Evidence of an intracellular interaction between the Escherichia coli enzymes EntC and EntB and identification of a potential electrostatic channeling surface.

Siderophores are high-affinity small-molecule chelators employed by bacteria to acquire iron from the extracellular environment. The Gram-negative bacterium Escherichia coli synthesizes and secretes enterobactin, a tris-catechol siderophore. Enterobactin is synthesized by six cytoplasmic enzyme activities: EntC, EntB (isochorismatase (IC) domain), EntA, EntE, EntB (aryl carrier protein (ArCP) domain), and EntF. While various pairwise protein-protein interactions have been reported between EntB, EntA, EntE, and EntF, evidence for an interaction between EntC and EntB has remained elusive. We have employed bacterial two-hybrid assays and in vivo crosslinking to demonstrate an intracellular EntC-EntB interaction. A T18-EntC/T25-EntB co-transformant exhibited a positive two-hybrid signal compared to a control T18-EntC/T25 co-transformant. In vivo formaldehyde crosslinking of E. coli cells co-expressing HA-tagged EntB and H6-tagged EntC resulted in an observable ∼80 kDa band on Western blots that cross-reacted with anti-HA and anti-H6, corresponding to one HA-EntB monomer (33 kDa) crosslinked with one H6-EntC monomer (45 kDa). This band disappeared upon sample boiling, confirming it to be a formaldehyde-crosslinked species. Bands of molecular masses greater than 80 kDa that cross-reacted with both antibodies were also observed. Automated docking of the crystal structures of monomeric EntC and dimeric EntB resulted in a top-ranked candidate docked ensemble in which the active sites of EntC and EntB were oriented in apposition and connected by an electropositive surface potentially capable of channeling negatively charged isochorismate. These research outcomes provide the first reported evidence of an EntC-EntB interaction, as well as the first experimental evidence of higher-order complexes containing EntC and EntB.

Dual Activity of Rose Bengal Functionalized to Albumin-Coated Lanthanide-Doped Upconverting Nanoparticles: Targeting and Photodynamic Therapy.

A modified version of a desolvation method was used to render lanthanide-doped upconverting nanoparticles NaGdF4:Yb3+/Er3+ (Ln-UCNPs) water-dispersible and biocompatible for photodynamic therapy. Bovine serum albumin (BSA) was used as surface coating with a direct conjugation to NaGdF4:Yb3+/Er3+ nanoparticles forming a ∼2 nm thick shell. It was estimated that approximately 112 molecules of BSA were present and cross-linked per NaGdF4:Yb3+/Er3+ nanoparticle. Analysis of the BSA structural behavior on the Ln-UCNP surfaces displayed up to 80% loss of α-helical content. Modification of the Ln-UCNPs with a BSA shell prevents luminescence quenching from solvent molecules (H2O) with high energy vibrations that can interact with the excited states of the optically active ions Er3+ and Yb3+ via dipole-dipole interactions. Additionally, the photosensitizer rose bengal (RB) was conjugated to albumin on the surface of the Ln-UCNPs. Emission spectroscopy under 980 nm excitation was carried out, and an energy transfer efficiency of 63% was obtained. In vitro cell studies performed using human lung cancer cells (A549 cell line) showed that Ln-UCNPs coated with BSA were not taken by the cells. However, when RB was conjugated to BSA on the surface of the nanoparticles, cellular uptake was observed, and cytotoxicity was induced by the production of singlet oxygen under 980 nm irradiation.

A novel set of vectors for Fur-controlled protein expression under iron deprivation in Escherichia coli.

BACKGROUND: In the presence of sufficient iron, the Escherichia coli protein Fur (Ferric Uptake Regulator) represses genes controlled by the Fur box, a consensus sequence near or within promoters of target genes. De-repression of Fur-controlled genes occurs upon iron deprivation. In the E. coli chromosome, there is a bidirectional intercistronic promoter region with two non-overlapping Fur boxes. This region controls Fur-regulated expression of entCEBAH in the clockwise direction and fepB in the anticlockwise direction. RESULTS: We cloned the E. coli bidirectional fepB/entC promoter region into low-copy-number plasmid backbones (pACYC184 and pBR322) along with downstream sequences encoding epitope tags and a multiple cloning site (MCS) compatible with the bacterial adenylate cyclase two-hybrid (BACTH) system. The vector pFCF1 allows for iron-controlled expression of FLAG-tagged proteins, whereas the pFBH1 vector allows for iron-controlled expression of HA-tagged proteins. We showed that E. coli knockout strains transformed with pFCF1-entA, pFCF1-entE and pFBH1-entB express corresponding proteins with appropriate epitope tags when grown under iron restriction. Furthermore, transformants exhibited positive chrome azurol S (CAS) assay signals under iron deprivation, indicating that the transformants were functional for siderophore biosynthesis. Western blotting and growth studies in rich and iron-depleted media demonstrated that protein expression from these plasmids was under iron control. Finally, we produced the vector pFCF2, a pFCF1 derivative in which a kanamycin resistance (KanR) gene was engineered in the direction opposite of the MCS. The entA ORF was then subcloned into the pFCF2 MCS. Bidirectional protein expression in an iron-deprived pFCF2-entA transformant was confirmed using antibiotic selection, CAS assays and growth studies. CONCLUSIONS: The vectors pFCF1, pFCF2, and pFBH1 have been shown to use the fepB/entC promoter region to control bidirectional in trans expression of epitope-tagged proteins in iron-depleted transformants. In the presence of intracellular iron, protein expression from these constructs was abrogated due to Fur repression. The compatibility of the pFCF1 and pFBH1 backbones allows for iron-controlled expression of multiple epitope-tagged proteins from a single co-transformant.

Intracellular co-localization of the Escherichia coli enterobactin biosynthetic enzymes EntA, EntB, and EntE.

Bacteria utilize small-molecule iron chelators called siderophores to support growth in low-iron environments. The Escherichia coli catecholate siderophore enterobactin is synthesized in the cytoplasm upon iron starvation. Seven enzymes are required for enterobactin biosynthesis: EntA-F, H. Given that EntB-EntE and EntA-EntE interactions have been reported, we investigated a possible EntA-EntB-EntE interaction in E. coli cells. We subcloned the E. coli entA and entB genes into bacterial adenylate cylase two-hybrid (BACTH) vectors allowing for co-expression of EntA and EntB with N-terminal fusions to the adenylate cyclase fragments T18 or T25. BACTH constructs were functionally validated using the CAS assay and growth studies. Co-transformants expressing T18/T25-EntA and T25/T18-EntB exhibited positive two-hybrid signals indicative of an intracellular EntA-EntB interaction. To gain further insights into the interaction interface, we performed computational docking in which an experimentally validated EntA-EntE model was docked to the EntB crystal structure. The resulting model of the EntA-EntB-EntE ternary complex predicted that the IC domain of EntB forms direct contacts with both EntA and EntE. BACTH constructs that expressed the isolated EntB IC domain fused to T18/T25 were prepared in order to investigate interactions with T25/T18-EntA and T25/T18-EntE. CAS assays and growth studies demonstrated that T25-IC co-expressed with the EntB ArCP domain could complement the E. coli entB(-) phenotype. In agreement with the ternary complex model, BACTH assays demonstrated that the EntB IC domain interacts with both EntA and EntE.

Subunit orientation in the Escherichia coli enterobactin biosynthetic EntA-EntE complex revealed by a two-hybrid approach.

The siderophore enterobactin is synthesized by the enzymes EntA-F and EntH in the Escherichia coli cytoplasm. We previously reported in vitro evidence of an interaction between tetrameric EntA and monomeric EntE. Here we used bacterial adenylate cyclase two-hybrid (BACTH) assays to demonstrate that the E. coli EntA-EntE interaction occurs intracellularly. Furthermore, to obtain information on subunit orientation in the EntA-EntE complex, we fused BACTH reporter fragments T18 and T25 to EntA and EntE in both N-terminal and C-terminal orientations. To validate functionality of our fusion proteins, we performed Chrome Azurol S (CAS) assays using E. coli entE(-) and entA(-) knockout strains transformed with our BACTH constructs. We found that transformants expressing N-terminal and C-terminal T18/T25 fusions to EntE exhibited CAS signals, indicating that these constructs could rescue the entE(-) phenotype. While expression of EntA with N-terminal T18/T25 fusions exhibited CAS signals, C-terminal fusions did not, presumably due to disruption of the EntA tetramer in vivo. Bacterial growth assays supported our CAS findings. Co-transformation of functional T18/T25 fusions into cya(-)E. coli BTH101 cells resulted in positive BACTH signals only when T18/T25 fragments were fused to the N-termini of both EntA and EntE. Co-expression of N-terminally fused EntA with C-terminally fused EntE resulted in no detectable BACTH signal. Analysis of protein expression by Western blotting confirmed that the loss of BACTH signal was not due to impaired expression of fusion proteins. Based on our results, we propose that the N-termini of EntA and EntE are proximal in the intracellular complex, while the EntA N-terminus and EntE C-terminus are distal. A protein-protein docking simulation using SwarmDock was in agreement with our experimental observations.

Identification of a surface glutamine residue (Q64) of Escherichia coli EntA required for interaction with EntE.

The enterobactin biosynthetic enzyme EntA forms a complex with EntE, the next enzyme in the pathway, to enhance activation of the enterobactin precursor 2,3-dihydroxybenzoate. Here we used phage display to identify an EntE-interacting region on the surface of EntA. Upon panning immobilized EntE with a random peptide phage library, we recovered 47 unique EntE-binding dodecamer peptide sequences that aligned to a region of the EntA primary sequence corresponding to helix α4. In order to further investigate this region, we mutagenized EntA Q64, a hydrogen-bonding residue found on the surface-exposed face α4. Far-UV circular dichroism, thermal denaturation experiments, and enzymatic assays showed that mutation of EntA residue Gln 64 to alanine (Q64A) had no deleterious effect on EntA structure or function. By following near-UV CD spectral changes, we found that the spectrum of wild-type EntA was altered in the presence of EntE, indicative of conformational changes in EntA aromatic chromophores upon formation of the EntA-EntE complex. However, EntE did not affect the CD spectrum of EntA variant Q64A, demonstrating that this variant did not interact with EntE in a manner similar to wild-type EntA. Analytical ultracentrifugation of wild-type and variant EntA proteins showed that EntA Q64A was predominantly dimeric at 20µM, unlike wild-type EntA which was predominantly tetrameric. Taken together, our findings establish that EntA α4 is required for efficient formation of the EntA-EntE as well as for EntA oligomerization.

The C-glycosyltransferase IroB from pathogenic Escherichia coli: identification of residues required for efficient catalysis.

Escherichia coli C-glycosyltransferase IroB catalyzes the formation of a CC bond between enterobactin and the glucose moiety of UDP-glucose, resulting in the production of mono-, di- and tri-glucosylated enterobactin (MGE, DGE, TGE). To identify catalytic residues, we generated a homology model of IroB from aligned structures of two similar C-glycosyltransferases as templates. Superposition of our homology model onto the structure of a TDP-bound orthologue revealed residue W264 as a possible stabilizer of UDP-glucose. D304 in our model was located near the predicted site of the glucose moiety of UDP-glucose. A loop containing possible catalytic residues (H65, H66, E67) was found at the predicted enterobactin-binding site. We generated IroB variants at positions 65-67, 264, and 304 and investigated variant protein conformations and enzymatic activities. Variants were found to have Tm values similar to wild-type IroB. Fluorescence emission spectra of H65A/H66A, E67A, and D304N were superimposable with wild-type IroB. However, the emission spectrum of W264L was blue-shifted, suggesting solvent exposure of W264. While H65A/H66A retained activity (92% conversion of enterobactin, with MGE as a major product), all other IroB variants were impaired in their abilities to glucosylate enterobactin: E67A catalyzed partial (29%) conversion of enterobactin to MGE; W264L converted 55% of enterobactin to MGE; D304N was completely inactive. Activity-impaired variants were found to bind enterobactin with affinities within 2.5-fold of wild-type IroB. Given our outcomes, we propose that IroB W264 and D304 are required for binding and orienting UDP-glucose, while E67, possibly supported by H65/H66, participates in enterobactin/MGE/DGE deprotonation.

Enzymatic adenylation of 2,3-dihydroxybenzoate is enhanced by a protein-protein interaction between Escherichia coli 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase (EntA) and 2,3-dihydroxybenzoate-AMP ligase (EntE).

The Escherichia coli siderophore enterobactin is synthesized in response to iron starvation. 2,3-Dihydro-2,3-dihydroxybenzoate dehydrogenase (EntA) produces 2,3-dihydroxybenzoate (DHB), a biosynthetic intermediate. 2,3-Dihydroxybenzoate-AMP ligase (EntE) adenylates DHB, activating it for attachment to the NRPS substrate holo-EntB. Using analytical ultracentrifugation, we found that EntA undergoes concentration-dependent dimer-tetramer self-association (K(D) = 12.3 µM). We further found that EntA can form a specific complex with EntE. Pull-down assays revealed that recombinant EntA bait pulled down EntE from E. coli lysates, whereas recombinant EntE bait could pull down EntA. Addition of the SMCC cross-linker to a mixture of EntA and EntE resulted in a cross-linked product with a molecular mass of >250 kDa, suggesting a complex stoichiometry of one EntA tetramer and four EntE monomers. The effect of EntA on EntE activity was also examined. Addition of a 4-fold excess of EntA to an EntE assay mixture resulted in a 6-fold stimulation of EntE activity. EntA was also found to perturb the FRET signal between EntE donor residues and EntE-bound DHB. By following the EntA-dependent decrease in the magnitude of the EntE-DHB FRET signal, EntA-EntE binding behavior was found to be sigmoidal, suggesting the presence of both low- and high-affinity binding sites. The EntA-EntE interaction was also directly measured by isothermal titration calorimetry at 10 °C. The resulting binding isotherm fit well to a model describing two binding sites, supporting our AUC and fluorescence data. Taken together, our data show that tetrameric EntA optimally interacts with EntE, resulting in an enhancement of EntE activity.

Ligand-induced conformational rearrangements promote interaction between the Escherichia coli enterobactin biosynthetic proteins EntE and EntB.

Siderophores are small-molecule iron chelators that many bacteria synthesize and secrete in order to survive in iron-depleted environments. Biosynthesis of enterobactin, the Escherichia coli catecholate siderophore, requires adenylation of 2,3-dihydroxybenzoic acid (2,3-DHB) by the cytoplasmic enzyme EntE. The DHB-AMP product is then transferred to the active site of holo-EntB subsequent to formation of an EntE-EntB complex. Here we investigate the binding of 2,3-DHB to EntE and how DHB binding affects EntE-EntB interaction. We overexpressed and purified recombinant forms of EntE and EntB with N-terminal hexahistidine tags (H6-EntE and H6-EntB). Isothermal titration calorimetry showed that 2,3-DHB binds to H6-EntE with a 1:1 stoichiometry and a K(d) of 7.4 microM. Fluorescence spectra revealed enhanced 2,3-DHB emission at 440 nm (lambda(ex)=280 nm) when bound to H6-EntE due to fluorescence resonance energy transfer (FRET) between EntE intrinsic fluorophore donors and bound 2,3-DHB acceptor. A FRET signal was not observed when H6-EntE was mixed with either 2,5-dihydroxybenzoic acid or 3,5-dihydroxybenzoic acid. The H6-EntE-2,3-DHB FRET signal was quenched by H6-EntB in a concentration-dependent manner. From these data, we were able to determine the EC(50) of EntE-EntB interaction to be approximately 1.5 microM. We also found by fluorescence and CD measurements that H6-EntB can bind 2,3-DHB, resulting in conformational changes in the protein. Additional alterations in H6-EntB near-UV and far-UV CD spectra were observed upon mixture with H6-EntE and 2,3-DHB, suggesting that further conformational rearrangements occur in EntB upon interaction with substrate-loaded EntE. We also found that H6-EntB as a bait protein pulled down a higher concentration of chromosomally expressed EntE in the presence of exogenous 2,3-DHB. Taken together, our results show that binding of 2,3-DHB to EntE and EntB primes these proteins for efficient complexation, thus facilitating direct channeling of the siderophore precursor 2,3-DHB-AMP.

A shared binding site for NAD+ and coenzyme A in an acetaldehyde dehydrogenase involved in bacterial degradation of aromatic compounds.

The meta-cleavage pathway for catechol is a central pathway for the bacterial dissimilation of a wide variety of aromatic compounds, including phenols, methylphenols, naphthalenes, and biphenyls. The last enzyme of the pathway is a bifunctional aldolase/dehydrogenase that converts 4-hydroxy-2-ketovalerate to pyruvate and acetyl-CoA via acetaldehyde. The structure of the NAD (+)/CoASH-dependent aldehyde dehydrogenase subunit is similar to that of glyceraldehyde-3-phosphate dehydrogenase, with a Rossmann fold-based NAD (+) binding site observed in the NAD (+)-enzyme complex [Manjasetty, B. A., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 6992-6997]. However, the location of the CoASH binding site was not determined. In this study, hydrogen-deuterium exchange experiments, coupled with peptic digest and mass spectrometry, were used to examine cofactor binding. The pattern of hydrogen-deuterium exchange in the presence of CoASH was almost identical to that observed with NAD (+), consistent with the two cofactors sharing a binding site. This is further supported by the observations that either CoASH or NAD (+) is able to elute the enzyme from an NAD (+) affinity column and that preincubation of the enzyme with NAD (+) protects against inactivation by CoASH. Consistent with these data, models of the CoASH complex generated using AUTODOCK showed that the docked conformation of CoASH can fully occupy the cavity containing the enzyme active site, superimposing with the NAD (+) cofactor observed in the X-ray crystal structure. Although CoASH binding Rossmann folds have been described previously, this is the first reported example of a Rossmann fold that can alternately bind CoASH or NAD (+) cofactors required for enzymatic catalysis.

Identification and functional characterization of a novel mutation in the calcium-sensing receptor gene in familial hypocalciuric hypercalcemia: modulation of clinical severity by vitamin D status.

CONTEXT: Familial hypocalciuric hypercalcemia (FHH) is a benign condition associated with heterogeneous inactivating mutations in the calcium-sensing receptor (CASR) gene. OBJECTIVE: The objective of the study was to identify and characterize a CASR mutation in a moderately hypercalcemic, hyperparathyroid individual and his family and assess the influence of vitamin D status on the clinical expression of the defect. SUBJECTS: We studied a kindred with FHH, in which the proband (a 34-yr-old male) was initially diagnosed with primary hyperparathyroidism due to frankly elevated serum PTH levels. METHODS: CASR gene mutation analysis was performed on genomic DNA of the proband and family members. The mutant CASR was functionally characterized by transient transfection studies in kidney cells in vitro. RESULTS: A novel heterozygous mutation (F180C, TTC>TGC) in exon 4 of the CASR gene was identified. Although the mutant receptor was expressed normally at the cell surface, it was unresponsive with respect to intracellular signaling (MAPK activation) to increases in extracellular calcium concentrations. The baby daughter of the proband presented with neonatal hyperparathyroidism with markedly elevated PTH. Vitamin D supplementation of both the proband and the baby resulted in reduction of serum PTH levels to the normal range. The serum calcium level remained at a constant and moderately elevated level. CONCLUSION: The identification of a novel CASR gene mutation established the basis of the hypercalcemia in the kindred. Concomitant vitamin D deficiency modulates the severity of the presentation of FHH.

Interactions between TonB from Escherichia coli and the periplasmic protein FhuD.

For uptake of ferrichrome into bacterial cells, FhuA, a TonB-dependent outer membrane receptor of Escherichia coli, is required. The periplasmic protein FhuD binds and transfers ferrichrome to the cytoplasmic membrane-associated permease FhuB/C. We exploited phage display to map protein-protein interactions in the E. coli cell envelope that contribute to ferrichrome transport. By panning random phage libraries against TonB and against FhuD, we identified interaction surfaces on each of these two proteins. Their interactions were detected in vitro by dynamic light scattering and indicated a 1:1 TonB-FhuD complex. FhuD residue Thr-181, located within the siderophorebinding site and mapping to a predicted TonB-interaction surface, was mutated to cysteine. FhuD T181C was reacted with two thiol-specific fluorescent probes; addition of the siderophore ferricrocin quenched fluorescence emissions of these conjugates. Similarly, quenching of fluorescence from both probes confirmed binding of TonB and established an apparent KD of approximately 300 nM. Prior saturation of the siderophorebinding site of FhuD with ferricrocin did not alter affinity of TonB for FhuD. Binding, further characterized with surface plasmon resonance, indicated a higher affinity complex with KD values in the low nanomolar range. Addition of FhuD to a preformed TonB-FhuA complex resulted in formation of a ternary complex. These observations led us to propose a novel mechanism in which TonB acts as a scaffold, directing FhuD to regions within the periplasm where it is poised to accept and deliver siderophore.

Structure of TonB in complex with FhuA, E. coli outer membrane receptor.

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.

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.

Identification of the gene responsible for methylmalonic aciduria and homocystinuria, cblC type.

Methylmalonic aciduria and homocystinuria, cblC type (OMIM 277400), is the most common inborn error of vitamin B(12) (cobalamin) metabolism, with about 250 known cases. Affected individuals have developmental, hematological, neurological, metabolic, ophthalmologic and dermatologic clinical findings. Although considered a disease of infancy or childhood, some individuals develop symptoms in adulthood. The cblC locus was mapped to chromosome region 1p by linkage analysis. We refined the chromosomal interval using homozygosity mapping and haplotype analyses and identified the MMACHC gene. In 204 individuals, 42 different mutations were identified, many consistent with a loss of function of the protein product. One mutation, 271dupA, accounted for 40% of all disease alleles. Transduction of wild-type MMACHC into immortalized cblC fibroblast cell lines corrected the cellular phenotype. Molecular modeling predicts that the C-terminal region of the gene product folds similarly to TonB, a bacterial protein involved in energy transduction for cobalamin uptake.

Siderophore transport through Escherichia coli outer membrane receptor FhuA with disulfide-tethered cork and barrel domains.

The hydroxamate siderophore receptor FhuA is a TonB-dependent outer membrane protein of Escherichia coli composed of a C-terminal 22-stranded beta-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.

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.

Kinetic analyses reveal multiple steps in forming TonB-FhuA complexes from Escherichia coli.

FhuA, an outer membrane receptor of Escherichia coli, facilitates transport of hydroxamate siderophores and siderophore-antibiotic conjugates. The cytoplasmic membrane complex TonB-ExbB-ExbD provides energy for transport via the proton motive force. This energy is transduced by protein-protein interactions between TonB and FhuA, but the molecular determinants of these interactions remain uncharacterized. Our analyses of FhuA and two recombinant TonB species by surface plasmon resonance revealed that TonB undergoes a kinetically limiting rearrangement upon initial interaction with FhuA: an intermediate TonB-FhuA complex of 1:1 stoichiometry was detected. The intermediate then recruits a second TonB protein. Addition of ferricrocin, a FhuA-specific ligand, enhanced amounts of the 2:1 complex but was not essential for its formation. To assess the role of the cork domain of FhuA in forming a 2:1 TonB-FhuA complex, we tested a FhuA deletion (residues 21-128) for its ability to interact with TonB. Analytical ultracentrifugation demonstrated that deletion of this region of the cork domain resulted in a 1:1 complex. Furthermore, the high-affinity 2:1 complex requires the N-terminal region of TonB. Together these in vitro experiments establish that TonB-FhuA interactions require sequential steps of kinetically limiting rearrangements. Additionally, domains that contribute to complex formation were identified in TonB and in FhuA.

Hemoglobin-binding protein HgbA in the outer membrane of Actinobacillus pleuropneumoniae: homology modelling reveals regions of potential interactions with hemoglobin and heme.

Analyses of the primary sequence of hemoglobin-binding protein HgbA from Actinobacillus pleuropneumoniae by comparative modelling and by a Hidden Markov Model identified its topological similarities to bacterial outer membrane receptors BtuB, FepA, FhuA, and FecA of Escherichia coli. The HgbA model has a globular N-terminal cork domain contained within a 22-stranded beta barrel domain, its folds being similar to the structures of outer membrane receptors that have been solved by X-ray crystallography. The barrel domain of the HgbA model superimposes onto the barrel domains of the four outer membrane receptors with rmsd values less than 1.0 A. This feature is consistent with a phylogenetic tree which indicated clustering of polypeptide sequences for three barrel domains. Furthermore, the HgbA model shares the highest structural similarity to BtuB, with the modelled HgbA barrel having approximately the same elliptical cross-section and height as that of BtuB. Extracellular loop regions of HgbA are predicted to be more extended than those of the E. coli outer membrane receptors, potentially facilitating a protein-protein interface with hemoglobin. Fold recognition modelling of the HgbA loop regions showed that 10 out of 11 predicted loops are highly homologous to known structures of protein loops that contribute to heme/iron or protein-protein interactions. Strikingly, HgbA loop 2 has structural homology to a loop in bovine endothelial nitric acid oxidase that is proximal to a heme-binding site; and HgbA loop 7 contains a histidine residue conserved in a motif that is involved in heme/hemoglobin interactions. These findings implicate HgbA loops 2 and 7 in recognition and binding of hemoglobin or the heme ligand.

Molecular cloning of haemoglobin-binding protein HgbA in the outer membrane of Actinobacillus pleuropneumoniae.

From the porcine pathogen Actinobacillus pleuropneumoniae cultivated in iron-deficient or haem-deficient media, haemoglobin (Hb)-agarose affinity purification was exploited to isolate an outer-membrane protein of approximately 105 kDa, designated HgbA. Internal peptide sequences of purified HgbA were used to design oligonucleotide primers for PCR amplification, yielding amplicons that showed partial sequences with homology to hgbA of Pasteurella multocida. Upon screening two genomic libraries of A. pleuropneumoniae serotype 1 strain 4074, positive clones were assembled into an ORF of 2838 bp. HgbA (946 aa) includes a signal peptide of 23 aa and the deduced HgbA sequence (104 890 Da) also demonstrated a possible Ton box. The promoter region of hgbA from A. pleuropneumoniae serotype 1 showed consensus for -35 and -10 sequences and a putative Fur-binding site. RT-PCR confirmed that hgbA of A. pleuropneumoniae is upregulated in response to diminished levels of iron in the culture medium. While an internally deleted hgbA mutant was unable to use pig Hb as sole source of iron for growth, flow cytometry confirmed its Hb binding; the internally deleted sequences may not be required for Hb binding, but appear necessary for the iron supply from Hb. HgbA is required for growth of A. pleuropneumoniae in the presence of Hb as sole iron source.