Lipocalin Blc is a potential heme-binding protein.
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ABSTRACT:
SUBMITTER: Bozhanova NG
PROVIDER: S-EPMC8177097 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Lipocalins, a widespread multifunctional family of small proteins (15-25kDa) have been first described in eukaryotes and more recently in Gram-negative bacteria. Bacterial lipocalins belonging to class I are outer membrane lipoproteins, among which Blc from E. coli is the better studied. Blc is expressed under conditions of starvation and high osmolarity, conditions known to exert stress on the cell envelope. The structure of Blc that we have previously solved (V. Campanacci, D. Nurizzo, S. Spinelli, C. Valencia, M. Tegoni, C. Cambillau, FEBS Lett. 562 (2004) 183-188.) suggested its possible role in binding fatty acids or phospholipids. Both physiological and structural data on Blc, therefore, point to a role in storage or transport of lipids necessary for membrane maintenance. In order to further document this hypothesis for Blc function, we have performed binding studies using fluorescence quenching experiments. Our results indicate that dimeric Blc binds fatty acids and phospholipids in a micromolar K(d) range. The crystal structure of Blc with vaccenic acid, an unsaturated C18 fatty acid, reveals that the binding site spans across the Blc dimer, opposite to its membrane anchored face. An exposed unfilled pocket seemingly suited to bind a polar group attached to the fatty acid prompted us to investigate lyso-phospholipids, which were found to bind in a nanomolar K(d) range. We discuss these findings in terms of a potential role for Blc in the metabolism of lysophospholipids generated in the bacterial outer membrane.
| S-EPMC5007124 | biostudies-literature
Project description:During blood-stage development, malaria parasites are challenged with the detoxification of enormous amounts of heme released during the proteolytic catabolism of erythrocytic hemoglobin. They tackle this problem by sequestering heme into bioinert crystals known as hemozoin. The mechanisms underlying this biomineralization process remain enigmatic. Here, we demonstrate that both rodent and human malaria parasite species secrete and internalize a lipocalin-like protein, PV5, to control heme crystallization. Transcriptional deregulation of PV5 in the rodent parasite Plasmodium berghei results in inordinate elongation of hemozoin crystals, while conditional PV5 inactivation in the human malaria agent Plasmodium falciparum causes excessive multidirectional crystal branching. Although hemoglobin processing remains unaffected, PV5-deficient parasites generate less hemozoin. Electron diffraction analysis indicates that despite the distinct changes in crystal morphology, neither the crystalline order nor unit cell of hemozoin are affected by impaired PV5 function. Deregulation of PV5 expression renders P. berghei hypersensitive to the antimalarial drugs artesunate, chloroquine, and atovaquone, resulting in accelerated parasite clearance following drug treatment in vivo. Together, our findings demonstrate the Plasmodium-tailored role of a lipocalin family member in hemozoin formation and underscore the heme biomineralization pathway as an attractive target for therapeutic exploitation.
| S-EPMC7368307 | biostudies-literature
Project description:We characterized heme binding in the bacterial iron response regulator (Irr) protein, which is a simple heme-regulated protein having a single "heme-regulatory motif", HRM, and plays a key role in the iron homeostasis of a nitrogen-fixing bacterium. The heme titration to wild-type and mutant Irr clearly showed that Irr has two heme binding sites: one of the heme binding sites is in the HRM, where (29)Cys is the axial ligand, and the other one, the secondary heme binding site, is located outside of the HRM. The Raman line for the Fe-S stretching mode observed at 333 cm(-1) unambiguously confirmed heme binding to Cys. The lower frequency of the Fe-S stretching mode corresponds to the weaker Fe-S bond, and the broad Raman line of the Fe-S bond suggests multiple configurations of heme binding. These structural characteristics are definitely different from those of typical hemoproteins. The unusual heme binding in Irr was also evident in the EPR spectra. The characteristic g-values of the 5-coordinate Cys-ligated heme and 6-coordinate His/His-ligated heme were observed, while the multiple configurations of heme binding were also confirmed. Such multiple heme configurations are not encountered for typical hemoproteins where the heme functions as the active center. Therefore, we conclude that heme binding to HRM in the heme-regulated protein, Irr, is quite different from that in conventional hemoproteins but characteristic of heme-regulated proteins using heme as the signaling molecule.
| S-EPMC3043289 | biostudies-literature
Project description:The Pseudomonas aeruginosa heme utilization (Phu) system encodes several proteins involved in the acquisition of heme as an iron source. Once internalized, heme is degraded by the iron-regulated heme oxygenase, HemO to biliverdin (BV) IXδ and β. In vitro studies have shown holo-PhuS transfers heme to the iron-regulated HemO. This protein-protein interaction is specific for HemO as PhuS does not interact with the α-regioselective heme oxygenase, BphO. Bacterial genetics and isotopic labeling ((13)C-heme) studies confirmed extracellular heme is converted to (13)C-BVIX δ and β through the catalytic action of HemO. In an effort to further understand the role of PhuS, similar studies were performed on the P. aeruginosa PAO1 ΔphuS and ΔphuS/ΔhemO strains. In contrast to wild-type strain, the absence of PhuS results in extracellular heme uptake and degradation via the catalytic action of HemO and BphO. At low heme concentrations, loss of PhuS leads to inefficient extracellular heme uptake supported by the fact the mRNA levels of PhuR, HemO, and BphO remain elevated when compared to the wild-type PAO1. On increasing extracellular heme concentrations, the elevated levels of PhuR, HemO, and BphO allow "leaky uptake" and degradation of heme via HemO and BphO. Similarly, in the ΔphuS/ΔhemO strain, the higher heme concentrations combined with elevated levels of PhuR and BphO leads to nonspecific heme uptake and degradation by BphO. Thus we propose heme flux into the cell is driven by the catalytic action of HemO with PhuS acting as a "control valve" to regulate extracellular heme flux.
| S-EPMC3748626 | biostudies-literature
Project description:The soluble monomeric domain of lipoprotein YxeF from the Gram positive bacterium B. subtilis was selected by the Northeast Structural Genomics Consortium (NESG) as a target of a biomedical theme project focusing on the structure determination of the soluble domains of bacterial lipoproteins. The solution NMR structure of YxeF reveals a calycin fold and distant homology with the lipocalin Blc from the Gram-negative bacterium E.coli. In particular, the characteristic ?-barrel, which is open to the solvent at one end, is extremely well conserved in YxeF with respect to Blc. The identification of YxeF as the first lipocalin homologue occurring in a Gram-positive bacterium suggests that lipocalins emerged before the evolutionary divergence of Gram positive and Gram negative bacteria. Since YxeF is devoid of the ?-helix that packs in all lipocalins with known structure against the ?-barrel to form a second hydrophobic core, we propose to introduce a new lipocalin sub-family named 'slim lipocalins', with YxeF and the other members of Pfam family PF11631 to which YxeF belongs constituting the first representatives. The results presented here exemplify the impact of structural genomics to enhance our understanding of biology and to generate new biological hypotheses.
| S-EPMC3367933 | biostudies-literature
Project description:The secreted Mycobacterium tuberculosis (Mtb) heme binding protein Rv0203 has been shown to play a role in Mtb heme uptake. In this work, we use spectroscopic (absorption, electron paramagnetic resonance, and magnetic circular dichrosim) methods to further characterize the heme coordination environments of His-tagged and native protein forms, Rv0203-His and Rv0203-notag, respectively. Rv0203-His binds the heme molecule through bis-His coordination and is low-spin in both ferric and ferrous oxidation states. Rv0203-notag is high-spin in both oxidation states and shares spectroscopic similarity with pentacoordinate oxygen-ligated heme proteins. Mutagenesis experiments determined that residues Tyr59, His63, and His89 are required for Rv0203-notag to efficiently bind heme, reinforcing the hypothesis based on our previous structural and mutagenesis studies of Rv0203-His. While Tyr59, His63, and His89 are required for the binding of heme to Rv0203-notag, comparison of the absorption spectra of the Rv0203-notag mutants suggests the heme ligand may be the hydroxyl group of Tyr59, although an exogenous hydroxide cannot be ruled out. Additionally, we measured the heme affinities of Rv0203-His and Rv0203-notag using stopped flow techniques. The rates for binding of heme to Rv0203-His and Rv0203-notag are similar, 115 and 133 ?M(-1) s(-1), respectively. However, the heme off rates differ quite dramatically, whereby Rv0203-His gives biphasic dissociation kinetics with fast and slow rates of 0.0019 and 0.0002 s(-1), respectively, and Rv0203-notag has a single off rate of 0.082 s(-1). The spectral and heme binding affinity differences between Rv0203-His and Rv0203-notag suggest that the His tag interferes with heme binding. Furthermore, these results imply that the His tag has the ability to stabilize heme binding as well as alter heme ligand coordination of Rv0203 by providing an unnatural histidine ligand. Moreover, the heme affinity of Rv0203-notag is comparable to that of other heme transport proteins, implying that Rv0203 may act as an extracellular heme transporter.
| S-EPMC3299815 | biostudies-literature
Project description:Hemoproteins are ubiquitous in biology and are commonly involved in critical processes such as electron transfer, oxidative phosphorylation, and signal transduction. Both the protein environment and the heme cofactor contribute to generate the range of chemical properties needed for these diverse functions. Using the heme nitric oxide/oxygen binding (H-NOX) protein from the thermophilic bacterium Thermoanaerobacter tengcongensis, we have shown that heme electronic properties can be modulated by porphyrin distortion within the same protein scaffold without changing the heme ligation state or heme environment. The degree of heme distortion was found to be directly correlated to the electron density at the heme iron, as evidenced by dramatic changes in the heme redox potential and pK(a) of the distal ligand ((-)OH vs H(2)O). Protein-induced porphyrin distortion represents a new strategy to rationally tune the electronic properties of protein-bound porphyrins and could be used to engineer proteins with desired reactivity or functionality.
| S-EPMC2939930 | biostudies-literature
Project description:BackgroundThe heme acquisition machinery in Streptococcus pyogenes is believed to consist of the surface proteins, Shr and Shp, and heme-specific ATP-binding cassette transporter HtsABC. Shp has been shown to rapidly transfer its heme to the lipoprotein component, HtsA, of HtsABC. The function of Shr and the heme source of Shp have not been established.ResultsThe objective of this study was to determine whether Shr binds heme and is a heme source of Shp. To achieve the objective, recombinant Shr protein was prepared. The purified Shr displays a spectrum typical of hemoproteins, indicating that Shr binds heme and acquires heme from Escherichia coli hemoproteins in vivo. Spectral analysis of Shr and Shp isolated from a mixture of Shr and heme-free Shp (apoShp) indicates that Shr and apoShp lost and gained heme, respectively; whereas Shr did not efficiently lose its heme in incubation with apoHtsA under the identical conditions. These results suggest that Shr directly transfers its heme to Shp. In addition, the rates of heme transfer from human hemoglobin to apoShp are close to those of simple ferric heme dissociation from hemoglobin, suggesting that methemoglobin does not directly transfer its heme to apoShp.ConclusionWe have demonstrated that recombinant Shr can acquire heme from E. coli hemoproteins in vivo and appears to directly transfer its heme to Shp and that Shp appears not to directly acquire heme from human methemoglobin. These results suggest the possibility that Shr is a source of heme for Shp and that the Shr-to-Shp heme transfer is a step of the heme acquisition process in S. pyogenes. Further characterization of the Shr/Shp/HtsA system would advance our understanding of the mechanism of heme acquisition in S. pyogenes.
| S-EPMC2266757 | biostudies-literature
Project description:The opportunistic pathogen Pseudomonas aeruginosa has evolved two outer membrane receptor-mediated uptake systems (encoded by the phu and has operons) by which it can utilize the hosts heme and hemeproteins as a source of iron. PhuS is a cytoplasmic heme binding protein encoded within the phu operon and has previously been shown to function in the trafficking of heme to the iron-regulated heme oxygenase (pa-HO). While the heme association rate for PhuS was similar to that of myoglobin, a markedly higher rate of heme dissociation (approximately 10(5) s(-1)) was observed, in keeping with a function in heme-trafficking. Additionally, the transfer of heme from PhuS to pa-HO was shown to be specific and unidirectional when compared to transfer to the non-iron regulated heme oxygenase (BphO), in which heme distribution between the two proteins merely reflects their relative intrinsic affinities for heme. Furthermore, the rate of transfer of heme from holo-PhuS to pa-HO of 0.11 +/- 0.01 s(-1) is 30-fold faster than that to apo-myoglobin, despite the significant higher binding affinity of apo-myoglobin for heme (kH = 1.3 x 10(-8) microM) than that of PhuS (0.2 microM). This data suggests that heme transfer to pa-HO is independent of heme affinity and is consistent with temperature dependence studies which indicate the reaction is driven by a negative entropic contribution, typical of an ordered transition state, and supports the notion that heme transfer from PhuS to pa-HO is mediated via a specific protein-protein interaction. In addition, pH studies, and reactions conducted in the presence of cyanide, suggest the involvement of spin transition during the heme transfer process, whereby the heme undergoes spin change from 6-c LS to 6-c HS either in PhuS or pa-HO. On the basis of the magnitudes of the activation parameters obtained in the presence of cyanide, whereby both complexes are maintained in a 6-c LS state, and the biphasic kinetics of heme transfer from holo-PhuS to pa-HO-wt, supports the notion that the spin-state crossover occur within holo-PhuS prior to the heme transfer step. Alternatively, the lack of the biphasic kinetic with pa-HO-G125V, 6-c LS, and with comparable rate of heme transfer as pa-HO is supportive of a mechanism in which the spin-change could occur within pa-HO. The present data suggests either or both of the two pathways proposed for heme transfer may occur under the present experimental conditions. The dissection of which pathway is physiologically relevant is the focus of ongoing studies.
| S-EPMC2631378 | biostudies-literature
Project description:Heme nitric oxide/oxygen (H-NOX)-binding proteins act as nitric oxide (NO) sensors among various bacterial species. In several cases, they act to mediate communal behavior such as biofilm formation, quorum sensing, and motility by influencing the activity of downstream signaling proteins such as histidine kinases (HisKa) in a NO-dependent manner. An H-NOX/HisKa regulatory circuit was recently identified in Vibrio cholerae, and the H-NOX protein has been spectroscopically characterized. However, the influence of the H-NOX protein on HisKa autophosphorylation has not been evaluated. This process may be important for persistence and pathogenicity in this organism. Here, we have expressed and purified the V. cholerae HisKa (HnoK) and H-NOX in its heme-bound (holo) and heme-free (apo) forms. Autophosphorylation assays of HnoK in the presence of H-NOX show that the holoprotein in the Fe(II)-NO and Fe(III) forms is a potent inhibitor of HnoK. Activity of the Fe(III) form and aerobic instability of the Fe(II) form suggested that Vibrio cholerae H-NOX may act as a sensor of the redox state as well as NO. Remarkably, the apoprotein also showed robust HnoK inhibition that was dependent on the oxidation of cysteine residues to form disulfide bonds at a highly conserved zinc site. The importance of cysteine in this process was confirmed by mutagenesis, which also showed that holo Fe(III), but not Fe(II)-NO, H-NOX relied heavily upon cysteine for activation. These results highlight a heme-independent mechanism for activation of V. cholerae H-NOX that implicates this protein as a dual redox/NO sensor.
| S-EPMC5016152 | biostudies-literature