A thiopyridine-bound mirror-image copper center in an artificial non-heme metalloenzyme.

Artificial metalloenzymes, in which a metal complex and protein matrix are combined, have been synthesized to catalyze stereoselective reactions using the chiral environment provided by the protein cavity. Artificial metalloenzymes can be engineered by the chemical modification and mutagenesis of the protein matrix. We developed artificial non-heme metalloenzymes using a cupin superfamily protein (TM1459) with a 4-His tetrad-metal-binding motif. The Cu-bound H52A/C106D mutant with 3-His triad showed a S-enantioselective Michael addition of nitromethane to α,ß-unsaturated ketone, 2-aza-chalcone 1. In this study, we demonstrated a chemical modification near the copper-binding site of this mutant to reverse its enantioselectivity. For chemical modification, the amino acid on the Si-face of the binding state of 1 to the copper center was replaced with Cys, followed by reaction with 4,4'-dithiopyridine (4-PDS) to form S-(pyridin-4-ylthio)cysteine (Cys-4py). Cu-bound I49C-4py/H52A/C106D showed reversal of the enantioselectivity from S-form to R-form (ee = 71%, (R)). The effect of steric hindrance of the amino acids at position 49 on enantioselectivity was investigated using I49X/H52A/C106D mutants (X = A, C, I, F, and W). Additionally, chemical modification with 2,2'-dithiopyridine (2-PDS) produced I49-2py/H52A/C106D, which showed lower R-enantioselectivity than I49-4py/H52A/C106D. Among the mutants, the 4py-modification on the Si-face was the most effective in reversing the enantioselectivity. By tuning the Re-face side, the H54A mutation introduced into the I49C-4py/H52A/C106D increased the R-enantioselectivity (ee = 88%, (R)). X-ray crystallography revealed a coordinated structure with ligation of thiopyridine in Cu-bound I49C-4py/H52A/H54A/C106D.

Copper-binding protein modelling by single-cell transcriptome and Bulk transcriptome to predict overall survival in lung adenocarcinoma patients.

Background: Copper and copper-binding proteins are key components of tumour progression as they play an important role in tumour invasion and migration, and abnormal accumulation of copper (Cu) may be intimately linked to with lung adenocarcinoma (LUAD). Methods: Data on lung adenocarcinoma were sourced from the Cancer Genome Atlas (TCGA) database and the National Centre for Biotechnology Information (GEO). 10x scRNA sequencing, which is from Bischoff P et al, was used for down-sequencing clustering and subgroup identification using TSNE. The genes for Copper-binding proteins (CBP) were acquired from the MSigDB database. LASSO-Cox analysis was subsequently used to construct a model for copper-binding proteins (CBPRS), which was then compared to lung adenocarcinoma models developed by others. External validation was carried out in the GSE31210 and GSE50081 cohorts. The effectiveness of immunotherapy was evaluated using the TIDE algorithm and the IMvigor210, GSE78220, and TCIA cohorts. Furthermore, differences in mutational profiles and the immune microenvironment between different risk groups were investigated. The CBPRS's key regulatory genes were screened using ROC diagnostic and KM survival curves. The differential expression of these genes was then verified by RT-qPCR. Results: The six CBP genes were identified as highly predictive of LUAD prognosis and significantly correlated with it. Multivariate analysis showed that patients in the low-risk group had a higher overall survival rate than those in the high-risk group, indicating that the model was an independent predictor of LUAD. The CBPRS demonstrated superior predictive ability compared to 11 previously published models. We constructed a column-line graph that includes CBPRS and clinical characteristics, which exhibits high predictive performance. Additionally, we observed significant differences in biological functions, mutational landscapes, and immune cell infiltration in the tumour microenvironment between the high-risk and low-risk groups. It is noteworthy that immunotherapy was also significant in both the high- and low-risk groups. These results suggest that the model has good predictive efficacy. Conclusions: The CBP model demonstrated good predictive performance, revealing characteristics of the tumour microenvironment. This provides a new method for assessing the efficacy of pre-immunisation and offers a potential strategy for future treatment of lung adenocarcinoma.

Crystallization of Ethylene Plant Hormone Receptor-Screening for Structure.

The plant hormone ethylene is a key regulator of plant growth, development, and stress adaptation. Many ethylene-related responses, such as abscission, seed germination, or ripening, are of great importance to global agriculture. Ethylene perception and response are mediated by a family of integral membrane receptors (ETRs), which form dimers and higher-order oligomers in their functional state as determined by the binding of Cu(I), a cofactor to their transmembrane helices in the ER-Golgi endomembrane system. The molecular structure and signaling mechanism of the membrane-integral sensor domain are still unknown. In this article, we report on the crystallization of transmembrane (TM) and membrane-adjacent domains of plant ethylene receptors by Lipidic Cubic Phase (LCP) technology using vapor diffusion in meso crystallization. The TM domain of ethylene receptors ETR1 and ETR2, which is expressed in E. coli in high quantities and purity, was successfully crystallized using the LCP approach with different lipids, lipid mixtures, and additives. From our extensive screening of 9216 conditions, crystals were obtained from identical crystallization conditions for ETR1 (aa 1-316) and ETR2 (aa 1-186), diffracting at a medium-high resolution of 2-4 Å. However, data quality was poor and not sufficient for data processing or further structure determination due to rotational blur and high mosaicity. Metal ion loading and inhibitory peptides were explored to improve crystallization. The addition of Zn(II) increased the number of well-formed crystals, while the addition of ripening inhibitory peptide NIP improved crystal morphology. However, despite these improvements, further optimization of crystallization conditions is needed to obtain well-diffracting, highly-ordered crystals for high-resolution structural determination. Overcoming these challenges will represent a major breakthrough in structurally determining plant ethylene receptors and promote an understanding of the molecular mechanisms of ethylene signaling.

Stabilization of a Cu-binding site by a highly conserved tryptophan residue.

Copper serves as an essential cofactor for nearly all living organisms. There are still many gaps remaining in our knowledge of how Gram-positive bacteria import copper and maintain homeostasis. To obtain a better understanding of how these processes work, here we focus on the ycnKJI operon responsible for regulating copper levels in the Gram-positive bacterium Bacillus subtilis. This operon encodes three Cu-related proteins: a copper-dependent transcriptional repressor (YcnK), a putative copper importer (YcnJ), and a copper-binding protein of unknown function (YcnI). We previously found that YcnI's extracellular Domain of Unknown Function 1775 (DUF1775) houses a monohistidine brace motif that coordinates a single Cu(II) ion. The Cu(II) binding site includes a highly conserved tryptophan residue. Here, we investigate the role of that tryptophan and the ability of the protein to interact with other oxidation states of Cu. We find that YcnI exhibits strong preference for binding Cu in the oxidized Cu(II) state, and that the conserved tryptophan residue is not essential for the interaction. We determine the structure of a tryptophan variant to 1.95 Å resolution that indicates that the tryptophan is needed to stabilize the metal binding interaction, and find that this variant has weaker affinity for Cu(II) than the wild-type protein. Together, these data provide further insights into the DUF1775 domain family and reveal the role of the conserved tryptophan residue.

Evaluation of Copper(II) Transfer between Amyloid-beta Peptides by Relaxation-Induced Dipolar Modulation Enhancement (RIDME).

In the brains of Alzheimer's disease patients, fibrillar aggregates containing amyloid-beta (Aß) peptides are found, along with elevated concentrations of Cu(II) ions. The aggregation pathways of Aß peptides can be modulated by Cu(II) ions and is determined by the formation and nature of the Cu(II)-Aß complex. If spin-labeled, the Cu(II)-Aß complex contains two dipolar coupled paramagnetic centers, the spin label and the Cu(II) ion. Measurement of the dipolar coupling between these paramagnetic centers by relaxation-induced dipolar modulation enhancement (RIDME) allows to monitor the complex formation and thus opens a way to follow the Cu(II) transfer between peptides if a mixture of wild-type and spin-labeled ones is used. We evaluate this approach for a specific Cu(II)-Aß complex, the aggregation-inert Component II. The kinetics of the Cu(II) transfer can be resolved by performing RIDME in a time-dependent manner. A temporal resolution of seconds has been achieved, with the potential to reach milliseconds, using a rapid-freeze quench device to stop the Cu(II) transfer in solution after defined incubation times.

Research Mechanism and Progress of the Natural Compound Curcumin in Treating Alzheimer´s Disease.

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases. AD patients usually present symptoms, such as cognitive dysfunction, progressive memory loss, and other manifestations. With the increasing number of AD cases worldwide, there is an urgent need to develop effective drug treatments. Currently, drugs targeting AD symptoms may not change or prevent the progression of the disease. Curcumin, a polyphenol extracted from the turmeric herb, has been used for the treatment of AD. In this review, we summarized both cellular and animal studies and described the mechanism of action of curcumin in altering the pathological features of AD. Curcumin attenuates the formation of amyloid-ß plaques and promotes its decomposition, reduces the phosphorylation of tau, improves its clearance rate, and binds with copper to reduce cholesterol. It changes the activity of microglia, suppresses acetylcholinesterase, regulates insulin signal transduction, and exhibits antioxidant properties. Studies have found that curcumin can promote nerve repair and has a significant effect on AD. However, the low bioavailability of curcumin may hinder its use as a therapeutic agent. If this limitation can be overcome, curcumin may emerge as a promising drug for the treatment of AD.

Ascorbate Peroxidase 2 (APX2) of Chlamydomonas Binds Copper and Modulates the Copper Insertion into Plastocyanin.

Recent phylogenetic studies have unveiled a novel class of ascorbate peroxidases called "ascorbate peroxidase-related" (APX-R). These enzymes, found in green photosynthetic eukaryotes, lack the amino acids necessary for ascorbate binding. This study focuses on the sole APX-R from Chlamydomonas reinhardtii referred to as ascorbate peroxidase 2 (APX2). We used immunoblotting to locate APX2 within the chloroplasts and in silico analysis to identify key structural motifs, such as the twin-arginine transport (TAT) motif for lumen translocation and the metal-binding MxxM motif. We also successfully expressed recombinant APX2 in Escherichia coli. Our in vitro results showed that the peroxidase activity of APX2 was detected with guaiacol but not with ascorbate as an electron donor. Furthermore, APX2 can bind both copper and heme, as evidenced by spectroscopic, and fluorescence experiments. These findings suggest a potential interaction between APX2 and plastocyanin, the primary copper-containing enzyme within the thylakoid lumen of the chloroplasts. Predictions from structural models and evidence from 1H-NMR experiments suggest a potential interaction between APX2 and plastocyanin, emphasizing the influence of APX2 on the copper-binding abilities of plastocyanin. In summary, our results propose a significant role for APX2 as a regulator in copper transfer to plastocyanin. This study sheds light on the unique properties of APX-R enzymes and their potential contributions to the complex processes of photosynthesis in green algae.

Transcriptomic Characterization of Copper-Binding Proteins for Predicting Prognosis in Glioma.

BACKGROUND: Copper and copper-binding proteins are key components of tumor progression as they play important roles in tumor invasion and migration, but their associations in gliomas remain unclear. METHODS: Transcriptomic datasets of glioblastoma, low-grade glioma, and normal brain cortex were derived from the TCGA and GTEX databases. Differentially expressed genes (DEGs) of copper-binding proteins were screened and used to construct a prognostic model based on COX and LASSO regression, which was further validated by the CGGA datasets. The expressions of risk-model genes were selectively confirmed via anatomic feature-based expression analysis and immunohistochemistry. The risk score was stratified by age, gender, WHO grade, IDH1 mutation, MGMT promoter methylation, and 1p/19q codeletion status, and a nomogram was constructed and validated. RESULTS: A total of 21 DEGs of copper-binding proteins were identified and a six-gene risk-score model was constructed, consisting of ANG, F5, IL1A, LOXL1, LOXL2, and STEAP3, which accurately predicted 1-, 3-, and 5-year overall survival rates, with the AUC values of 0.87, 0.88, and 0.82, respectively. The high-risk group had a significantly shorter OS (p < 0.0001) and was associated with old age, wild-type IDH1, a high WHO grade, an unmethylated MGMT promoter, and 1p/19q non-codeletion and had higher levels of immune cell infiltration, cancer-immunity suppressor, and immune checkpoint gene expression as well as a higher TMB. CONCLUSIONS: The model based on the genes of copper-binding proteins could contribute to prognosis prediction and provide potential targets against gliomas.

ABCE1 selectively promotes HIF-1α transactivation of angiogenic gene expression.

BACKGROUND: Copper (Cu), by inhibiting the factor inhibiting HIF-1 (FIH-1), promotes the transcriptional activity of hypoxia-inducible factor-1 (HIF-1). OBJECTIVE: The present study was undertaken to understand the molecular mechanism by which Cu inhibits FIH-1. METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with dimethyloxalylglycine (DMOG) resulting in HIF-1α accumulation and the FIH-1 protein complexes were pulled down for candidate protein analysis. The metal binding sites were predicted by both MetalDetector V2.0 and Metal Ion-Binding Site Prediction Server, and then the actual ability to bind to Cu in vitro was tested by both Copper-Immobilized metal affinity chromatography (Cu-IMAC) and Isothermal Titration Calorimetry (ITC). Subsequently, subcellular localization was monitored by immunocytochemistry, GFP-fusion protein expression plasmid and Western blotting in the nuclear extract. The interaction of candidate protein with HIF-1α and FIH-1 was validated by Co-Immunoprecipitation (Co-IP). Finally, the effect of candidate protein on the FIH-1 structure and HIF-1α transcriptional activity was analyzed by the InterEvDock3 web server and real-time quantitative RT-PCR. RESULTS: ATP-binding cassette E1 (ABCE1) was present in the FIH-1 complexes and identified as a leading Cu-binding protein as indicated by a number of possible Cu binding sites. The ability of ABCE1 to bind Cu was demonstrated in vitro. ABCE1 entered the nucleus along with FIH-1 under hypoxic conditions. Protein interaction analysis revealed that ABCE1 prevented FIH-1 to bind iron ions, inhibiting FIH-1 enzymatic activity. ABCE1 silencing suppressed the expression of Cu-dependent HIF-1 target gene BNIP3, not that of Cu-independent IGF-2. CONCLUSION: The results demonstrate that ABCE1, as a Cu-binding protein, enters the nucleus under hypoxic conditions and inhibits FIH-1degradation of HIF-1α, thus promoting HIF-1 transactivation of angiogenic gene expression.

Computational Evaluation of the Potential Pharmacological Activity of Salen-Type Ligands in Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is the most common form of dementia representing from 60% to 70% of the cases globally. It is a multifactorial disease that, among its many pathological characteristics, has been found to provoke the metal ion dysregulation in the brain, along with an increase in the oxidative stress. There is proof that metallic complexes formed by the amyloid-ß peptide (Aß) and extraneuronal copper can catalyze the production of reactive oxygen species, leading to an increase in oxidative stress, promoting neuronal death. Due to this interaction, bioavailable copper has become an important redox active target to consider within the search protocols of multifunctional agents for AD's treatment. OBJECTIVE: In this study, we examined by using bioinformatics and electronic structure calculations the potential application of 44 salen-type copper chelating ligands and 12 further proposed molecules as possible multifunctional agents in the context of AD. METHODS: The candidates were evaluated by combining bioinformatic tools and electronic structure calculations, which allowed us to classify the molecules as potential antioxidants, redistributor-like compounds, and the newly proposed suppressor mechanism. RESULTS: This evaluation demonstrate that salen-type ligands exhibit properties suitable for interfering in the chain of copper-induced oxidative stress reactions present in AD and potential redistributor and suppressor activity for copper ions. Finally, a novel set of plausible candidates is proposed and evaluated. CONCLUSION: According to the evaluated criteria, a subset of 13 salen-type candidates was found to exhibit promissory pharmacological properties in the AD framework and were classified according to three plausible action mechanisms.

An Electrochemistry and Computational Study at an Electrified Liquid-Liquid Interface for Studying Beta-Amyloid Aggregation.

Amphiphilic peptides, such as Aß amyloids, can adsorb at an interface between two immiscible electrolyte solutions (ITIES). Based on previous work (vide infra), a hydrophilic/hydrophobic interface is used as a simple biomimetic system for studying drug interactions. The ITIES provides a 2D interface to study ion-transfer processes associated with aggregation, as a function of Galvani potential difference. Here, the aggregation/complexation behaviour of Aß(1-42) is studied in the presence of Cu (II) ions, together with the effect of a multifunctional peptidomimetic inhibitor (P6). Cyclic and differential pulse voltammetry proved to be particularly sensitive to the detection of the complexation and aggregation of Aß(1-42), enabling estimations of changes in lipophilicity upon binding to Cu (II) and P6. At a 1:1 ratio of Cu (II):Aß(1-42), fresh samples showed a single DPV (Differential Pulse Voltammetry) peak half wave transfer potential (E1/2) at 0.40 V. Upon increasing the ratio of Cu (II) two-fold, fluctuations were observed in the DPVs, indicating aggregation. The approximate stoichiometry and binding properties of Aß(1-42) during complexation with Cu (II) were determined by performing a differential pulse voltammetry (DPV) standard addition method, which showed two binding regimes. A pKa of 8.1 was estimated, with a Cu:Aß1-42 ratio~1:1.7. Studies using molecular dynamics simulations of peptides at the ITIES show that Aß(1-42) strands interact through the formation of ß-sheet stabilised structures. In the absence of copper, binding/unbinding is dynamic, and interactions are relatively weak, leading to the observation of parallel and anti-parallel arrangements of ß-sheet stabilised aggregates. In the presence of copper ions, strong binding occurs between a copper ion and histidine residues on two peptides. This provides a convenient geometry for inducing favourable interactions between folded ß-sheet structures. Circular Dichroism spectroscopy (CD spectroscopy) was used to support the aggregation behaviour of the Aß(1-42) peptides following the addition of Cu (II) and P6 to the aqueous phase.

Complexation of copper algaecide and algal organic matter in algae-laden water: Insights into complex metal-organic interactions.

Copper-based algicides have been widely used to suppress algae blooms; however, the release of algal organic matter (AOM) on account of cell lysis may cause significant changes in the mitigation, transformation, and bioavailability of Cu(II). In the present work, the binding characteristics of Cu(II) with AOM were explored via combinative characterization methods, such as high-performance size exclusion chromatography, differential absorption spectra analysis, and joint applications of two-dimensional correlation spectroscopy (2D-COS), as well as heterospectral 2D-COS and moving window 2D-COS analyses of UV, synchronous fluorescence, and FTIR spectra. Carboxyl groups displayed a preferential interaction to Cu(II) binding, followed by polysaccharides. The spectral changes of C]O stretching occur after the change of chromophores in complexation with Cu(II). The AOM chromophores exhibit obvious conformations at Cu(II) concentrations higher than 120 µM, while AOM fluorophores and functional groups exhibit the greatest changes at Cu(II) concentrations lower than 20 µM. All these observations have verified the presence of binding heterogeneity and indicate that AOM could interact with Cu(II) through diverse functional moieties. Therefore, our study contributes to the better understanding of the fate of Cu(II)-AOM complexes in aquatic systems.

Metal-binding and circular permutation-dependent thermodynamic and kinetic stability of azurin.

Native topology is known to determine the folding kinetics and the energy landscape of proteins. Furthermore, the circular permutation (CP) of proteins alters the order of the secondary structure connectivity while retaining the three-dimensional structure, making it an elegant and powerful approach to altering native topology. Previous studies elucidated the influence of CP in proteins with different folds such as Greek key ß-barrel, ß-sandwich, ß-α-ß, and all α-Greek key. CP mainly affects the protein stability and unfolding kinetics, while folding kinetics remains mostly unaltered. However, the effect of CP on metalloproteins is yet to be elaborately studied. The active site of metalloproteins poses an additional complexity in studying protein folding. Here, we investigate a CP variant (cpN42) of azurin-in both metal-free and metal-bound (holo) forms. As observed earlier in other proteins, apo-forms of wild-type (WT) and cpN42 fold with similar rates. In contrast, zinc-binding accelerates the folding of WT but decelerates the folding of cpN42. On zinc-binding, the spontaneous folding rate of WT increases by >250 times that of cpN42, which is unprecedented and the highest for any CP to date. On the other hand, zinc-binding reduces the spontaneous unfolding rate of cpN42 by ~100 times, making the WT and CP azurins unfold at similar rates. Our study demonstrates metal binding as a novel way to modulate the unfolding and folding rates of CPs compared to their WT counterparts. We hope our study increases the understanding of the effect of CP on the folding mechanism and energy landscape of metalloproteins.

Copper coordination states affect the flexibility of copper Metallochaperone Atox1: Insights from molecular dynamics simulations.

Copper is an essential element in nature but in excess, it is toxic to the living cell. The human metallochaperone Atox1 participates in copper homeostasis and is responsible for copper transmission. In a previous multiscale simulation study, we noticed a change in the coordination state of the Cu(I) ion, from 4 bound cysteine residues to 3, in agreement with earlier studies. Here, we perform and analyze classical molecular dynamic simulations of various coordination states: 2, 3, and 4. The main observation is an increase in protein flexibility as a result of a decrease in the coordination state. In addition, we identified several populated conformations that correlate well with double electron-electron resonance distance distributions or an X-ray structure of Cu(I)-bound Atox1. We suggest that the increased flexibility might benefit the process of ion transmission between interacting proteins. Further experiments can scrutinize this hypothesis and shed additional light on the mechanism of action of Atox1.

Memo1 binds reduced copper ions, interacts with copper chaperone Atox1, and protects against copper-mediated redox activity in vitro.

The protein mediator of ERBB2-driven cell motility 1 (Memo1) is connected to many signaling pathways that play key roles in cancer. Memo1 was recently postulated to bind copper (Cu) ions and thereby promote the generation of reactive oxygen species (ROS) in cancer cells. Since the concentration of Cu as well as ROS are increased in cancer cells, both can be toxic if not well regulated. Here, we investigated the Cu-binding capacity of Memo1 using an array of biophysical methods at reducing as well as oxidizing conditions in vitro. We find that Memo1 coordinates two reduced Cu (Cu(I)) ions per protein, and, by doing so, the metal ions are shielded from ROS generation. In support of biological relevance, we show that the cytoplasmic Cu chaperone Atox1, which delivers Cu(I) in the secretory pathway, can interact with and exchange Cu(I) with Memo1 in vitro and that the two proteins exhibit spatial proximity in breast cancer cells. Thus, Memo1 appears to act as a Cu(I) chelator (perhaps shuttling the metal ion to Atox1 and the secretory path) that protects cells from Cu-mediated toxicity, such as uncontrolled formation of ROS. This Memo1 functionality may be a safety mechanism to cope with the increased demand of Cu ions in cancer cells.

Refolding and biophysical characterization of the Caulobacter crescentus copper resistance protein, PcoB: An outer membrane protein containing an intrinsically disordered domain.

Copper cations play fundamental roles in biological systems, such as protein folding and stabilization, or enzymatic reactions. Although copper is essential to the cell, it can become cytotoxic if present in too high concentration. Organisms have therefore developed specific regulation mechanisms towards copper. This is the case of the Pco system present in the bacterium Caulobacter crescentus, which is composed of two proteins: a soluble periplasmic protein PcoA and an outer membrane protein PcoB. PcoA oxidizes Cu+ to Cu2+, whereas PcoB is thought to be an efflux pump for Cu2+. While the PcoA protein has already been studied, very little is known about the structure and function of PcoB. In the present work, PcoB has been overexpressed in high yield in E. coli strains and successfully refolded by the SDS-cosolvent method. Binding to divalent cations has also been studied using several spectroscopic techniques. In addition, a three-dimensional structure model of PcoB, experimentally supported by circular dichroism, has been constructed, showing a ß-barrel conformation with a N-terminal disordered chain. This peculiar intrinsic disorder property has also been confirmed by various bioinformatic tools.

Copper Binding and Oligomerization Studies of the Metal Resistance Determinant CrdA from Helicobacter pylori.

Within this research, the CrdA protein from Helicobacter pylori (HpCrdA), a putative copper-binding protein important for the survival of bacterium, was biophysically characterized in a solution, and its binding affinity toward copper was experimentally determined. Incubation of HpCrdA with Cu(II) ions favors the formation of the monomeric species in the solution. The modeled HpCrdA structure shows a conserved methionine-rich region, a potential binding site for Cu(I), as in the structures of similar copper-binding proteins, CopC and PcoC, from Pseudomonas syringae and from Escherichia coli, respectively. Within the conserved amino acid motif, HpCrdA contains two additional methionines and two glutamic acid residues (MMXEMPGMXXMXEM) in comparison to CopC and PcoC but lacks the canonical Cu(II) binding site (two His) since the sequence has no His residues. The methionine-rich site is in a flexible loop and can adopt different geometries for the two copper oxidation states. It could bind copper in both oxidation states (I and II), but with different binding affinities, micromolar was found for Cu(II), and less than nanomolar is proposed for Cu(I). Considering that CrdA is a periplasmic protein involved in chaperoning copper export and delivery in the H. pylori cell and that the affinity of the interaction corresponds to a middle or strong metal-protein interaction depending on the copper oxidation state, we conclude that the interaction also occurs in vivo and is physiologically relevant for H. pylori.

Computational identification of putative copper-binding proteins in pomegranate bacterial blight pathogen Xanthomonas citri pv. punicae.

Xanthomonas citri pv. punicae (Xcp) causes bacterial blight in pomegranate, and an effective management strategy to control this devastating disease is yet to be formulated. Copper is a vital micronutrient that plays a significant role in bacterial physiology and virulence mechanism. It acts as a cofactor and conjugates with proteins, catalyse various biological processes, and contributes to the structural integrity of proteins. In this study, we have screened copper-binding proteins of Xcp and their plausible role in the pathogenesis of pomegranate bacterial blight disease by adopting advanced in silico tools. We have identified 46 putative copper-binding proteins (PCBPs) from Xcp, approximately 0.85% of the Xcp proteome, of which 34 and 25 PCBPs are essential and pathogen-host interaction (PHI) responsible proteins, respectively. Of the 25 PHI-responsible proteins, 9 are classified into classical secretory proteins and 8 are classified into non-classical secretory proteins. These PHI-responsible PCBPs are involved in diverse processes, including metabolism, response to oxidative stress, transport, protein folding, signalling, and virulence mechanism. Our study identified 16 drug target proteins among the PHI-responsible PCBPs, which can be used as an ideal target for various antimicrobial agents to control pomegranate blight disease. Our observations pave the way to the understanding of copper homeostasis in Xcp and its possible involvement in the disease-causing process.

New insights into the mechanism of iron transport through the bacterial Ftr system present in pathogens.

Iron is an essential nutrient in bacteria. Its ferrous form, mostly present in low oxygen and acidic pH environments, can be imported using the specific Ftr-type transport system, which encompasses the conserved FtrABCD system found in pathogenic bacteria such as Bordetella, Brucella and Burkholderia. The nonpathogenicity and versatile metabolism of Rubrivivax gelatinosus make it an ideal model to study the FtrABCD system. Here, we report a new aspect of its regulation and the role of the periplasmic proteins FtrA and FtrB using in vivo and in vitro approaches. We investigated the metal binding mode and redox state of copper and iron to FtrA by crystallography and biophysical methods. An 'as isolated' FtrA protein from the bacterial periplasm contained a copper ion (Cu+ ) identified by electron paramagnetic resonance (EPR). Copper is coordinated by four conserved side chains (His and Met) in the primary metal site. Structural analysis of R. gelatinosus FtrA and FtrA homologues revealed that copper binding induces a rearrangement of the His95 imidazole ring, releasing thereafter space, as well as both Asp45 and Asp92 side chains, for iron binding in the secondary metal site. EPR highlighted that FtrA can oxidize the bound ferrous ion into the ferric form by reducing the bound Cu2+ into Cu+ , both metal sites being separated by 7 Å. Finally, we showed that FtrB binds iron and not copper. These results provide new insights into the mechanism of ferrous iron utilization by the conserved FtrABCD iron transporter for which we propose a new functional model.

New insights in copper handling strategies in the green alga Chlamydomonas reinhardtii under low-iron condition.

Copper (Cu) is a redox-active transition element critical to various metabolic processes. These functions are accomplished in tandem with Cu-binding ligands, mainly proteins. The main goal of this work was to understand the mechanisms that govern the intracellular fate of Cu in the freshwater green alga, Chlamydomonas reinhardtii, and more specifically to understand the mechanisms underlying Cu detoxification by algal cells in low-Fe conditions. We show that Cu accumulation was up to 51-fold greater for algae exposed to Cu in low-Fe medium as compared to the replete-Fe growth medium. Using the stable isotope 65Cu as a tracer, we studied the subcellular distribution of Cu within the various cell compartments of C. reinhardtii. These data were coupled with metallomic and proteomic approaches to identify potential Cu-binding ligands in the heat-stable proteins and peptides fraction of the cytosol. Cu was mostly found in the organelles (78%), and in the heat-stable proteins and peptides (21%) fractions. The organelle fraction appeared to also be the main target compartment of Cu accumulation in Fe-depleted cells. As Fe levels in the medium were shown to influence Cu homeostasis, we found that C. reinhardtii can cope with this additional stress by utilizing different Cu-binding ligands. Indeed, in addition to expected Cu-binding ligands such as glutathione and phytochelatins, 25 proteins were detected that may also play a role in the Cu-detoxification processes in C. reinhardtii. Our results shed new light on the coping mechanisms of C. reinhardtii when exposed to environmental conditions that induce high rates of Cu accumulation.