivatives which were not affected by Llp were, according to a previously published FhuA transmembrane topology model, located in periplasmic turns and in the TonB box close to the periplasm. Since the ligands bind to the cell surface, interaction of FhuA with Llp in the periplasm may induce a FhuA conformation which impairs binding of the ligands. This conclusion was supported by the increase rather than decrease of colicin M sensitivity of two mutants in the presence of Llp. The only Llp-resistant FhuA derivatives with mutations at the cell surface contained insertions of 16 residues in the loop that determines the permeability of the FhuA channel and serves as the principal binding site for all FhuA ligands. This region may be inactivated by steric hindrance in that a portion of Llp penetrates into the channel. Outer membranes prepared with 0.25% Triton X-100 from cells expressing Llp contained inactivated FhuA, suggesting Llp to be an outer membrane protein whose interaction with FhuA was not abolished by Triton X-100. Llp solubilized in 1.1% octylglucoside prevented T5 inactivation by FhuA dissolved in octylglucoside.     

Gibbs motif sampling: detection of bacterial outer membrane protein repeats.

The detection and alignment of locally conserved regions (motifs) in multiple sequences can provide insight into protein structure, function, and evolution. A new Gibbs sampling algorithm is described that detects motif-encoding regions in sequences and optimally partitions them into distinct motif models; this is illustrated using a set of immunoglobulin fold proteins. When applied to sequences sharing a single motif, the sampler can be used to classify motif regions into related submodels, as is illustrated using helix-turn-helix DNA-binding proteins. Other statistically based procedures are described for searching a database for sequences matching motifs found by the sampler. When applied to a set of 32 very distantly related bacterial integral outer membrane proteins, the sampler revealed that they share a subtle, repetitive motif. Although BLAST (Altschul SF et al., 1990, J Mol Biol 215:403-410) fails to detect significant pairwise similarity between any of the sequences, the repeats present in these outer membrane proteins, taken as a whole, are highly significant (based on a generally applicable statistical test for motifs described here). Analysis of bacterial porins with known trimeric beta-barrel structure and related proteins reveals a similar repetitive motif corresponding to alternating membrane-spanning beta-strands. These beta-strands occur on the membrane interface (as opposed to the trimeric interface) of the beta-barrel. The broad conservation and structural location of these repeats suggests that they play important functional roles.