Iron-binding Sites Research Articles

Biosynthesis of iron-chelating terramides A-C and their role in Aspergillus terreus infection

Fungal natural products from various species often feature hydroxamic acid motifs that have the ability to chelate iron. These compounds have an array of medicinally and ecologically relevant activities. Through genome mining, gene deletion in the host Aspergillus terreus, and heterologous expression experiments, this study has revealed that a nonribosomal peptide synthetase (NRPS) TamA and a specialized cytochrome P450 monooxygenase TamB catalyze the sequential biosynthetic reactions in the formation of terramides A-C, a series of diketopiperazines (DKPs) with hydroxamic acid motifs. Feeding experiments showed that TamB catalyzes an unprecedented di-hydroxylation of the amide nitrogens in the diketopiperazine core. This tailoring reaction led to the formation of two bidentate iron-binding sites per molecule with an unusual iron-binding stoichiometry. The structure of the terramide A-Fe complex was characterized by liquid chromatography-mass spectrometry (LC-MS), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy and electron paramagnetic resonance spectroscopy (EPR). Antimicrobial assays showed that the iron-binding motifs are crucial for the activity against bacteria and fungi. Murine infection experiments indicated that terramide production is crucial for the virulence of A. terreus and could be a potential antifungal drug target.

Escherichia coli alcohol dehydrogenase YahK is a protein that binds both iron and zinc.

Previous studies have highlighted the catalytic activity of Escherichia coli alcohol dehydrogenase YahK in the presence of coenzyme nicotinamide adenine dinucleotide (NAD) and metal zinc. Notably, competitive interaction between iron and zinc ligands has been shown to influence the catalytic efficiency of several key proteases. This study aims to unravel the intricate mechanisms underlying YahK's catalytic action, with a particular focus on the pivotal roles played by metal ions zinc and iron. The purified YahK protein from E. coli cells cultivated in LB medium was utilized to investigate its metal-binding properties through UV-visible absorption measurements and determination of metal content. Subsequently, the effects of excess zinc and iron on the metal-binding ability and alcohol dehydrogenase activity of the YahK protein were explored using M9 minimal medium. Furthermore, site-directed mutagenesis technology was employed to determine the iron-binding site location within the YahK protein. Polyacrylamide gel electrophoresis was conducted to examine the relationship between iron and zinc with respect to the YahK protein. The study confirmed the presence of iron and zinc in the YahK protein, with the zinc-bound form exhibiting enhanced catalytic activity in alcohol dehydrogenation reactions. Conversely, the presence of iron appears to play a pivotal role in maintaining overall stability of the YahK protein. Furthermore, experimental findings indicate that excessive zinc within M9 minimal medium can competitively bind to iron-binding sites on YahK, thereby augmenting its alcohol dehydrogenase activity. The dynamic binding of YahK to iron and zinc unveils its intricate regulatory mechanism as an alcohol dehydrogenase, thereby highlighting the possible physiological role of YahK in E. coli and its significance in governing cellular metabolic processes. This discovery provides a novel perspective for further investigating the specific impact of metal ion binding on YahK and E. coli cell metabolism.

Hemoglobin Hydrolyzate Promotes Iron Absorption in the Small Intestine through Iron Binding Peptides.

Hemoglobin is an excellent source of iron supplements, and its hydrolyzate spontaneously binds iron during digestion and promotes iron absorption in vivo. However, the underlying mechanisms of what peptides bind and how they bind iron ions remain unclear. This study prepared the porcine hemoglobin hydrolyzate through enzymatic hydrolysis and acid treatment and investigated the mechanisms of hemoglobin hydrolyzate on iron absorption through the determination of iron levels in dietary intervention mice, iron binding site analyses, peptide digestion analyses, molecular simulation docking, and INT407 cell validation. The results showed that ingestion of the hemoglobin hydrolyzate diets increased iron levels in the blood of mice, accompanied by the upregulation of duodenal iron circulation-related genes such as ferritin, PCBP1, and HP. Carboxyl, imidazole groups, and aromatic amino acid residues were iron binding sites of hemoglobin hydrolyzate during digestion. VDEVGGEA and VDEVGGE were found to involve the spontaneous and efficient binding of hemoglobin hydrolyzate to iron ions in the intestinal cavity. In particular, the DEVGGE peptide was the typical sequence for hemoglobin hydrolytic peptides to exert iron binding activity.

In Situ Magnetometry of Iron in Human Dopaminergic Neurons Using Superresolution MRI and Ion-Beam Microscopy

Paramagnetic transition metals play a crucial role as cofactors in various cellular catalytic processes. However, their high concentrations in reactive oxidation states can induce oxidative stress, resulting in cell dysfunction or death. Hence, it is vital to have methods to monitor metal concentrations and paramagnetic properties in cells for medicine and cell biology. Here we present a novel multimodal method for in-cell magnetometry enabling direct measurement of metal magnetic properties within individual cells in tissue, without prior isolation and at room temperature. Individual cell magnetic moments are measured using superresolution magnetic resonance imaging (MRI) microscopy at 9.4 T by detecting microscopic magnetic-field perturbations around the cells. The cellular metal content is quantified using ion-beam microscopy or synchrotron micro-x-ray fluorescence for the same cells. The metal magnetic susceptibility at 9.4 T is then obtained from the slope of the cell magnetic moments’ dependence on cell metal content. To estimate the susceptibility at lower fields, multifield MR relaxometry and biophysical modeling are employed, extrapolating the 9.4-T susceptibility values to fields as low as 3 T. We apply the new method to determine the susceptibility of iron accumulated in human dopaminergic neurons inside neuromelanin, the by-product of dopamine synthesis. The susceptibility of iron in neuromelanin is measured to be χρ=(2.98±0.19)×10−6 m3/kg providing unique insights into the biochemistry of iron inside dopaminergic neurons. The obtained value reveals a predominant monoatomic low-affinity iron-binding site within neuromelanin, indicating a higher neurotoxicity of iron than previously suggested. Furthermore, the measured susceptibility value establishes a quantitative relationship between cellular iron concentration and iron-sensitive MRI parameters, which can be noninvasively measured . This breakthrough paves the way for the detection of dopaminergic neuron density and iron load, requiring a standard clinical MRI scanner only. It promises to facilitate early diagnosis of Parkinson’s disease. In conclusion, our presented novel method enables the direct measurements of magnetic properties of paramagnetic metals within single cells with high sensitivity and across large cell groups within a macroscopic volume, providing invaluable information about the cellular biology of metals. Published by the American Physical Society 2024

The binding pattern of ferric iron and iron-binding protein in Botrytis cinerea

Iron-binding protein (Ibp) has protective effect on pathogen exposed to H2O2 in defense response of plants. Ibp in Botrytis cinerea (BcIbp) is related to its virulence. Bcibp mutation lead to virulence deficiencies in B. cinerea. BcIbp is involved in the Fe3+ homeostasis regulation. Recognition the binding site and binding pattern of ferric iron and iron-binding protein in B. cinerea are vital to understand its function. In this study, molecular dynamics (MD) simulations, gaussian accelerated molecular dynamics (GaMD) simulations, dynamic cross correlation analysis and quantum chemical energy calculation were used to explore binding pattern of ferric iron. MD results showed that the C-terminal region had little effect on the stability of residues in the Fe3+-binding pocket. Energy calculations suggested the most likely coordination pattern for ferric iron in iron-binding protein. These results will help to understand the binding of ferric iron to iron-binding protein and provide new ideas for regulating the virulence of B. cinerea.

Alternative splicing generates a novel ferroportin isoform with a shorter C-terminal and an intact iron- and hepcidin-binding property.

Ferroportin (FPN) is a transmembrane protein and is the only known iron exporter that helps in maintaining iron homeostasis in vertebrates. To maintain stable iron equilibrium in the body, ferroportin works in conjunction with a peptide called hepcidin. In this study, we have identified an alternatively spliced novel isoform of the human SLC40A1 gene, which encodes for the FPN protein and is found to be expressed in different tissues. The novel transcript has an alternate last exon and encodes 31-amino acid long peptide sequence that replaces 104 amino acids at C-terminal in the novel transcript. Molecular modelling and molecular dynamics (MD) simulation studies revealed key structural features of the novel isoform (FPN-N). FPN-N was predicted to have 12 transmembrane domains similar to the reported isoform (FPN), despite being much smaller in size. FPN-N was found to interact with hepcidin, a key regulator of ferroportin activity. Also, the iron-binding sites were retained in the novel isoform as revealed by the MD simulation of FPN-N in bilipid membrane. The novel isoform identified in this study may play important role in iron homeostasis. However, further studies are required to characterize the FPN-N isoform and decipher its role inside the cell.

Molecular Dynamics Simulation of an Iron(III) Binding Site on the Fc Domain of IgG1 Relevant for Visible Light-Induced Protein Fragmentation.

Molecular dynamics simulations were employed to investigate the interaction between Fe(III) and an iron-binding site composed of THR259, ASP252, and GLU261 on the Fc domain of an IgG1. The goal was to provide microscopic mechanistic information for the photochemical, iron-dependent site-specific oxidative fragmentation of IgG1 at THR259 reported in our previous paper. The distance between Fe(III) and residues of interest as well as the binding pocket size was examined for both protonated and deprotonated THR259. The Fe(III) binding free energy (ΔG) was estimated by using an umbrella sampling approach. The pKa shift of the THR259 hydroxyl group caused by the presence of nearby Fe(III) was estimated based on a thermodynamic cycle. The simulation results show that Fe(III) resides inside the proposed binding pocket and profoundly changes the pocket configuration. The ΔG values indicate that the pocket possesses a strong binding affinity for Fe(III). Furthermore, Fe(III) profoundly lowers the pKa value of the THR259 hydroxyl group by 5.4 pKa units.

Unveiling labile iron speciation in aquatic systems: Depth binding profiles in chelex-based resin hydrogels using diffusive gradients in thin films - Beam deflection spectrometry

The paper presents the characterization of the Chelex-based resin hydrogels used as a passive sampling technique of diffusive gradients in thin film (DGT) to determine the binding profiles of iron in aquatic environments. The non-destructive photothermal beam deflection spectrometry is employed to carry out the analysis. Under non-advective conditions, gels’ capacity and optimal experimental parameters for depth profiling have been established. Depth profiles reveal a layer-by-layer interaction between dissolved iron and resin binding sites. The obtained accuracy of the measurements when applied for iron determination in natural waters is obtained as 100 ± 5 % of the nominal concentration, whereas the precision under 5 % demonstrates good repeatability and reproducibility of the measurements.

Chelating peptides from rapeseed meal protein hydrolysates: identification and evaluation of their capacity to inhibit lipid oxidation.

An optimized proteolysis process was applied to rapeseed meal proteins (RP) and the hydrolysate was separated by membrane filtration allowing the production of highly metal-chelating peptides in the permeate. In order to identify the chemical structure of the most active obtained metal-chelating peptides, immobilized metal affinity chromatography (IMAC) was applied. The RP-IMAC peptide fraction was mainly composed of small peptides from 2 to 20 amino acids. Using the Ferrozine assay, RP-IMAC peptides showed a significant chelating efficiency higher than sodium citrate and close to that of EDTA. The peptide sequences were identified by UHPLC-MS and several possible iron binding sites were found. β-carotene oxidation assay and lipid oxidation in bulk oils or emulsion were carried out to evaluate the potential of such peptides as efficient antioxidants to protect lipids from oxidation. While chelating peptides showed a limited efficiency in bulk oil, they performed more efficiently in emulsion.

Conformational multiplicity of bacterial ferric binding protein revealed by small angle x-ray scattering and molecular dynamics calculations.

This study combines molecular dynamics (MD) simulations with small angle x-ray scattering (SAXS) measurements to investigate the range of conformations that can be adopted by a pH/ionic strength (IS) sensitive protein and to quantify its distinct populations in solution. To explore how the conformational distribution of proteins may be modified in the environmental niches of biological media, we focus on the periplasmic ferric binding protein A (FbpA) from Haemophilus influenzae involved in the mechanism by which bacteria capture iron from higher organisms. We examine iron-binding/release mechanisms of FbpA in varying conditions simulating its biological environment. While we show that these changes fall within the detectable range for SAXS as evidenced by differences observed in the theoretical scattering patterns calculated from the crystal structure models of apo and holo forms, detection of conformational changes due to the point mutation D52A and changes in ionic strength (IS) from SAXS scattering profiles have been challenging. Here, to reach conclusions, statistical analyses with SAXS profiles and results from different techniques were combined in a complementary fashion. The SAXS data complemented by size exclusion chromatography point to multiple and/or alternative conformations at physiological IS, whereas they are well-explained by single crystallographic structures in low IS buffers. By fitting the SAXS data with unique conformations sampled by a series of MD simulations under conditions mimicking the buffers, we quantify the populations of the occupied substates. We also find that the D52A mutant that we predicted by coarse-grained computational modeling to allosterically control the iron binding site in FbpA, responds to the environmental changes in our experiments with conformational selection scenarios that differ from those of the wild type.

Increased ionic strength triggers multiple conformations in both apo and holo forms of bacterial ferric binding protein.

This study combines molecular dynamics (MD) simulations with solution X-ray scattering measurements to investigate the range of conformations that can be achieved by a pH/ionic strength (IS) sensitive protein and to quantify its distinct populations. To explore how the conformational multiplicity of proteins might be modified in the environmental niches of biological media, we focus on the periplasmic protein FbpA from H. influenzae which is a part of the mechanism developed by bacteria to capture iron from higher organisms. We examine iron-binding/release mechanisms of FbpA in varying conditions simulating its unique biological environment. The collected SAXS data complemented by SEC analyses and binding assays point to multiple conformations at physiological IS while they are well-explained by single x-ray structures in a 15 mM buffer. Moreover, by fitting the SAXS data with unique conformations sampled by a series of MD simulations carried out under conditions mimicking the buffers, we quantify with high accuracy the populations of the occupied substates. Furthermore, we find the D52A mutant that we predicted by coarse-grained computational approaches to allosterically control the iron binding site in FbpA responds to the environmental changes in our experiments with varied conformational selection scenarios. We show an application of the environment dependence of the conformers by designing a genetically encoded iron biosensor as a chimera of FbpA and green fluorescent protein using the D52 region as the insertion point. This work exemplifies how unifying a range of experimental and computational techniques provides platforms for achieving the next generation of applications that put protein dynamics rather than the static structure at the centerstage. Support by Turkish Atomic Energy Authority, TUBITAK grant no 121Z329 and a scholarship to GL through 2214-A program. Measurements conducted at EMBL-Hamburg.

The Antimicrobial Bifunctional Camel Lactoferrin: In Silico and Molecular Dynamic Perspective.

Lactoferrin (LF) is a major natural antimicrobial agent secreted in body fluids as a natural innate immunity protein. The action and structure of LF are closely related to its iron-binding capacity with structural reporting in open and closed conformations. This study looked at how lactoferrin structures change in camel (cLF), bovine (bLF), and human (hLF) lactoferrin closed forms after iron is removed from their binding sites. Initially, the sequence comparison between cLF and the LFs of marine mammals, bats, and domestic animals was the most intriguing conclusion. Camel LF is revealed to be more closely related to marine animals (~80.36% identity) and bats (~79.3% identity) than to terrestrial mammal species (~75.5% identity). Results indicated that cLF was more dynamic in nature than bLF and hLF by showing higher RMSD values. The cLF is known to be half lactoferrin half transferrin; in this study, we show that there are different MD behavior of both iron-binding sites. While LF contains two lobes (C- and N-lobes), the C-lobe showed high fluctuations as N-lobe was more stable in the absence of ferric ions. The C-lobe and N-lobe of cLF react differently at physiological pH, revealing distinct molecular interactions between these components. In addition, cLF showed higher system flexibility derived from its larger RMSD, RMSF, lower intermolecular hydrogen bonds, and higher solvent accessible surface area (SASA).

Contact Lens Wear Induces Alterations of Lactoferrin Functionality in Human Tears.

The tear film is a complex matrix composed of several molecular classes, from small metal ions to macromolecules. Contact lens (CL) wear can affect the protein homeostasis of the tear film, by accumulating deposits on the CL surface and/or altering their structural and functional properties. This work investigates the effect of CL wear on lactoferrin (Lf), one of the most abundant tear proteins, known as an unspecific biomarker of inflammation. Tears from eight volunteers were collected and analyzed after alternated periods of CL wear and without CL. The experimental approach is to probe Lf into unprocessed human tears by the peculiar fluorescence emission originating from complex formation of Lf with terbium (Tb3+) at the iron-binding sites. The experimental data indicate that CL wear does not significantly affect the total amount of Lf. On the other hand, Lf affinity for Tb3+ is reduced upon CL wear, suggesting relevant changes in Lf structure and possible alterations of protein functionality. Future studies based on this approach will help define CL features (material, lens-care solution, wearing time, etc.) with minimal effects on tear protein activity, in order to obtain more biocompatible and comfortable devices.

Absorption of iron from Tegillarca granosa using an in vitro simulated digestion and Caco-2/HepG2 co-culture system.

Iron-deficiency anemia is one severe micronutrient malnutrition and has captured worldwide attention. This study evaluated the in vitro iron absorption of two iron-binding proteins (hemoglobin and ferritin) from Tegillarca granosa. In addition, the protein structure-iron absorption relationship and the regulatory effect of hepcidin on cellular iron absorption were explored. Our findings revealed that both hemoglobin and ferritin extracted from T.granosa contained abundant iron-binding sites, as evidenced by stronger peaks in amide I and II regions compared with the two proteins from humans. Less β-sheet (27.67%) structures were found in hemoglobin compared with ferritin (36.40%), probably contributing to its greater digestibility and more release of available iron. This was confirmed by the results of Caco-2/HepG2 cell culture system that showed iron absorption of hemoglobin was 26.10-39.31% higher than that of ferritin with an iron content of 50-150 μmol L-1 . This high iron absorption of hemoglobin (117.86-174.10ng mg-1 ) could also be due to more hepcidin produced by HepG2 cells, thereby preventing ferroportin-mediated iron efflux from Caco-2 cells. In addition, the possible risk of oxidative stress was evaluated in cells post-iron exposure. In comparison with ferrous sulfate, a common iron supplement, Caco-2 cells treated with the iron-binding proteins had a 9.50-25.73% lower level of intracellular reactive oxygen species, indicating the safety of hemoglobin and ferritin. Collectively, the data of this research would be helpful for understanding the key features and potential of developing hemoglobin and ferritin from T.granosa as novel iron supplements. © 2022 Society of Chemical Industry.

A hypothesis concerning the role of PPAR family on cardiac energetics in Adriamycin-induced cardiomyopathy.

Adriamycin is an effective anti-neoplastic drug against a variety of cancer types. However, the drug causes adverse side effects in a number of organ systems. Cardiomyopathy is one of the life-threatening side effects of Adriamycin. In the current work, we have derived a hypothesis with possible involvement of PPAR family members in the development of Adriamycin-induced cardiomyopathy. Dysregulation of PPAR family by Adriamycin causes impairment in the transport and β-oxidation of fatty acids, the key substrate for ATP synthesis in heart. Evidences suggest that dysregulation of PPAR family alters the recruitment of glucose transporters. Furthermore, heme oxygenase-1 is a crucial enzyme regulating the iron homeostasis in the heart whose expression is regulated by PPAR family. Inverse relationship exists between the expression levels of PPARγ and heme oxygenase-1. Adriamycin upregulates the expression of heme oxygenase-1 which in turn disrupts the iron homeostasis in cardiomyocytes. Our molecular docking results show that Adriamycin has a high affinity for iron-binding sites of heme oxygenase-1, thereby hindering formation of iron-sulfur complex. The lack of iron-sulfur complex impairs the electron transport chain. In addition, succinate dehydrogenase subunit A is downregulated by Adriamycin. The lack of this subunit uncouples Krebs cycle from ETC. Further, lack of this subunit increases the concentration of succinate, which further alters the mitochondrial membrane potential. Overall, in the present work, we hypothesize that alteration in the expression of PPAR family members is one of the major causes of metabolic chaos and oxidative stress caused by Adriamycin during the development of cardiomyopathy.

Dynamics of Human Serum Transferrin in Varying Physicochemical Conditions Explored by Using Molecular Dynamics Simulations.

Conformational stability of human serum transferrin (Tf) at varying pH values and salt and excipient concentrations were investigated using molecular dynamics (MD) simulations, and the results are compared with previously published small-angle X-ray scattering (SAXS) experiments. SAXS study showed that at pH 5, Tf is predominantly present in a partially open (PO) form, and the factions of PO differ based on the physicochemical condition and drift toward the closed form (HO) as the pH increases. Tf is a bilobal glycoprotein that is composed of homologous halves termed the N- and C-lobes. The current study shows that the protonation of Y188 and K206 at pH 5 is the primary conformational drive into PO, which shifts toward the closed (HO) conformer as the pH increases. Furthermore, at pH 6.5, PO is unfavorable due to negative charge-charge repulsion at the N/C-lobe interface linker region causing increased hinge distance when compared to HO, which has favorable attractive electrostatic interactions in this region. Subsequently, the effect of salt concentration was studied at 70 and 140 mM NaCl. At 70 mM NaCl and pH 5, chloride ions bind strongly in the N-lobe iron-binding site, whereas these interactions are weak at pH 6.5. With increasing salt concentration at pH 5, the regions surrounding the N-lobe iron-binding site are saturated, and as a consequence, sodium and chloride ions accumulate into the bulk. Additionally, protein-excipient interactions were investigated. At pH 5, the excipients interact in similar loop regions, E89-T93, and D416-D420, located in the N- and C-lobes of the HO conformer, respectively. It is anticipated that interactions of additives in these two loop regions cause conformational changes that lead to iron-coordinating residues in the N-lobe to drift away from iron and thus drive HO to PO conversion. Furthermore, at pH 6.5 and 140 mM histidine, these interactions are negligible leading to the stabilization of HO.

Structure and Mechanism of Mammalian Stearoyl‐CoA Desaturase‐1 and its Redox Partners

Mammalian stearoyl‐CoA desaturase‐1 (SCD1) is a membrane‐embedded diiron enzyme that introduces the first double‐bond to a saturated long‐chain fatty acid (in the form of an acyl‐CoA). Structures of mouse and human SCD1 were reported previously, however, these studies found that the heterologously expressed mammalian SCD1 contains two zinc ions (Zn2+), likely mis‐incorporated during the expression, and that the enzyme is inactive. We developed a protocol to enhance iron incorporation and obtained fully functional SCD1. We also solved the structure of mouse SCD1 with a diiron center. The diiron center in SCD1 has an Fe‐Fe distance of 6.4 Å and are coordinated entirely by histidine residues. This configuration is different from all previously characterized diiron centers, in which carboxylate/oxygen groups bridge two irons and the Fe‐Fe distance is usually less than 4 Å. The longer distance and different coordination of the diiron center in SCD1 prompted us to further examine its redox chemistry.The redox cycle of the diiron center in SCD1 requires reducing equivalents from NADH and the electrons are transferred via cytochrome b5 reductase (b5R) and cytochrome b5 (cyt b5), both of which have a single transmembrane (TM) anchor. Although proximity of these proteins is expected, it is not clear how b5R, cyt b5and SCD1 could interact on membranes. We showed that b5R, cyt b5 and SCD1 form a ternary complex in cells, and that the complex is stabilized mainly by interactions of the TM domains. We also showed that b5R and cyt b5 form stable binary complex, and so are cyt b5 and SCD1. These results will enable further structural and functional analyses to understand the mechanism of electron transfer in SCD1, and since b5R and cyt b5are partners to several other redox enzymes, our discoveries of stable binary and ternary complex may also be applicable in these enzymes as well.We assembled purified b5R, cyt b5 and SCD1 proteins in a test tube and enabled in‐vitro study of SCD1’s activity. We found that SCD1 loses activity within ten turnovers. This deactivation is due to the loss of a single iron ion in the diiron center. Addition of ferrous ions in the reaction mixture prevents such deactivation. These results suggest that structural changes during the enzymatic cycle is significant enough to weaken the coordination of one of the iron ions. We then measured electron paramagnetic resonance (EPR) spectra of SCD1, and found strong signals around g = 4 – 6 from high spin iron(s), and a broad symmetric feature centered at g = 2, which may be due to exchange‐ or dipolar‐coupled ferric irons. The broad feature at g = 2 disappeared in SCD1 mutants that disrupt one of the iron binding sites, and in the wild‐type SCD1 devoid of an iron after reaction cycles. Although further experiments are required to interpret and understand the EPR spectra, these results helped identify the iron prone to dissociation during the reaction cycle. In summary, results from this study enhanced our knowledge of the diiron center in SCD1, and established an avenue of research for understanding the redox chemistry of the novel diiron center in SCD1.

The Conformation of the N-Terminal Tails of Deinococcus grandis Dps Is Modulated by the Ionic Strength

DNA-binding proteins from starved cells (Dps) are homododecameric nanocages, with N- and C-terminal tail extensions of variable length and amino acid composition. They accumulate iron in the form of a ferrihydrite mineral core and are capable of binding to and compacting DNA, forming low- and high-order condensates. This dual activity is designed to protect DNA from oxidative stress, resulting from Fenton chemistry or radiation exposure. In most Dps proteins, the DNA-binding properties stem from the N-terminal tail extensions. We explored the structural characteristics of a Dps from Deinococcus grandis that exhibits an atypically long N-terminal tail composed of 52 residues and probed the impact of the ionic strength on protein conformation using size exclusion chromatography, dynamic light scattering, synchrotron radiation circular dichroism and small-angle X-ray scattering. A novel high-spin ferrous iron-binding site was identified in the N-terminal tails, using Mössbauer spectroscopy. Our data reveals that the N-terminal tails are structurally dynamic and alter between compact and extended conformations, depending on the ionic strength of the buffer. This prompts the search for other physiologically relevant modulators of tail conformation and hints that the DNA-binding properties of Dps proteins may be affected by external factors.

Functional disruption of transferrin expression alters reproductive physiology in Anopheles culicifacies.

BackgroundIron metabolism is crucial to maintain optimal physiological homeostasis of every organism and any alteration of the iron concentration (i.e. deficit or excess) can have adverse consequences. Transferrins are glycoproteins that play important role in iron transportation and have been widely characterized in vertebrates and insects, but poorly studied in blood-feeding mosquitoes.ResultsWe characterized a 2102 bp long transcript AcTrf1a with complete CDS of 1872bp, and 226bp UTR region, encoding putative transferrin homolog protein from mosquito An. culicifacies. A detailed in silico analysis predicts AcTrf1a encodes 624 amino acid (aa) long polypeptide that carries transferrin domain. AcTrf1a also showed a putative N-linked glycosylation site, a characteristic feature of most of the mammalian transferrins and certain non-blood feeding insects. Structure modelling prediction confirms the presence of an iron-binding site at the N-terminal lobe of the transferrin. Our spatial and temporal expression analysis under altered pathophysiological conditions showed that AcTrf1a is abundantly expressed in the fat-body, ovary, and its response is significantly altered (enhanced) after blood meal uptake, and exogenous bacterial challenge. Additionally, non-heme iron supplementation of FeCl3 at 1 mM concentration not only augmented the AcTrf1a transcript expression in fat-body but also enhanced the reproductive fecundity of gravid adult female mosquitoes. RNAi-mediated knockdown of AcTrf1a causes a significant reduction in fecundity, confirming the important role of transferrin in oocyte maturation.ConclusionAll together our results advocate that detailed characterization of newly identified AcTrf1a transcript may help to select it as a unique target to impair the mosquito reproductive outcome.

Verticillium dahliae CFEM proteins manipulate host immunity and differentially contribute to virulence

BackgroundVerticillium dahliae is a fungal pathogen that causes a vascular wilt on many economically important crops. Common fungal extracellular membrane (CFEM) domain proteins including secreted types have been implicated in virulence, but their roles in this pathogen are still unknown.ResultsNine secreted small cysteine-rich proteins (VdSCPs) with CFEM domains were identified by bioinformatic analyses and their differential suppression of host immune responses were evaluated. Two of these proteins, VdSCP76 and VdSCP77, localized to the plant plasma membrane owing to their signal peptides and mediated broad-spectrum suppression of all immune responses induced by typical effectors. Deletion of either VdSCP76 or VdSCP77 significantly reduced the virulence of V. dahliae on cotton. Furthermore, VdSCP76 and VdSCP77 suppressed host immunity through the potential iron binding site conserved in CFEM family members, characterized by an aspartic acid residue in seven VdSCPs (Asp-type) in contrast with an asparagine residue (Asn-type) in VdSCP76 and VdSCP77. V. dahliae isolates carrying the Asn-type CFEM members were more virulent on cotton than those carrying the Asp-type.ConclusionsIn the iron-insufficient xylem, V. dahliae is likely to employ the Asp-type CFEM members to chelate iron, and Asn-type CFEM members to suppress immunity, for successful colonization and propagation in host plants.