Nickel-binding Protein Research Articles

Quantifying metal-binding specificity of CcNikZ-II from Clostridium carboxidivorans in the presence of competing metal ions

Many proteins bind transition metal ions as cofactors to carry out their biological functions. Despite binding affinities for divalent transition metal ions being predominantly dictated by the Irving-Williams series for wild-type proteins, in vivo metal ion binding specificity is ensured by intracellular mechanisms that regulate free metal ion concentrations. However, a growing area of biotechnology research considers the use of metal-binding proteins in vitro to purify specific metal ions from wastewater, where specificity is dictated by the protein's metal binding affinities. A goal of metalloprotein engineering is to modulate these affinities to improve a protein's specificity towards a particular metal; however, the quantitative relationship between the affinities and the equilibrium metal-bound protein fractions depends on the underlying binding mechanisms. Here we demonstrate a high-throughput intrinsic tryptophan fluorescence quenching method to validate binding models in multi-metal solutions for CcNikZ-II, a nickel-binding protein from Clostridium carboxidivorans. Using our validated models, we quantify the relationship between binding affinity and specificity in different classes of metal-binding models for CcNikZ-II. We further illustrate the potential relevance of data-informed models to predicting engineering targets for improved specificity.

Deletion of genes linked to the C1-fixing gene cluster affects growth, by-products, and proteome of Clostridium autoethanogenum.

Gas fermentation has emerged as a sustainable route to produce fuels and chemicals by recycling inexpensive one-carbon (C1) feedstocks from gaseous and solid waste using gas-fermenting microbes. Currently, acetogens that utilise the Wood-Ljungdahl pathway to convert carbon oxides (CO and CO2) into valuable products are the most advanced biocatalysts for gas fermentation. However, our understanding of the functionalities of the genes involved in the C1-fixing gene cluster and its closely-linked genes is incomplete. Here, we investigate the role of two genes with unclear functions-hypothetical protein (hp; LABRINI_07945) and CooT nickel binding protein (nbp; LABRINI_07950)-directly adjacent and expressed at similar levels to the C1-fixing gene cluster in the gas-fermenting model-acetogen Clostridium autoethanogenum. Targeted deletion of either the hp or nbp gene using CRISPR/nCas9, and phenotypic characterisation in heterotrophic and autotrophic batch and autotrophic bioreactor continuous cultures revealed significant growth defects and altered by-product profiles for both ∆hp and ∆nbp strains. Variable effects of gene deletion on autotrophic batch growth on rich or minimal media suggest that both genes affect the utilisation of complex nutrients. Autotrophic chemostat cultures showed lower acetate and ethanol production rates and higher carbon flux to CO2 and biomass for both deletion strains. Additionally, proteome analysis revealed that disruption of either gene affects the expression of proteins of the C1-fixing gene cluster and ethanol synthesis pathways. Our work contributes to a better understanding of genotype-phenotype relationships in acetogens and offers engineering targets to improve carbon fixation efficiency in gas fermentation.

Ynt is the primary nickel import system used by Proteus mirabilis and specifically contributes to fitness by supplying nickel for urease activity.

Proteus mirabilis is a Gram-negative uropathogen and frequent cause of catheter-associated urinary tract infection (CAUTI). One important virulence factor is its urease enzyme, which requires nickel to be catalytically active. It is, therefore, hypothesized that nickel import is critical for P. mirabilis urease activity and pathogenesis during infection. P. mirabilis strain HI4320 encodes two putative nickel import systems, designated Nik and Ynt. By disrupting the substrate-binding proteins from each import system (nikA and yntA), we show that Ynt is the primary nickel importer, while Nik only compensates for loss of Ynt at high nickel concentrations. We further demonstrate that these are the only binding proteins capable of importing nickel for incorporation into the urease enzyme. Loss of either nickel-binding protein results in a significant fitness defect in a murine model of CAUTI, but YntA is more crucial as the yntA mutant was significantly outcompeted by the nikA mutant. Furthermore, despite the importance of nickel transport for hydrogenase activity, the sole contribution of yntA and nikA to virulence is due to their role in urease activity, as neither mutant exhibited a fitness defect when disrupted in a urease-negative background.

The oxygen reactivity of an artificial hydrogenase designed in a reengineered copper storage protein

The O2 reactivity of an artificial biomolecular hydrogenase, the nickel binding protein (NBP) is investigated. Kinetic analyses revealed a complete 4e- reduction of O2 to H2O under catalytic conditions with associated k0 for ET in the order of 10-6 cm s-1. Protein destabilization and S oxygenation are contributing factors to the deactivation of NBP under oxic conditions. Computational studies provided insight into the S oxygenation and the reaction intermediates of a proposed mechanistic pathway for O2 activation by NBP.

The Metallochaperone Encoding Gene hypA Is Widely Distributed among Pathogenic Aeromonas spp. and Its Expression Is Increased under Acidic pH and within Macrophages.

Metallochaperones are essential proteins that insert metal ions or metal cofactors into specific enzymes, that after maturation will become metalloenzymes. One of the most studied metallochaperones is the nickel-binding protein HypA, involved in the maturation of nickel-dependent hydrogenases and ureases. HypA was previously described in the human pathogens Escherichia coli and Helicobacter pylori and was considered a key virulence factor in the latter. However, nothing is known about this metallochaperone in the species of the emerging pathogen genus Aeromonas. These bacteria are native inhabitants of aquatic environments, often associated with cases of diarrhea and wound infections. In this study, we performed an in silico study of the hypA gene on 36 Aeromonas species genomes, which showed the presence of the gene in 69.4% (25/36) of the Aeromonas genomes. The similarity of Aeromonas HypA proteins with the H. pylori orthologous protein ranged from 21−23%, while with that of E. coli it was 41−45%. However, despite this low percentage, Aeromonas HypA displays the conserved characteristic metal-binding domains found in the other pathogens. The transcriptional analysis enabled the determination of hypA expression levels under acidic and alkaline conditions and after macrophage phagocytosis. The transcriptional regulation of hypA was found to be pH-dependent, showing upregulation at acidic pH. A higher upregulation occurred after macrophage infection. This is the first study that provided evidence that the HypA metallochaperone in Aeromonas might play a role in acid tolerance and in the defense against macrophages.

Real time quantification of intracellular nickel using genetically encoded FRET-based nanosensor

Nickel (Ni2+) is an essential mineral nutrient that is required in trace quantities, but exposure to excess amounts can be toxic or carcinogenic. Mechanisms underlying Ni2+ imbalance and toxicity are poorly understood because most analytical methods currently used to probe this metal are invasive or also have toxic effects. To address these problems, a genetically encoded FRET-based probe for nickel metal (FProNiM) was constructed for real-time detection of Ni2+ in live cells. FProNiM was constructed by sandwiching the bacterial periplasmic nickel-binding protein NikA between the fluorescent protein variants ECFP and Venus. Ni2+ binding to FProNiM induced a conformational change that could be detected by a change in fluorescence resonance energy transfer (FRET) ratio (acceptor/donor fluorescence). In vitro studies demonstrated that FProNiM is specific for Ni2+, insensitive to changes in pH from 5.0 to 8.0, and has an affinity (Kd) of 21.6 μM, which allows detection of Ni2+ concentrations from 0.1 to 5000 μM. The sensor variant FProNiM-5 was also expressed in Escherichia coli (E. coli), yeast, and mammalian cells. In each cell type, changes in Ni2+ concentrations were detected with subcellular resolution using confocal microscopy.

Analysis of Nickel-Binding Proteins from Various Animal Sera

Abstract Nickel-binding proteins play an important role in the biological processes and can also be utilized in several fields of biotechnology. This study was focused on analysing the nickel-binding proteins from the blood sera of humans (Homo sapiens), cattle (Bos taurus), sheep (Ovis aries), red deer (Cervus elaphus), mouflon (Ovis orientalis), fallow deer (Dama dama), horses (Equus ferus caballus), pigs (Sus scrofa domesticus), wildboars (Sus scrofa), brown bears (Ursus arctos) and pheasants (Phasianus colchicus). The presence of higher abundance proteins in the blood serum, such as albumins, may mask the detection of lower abundance proteins. The samples were depleted from these higher abundance proteins to facilitate the detection of those with lower abundance. For the characterization of these proteins, nickel cations bound to tetradentate ligand nitrilotriacetic acid(Ni-NTA)immobilized on agarose beads were incubated with animal sera to capture nickel-binding proteins and subsequently the proteins were eluted and fractionated on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed a set of nickel-binding proteins with various molecular weights within different animal species. A unique ~42 kDa nickel-binding protein in the brown bear serum, which was not present in any of the other species, was further characterized and identified by matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS). This protein was identified as ahaptoglobin-like protein. This result may provide some valuable clue for the physiological difference in the metal binding proteins in the serum of Ursus arctos and other animals.

CXCL4 is a novel nickel-binding protein and augments nickel allergy.

Nickel (Ni) is the most frequent metal allergen and induces a TH1 -dependent type-IV allergy. Although Ni2+ is considered to bind to endogenous proteins, it currently remains unclear whether these Ni-binding proteins are involved in Ni allergy invivo. We previously reported the adjuvant effects of lipopolysaccharide (LPS) in a Ni allergy mouse model. As LPS induces a number of inflammatory mediators, we hypothesized that Ni-binding protein(s) are also induced by LPS. The objective of this study was to purify and identify Ni-binding protein(s) from serum taken from LPS-injected mice (referred as LPS serum) and examined the augmenting effects of these Ni-binding protein(s) on Ni allergy in an invivo model. BALB/cA mice were sensitized with an i.p. injection of NiCl2 and LPS. Ten days after sensitization, mice were challenged with NiCl2 by an i.d. injection into ear pinnae. Ni-binding protein(s) were purified by Ni-affinity column chromatography and gel filtration. Lipopolysaccharide serum, but not serum taken from saline-injected mice, augmented ear swelling induced by Ni-allergic inflammation. Ni-binding, but not non-binding fraction, purified from LPS serum augmented Ni-allergic inflammation. Mass spectrometry and Western blotting detected CXCL4 in the active fraction. A batch analysis with Ni-sepharose and a surface plasmon resonance analysis revealed direct binding between CXCL4 and Ni2+ . Recombinant CXCL4 augmented Ni-allergic inflammation and exerted adjuvant effects at the sensitization phase. These results indicate that CXCL4 is a novel Ni-binding protein that augments Ni allergy at the elicitation and sensitization phases. This is the first study to demonstrate that the Ni-binding protein augments Ni allergy invivo.

The CO dehydrogenase accessory protein CooT is a novel nickel-binding protein.

In Rhodospirillum rubrum, maturation of Carbon Monoxide Dehydrogenase (CODH) requires three accessory proteins, CooC, CooT and CooJ, dedicated to nickel insertion into the active site, which is constituted by a distorted [NiFe3S4] cubane coordinated with a mononuclear Fe site. CooC is an ATPase proposed to provide the energy required for the maturation process, while CooJ is described as a metallochaperone with 16 histidines and 2 cysteines at the C-terminus, likely involved in metal binding and/or storage. Prior to the present study, no information was available on CooT at the molecular level. Here, the X-ray structure of RrCooT was obtained, which revealed that this protein is a homodimer featuring a fold that resembles an Sm-like domain, suggesting a role in RNA metabolism that was however not supported by experimental observations. Biochemical and biophysical evidence based on circular dichroism spectroscopy, light scattering, isothermal titration calorimetry and site-directed mutagenesis showed that RrCooT specifically binds a single Ni(ii) per dimer, with a dissociation constant of 9 nM, through the pair of Cys2, highly conserved residues, located at the dimer interface. Despite its role in the activation of RrCODH in vivo, CooT was thought to be a unique protein, found only in R. rubrum, with an unclear function. In this study, we extended the biological impact of CooT, establishing that this protein is a member of a novel Ni(ii)-binding protein family with 111 homologues, linked to anaerobic metabolism in bacteria and archaea, and in most cases to the presence of CODH.

Streptomyces coelicolor SCO4226 is a nickel binding protein.

The open reading frame SCO4226 of Streptomyces coelicolor A3(2) encodes an 82-residue hypothetical protein. Biochemical assays revealed that each SCO4226 dimer binds four nickel ions. To decipher the molecular function, we solved the crystal structures of SCO4226 in both apo- and nickel-bound (Ni-SCO4226) forms at 1.30 and 2.04 Å resolution, respectively. Each subunit of SCO4226 dimer adopts a canonical ferredoxin-like fold with five β-strands flanked by two α-helices. In the structure of Ni-SCO4226, four nickel ions are coordinated at the surface of the dimer. Further biochemical assays suggested that the binding of Ni2+ triggers the self-aggregation of SCO4226 in vitro. In addition, RT-qPCR assays demonstrated that the expression of SCO4226 gene in S. coelicolor is specifically up-regulated by the addition of Ni2+, but not other divalent ions such as Cu2+, Mn2+ or Co2+. All these results suggested that SCO4226 acts as a nickel binding protein, probably required for nickel sequestration and/or detoxification.

Promiscuous Nickel Import in Human Pathogens: Structure, Thermodynamics, and Evolution of Extracytoplasmic Nickel-Binding Proteins

In human pathogenic bacteria, nickel is required for the activation of two enzymes, urease and [NiFe]-hydrogenase, necessary for host infection. Acquisition of Ni(II) is mediated by either permeases or ABC-importers, the latter including a subclass that involves an extracytoplasmic nickel-binding protein, Ni-BP. This study reports on the structure of three Ni-BPs from a diversity of human pathogens and on the existence of three new nickel-binding motifs. These are different from that previously described for Escherichia coli Ni-BP NikA, known to bind nickel via a nickelophore, and indicate a variegated ligand selectivity for Ni-BPs. The structures are consistent with ligand affinities measured in solution by calorimetry and challenge the hypothesis of a general requirement of nickelophores for nickel uptake by canonical ABC importers. Phylogenetic analyses showed that Ni-BPs have different evolutionary origins and emerged independently from peptide-binding proteins, possibly explaining the promiscuous behavior of this class of Ni(II) carriers.

Molecular function of the prolyl cis/trans isomerase and metallochaperone SlyD

SlyD is a bacterial two-domain protein that functions as a molecular chaperone, a prolyl cis/trans isomerase, and a nickel-binding protein. This review summarizes recent findings about the molecular enzyme mechanism of SlyD. The chaperone function located in one domain of SlyD is involved in twin-arginine translocation and increases the catalytic efficiency of the prolyl cis/trans isomerase domain in protein folding by two orders of magnitude. The C-terminal tail of SlyD binds Ni2+ ions and supplies them for the maturation of [NiFe] hydrogenases. A combined biochemical and biophysical analysis revealed the molecular basis of the delicate interplay of the different domains of SlyD for optimal function.

The structure of the periplasmic nickel-binding protein NikA provides insights for artificial metalloenzyme design

Understanding the interaction of a protein with a relevant ligand is crucial for the design of an artificial metalloenzyme. Our own interest is focused on the synthesis of artificial monooxygenases. In an initial effort, we have used the periplasmic nickel-binding protein NikA from Escherichia coli and iron complexes in which N(2)Py(2) ligands (where Py is pyridine) have been varied in terms of charge, aromaticity, and size. Six "NikA/iron complex" hybrids have been characterized by X-ray crystallography, and their interactions and solution properties have been studied. The hybrids are stable as indicated by their K (d) values, which are all in the micromolar range. The X-ray structures show that the ligands interact with NikA through salt bridges with arginine residues and π-stacking with a tryptophan residue. We have further characterized these interactions using quantum mechanical calculations and determined that weak CH/π hydrogen bonds finely modulate the stability differences between hybrids. We emphasize the important role of the tryptophan residues. Thus, our study aims at the complete characterization of the factors that condition the interaction of an artificial ligand and a protein and their implications for catalysis. Besides its potential usefulness in the synthesis of artificial monooxygenases, our approach should be generally applicable in the field of artificial metalloenzymes.

Molecular Reproduction & Development Volume 78, Issue 2 cover image

Molecular Reproduction and DevelopmentVolume 78, Issue 2 p. C1-C1 Cover ImageFree Access Molecular Reproduction & Development Volume 78, Issue 2 cover image First published: 17 February 2011 https://doi.org/10.1002/mrd.21294AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Exposure to metals, such as nickel, can contribute to male subfertility, but the mechanisms involved are poorly understood. Glutamate-ammonia ligase (GLUL) was identified by Sun et al. (in this issue) as a prominent testicular nickel-binding protein whose activity is inhibited by nickel. Here, GLUL (green) is found in interstitial cells of the seminiferous tubules. Section was counterstained for nuclear DNA (DAPI, blue) and sperm (acetylated tubulin in tails, red). Volume78, Issue2February 2011Pages C1-C1 RelatedInformation

Binding of nickel to testicular glutamate-ammonia ligase inhibits its enzymatic activity

Exposure to nickel has been shown to cause damage to the testis in several animal models. It is not known if the testis expresses protein(s) that can bind nickel. To test this, we used a nickel-binding assay to isolate testicular nickel-binding proteins. We identified glutamate-ammonia ligase (GLUL) as a prominent nickel-binding protein by mass spectrometry. Protein analysis and reverse transcriptase polymerase chain reaction showed that GLUL is expressed in the testis, predominantly in interstitial cells. We determined that GLUL has a higher affinity for nickel than for its regular co-factor manganese. We produced an enzymatically active, recombinant GLUL protein. Upon binding, nickel interferes with the manganese-catalyzed enzymatic activity of recombinant GLUL protein. We also determined that GLUL activity in testes of animals exposed to nickel sulfate is reduced. Our results identify testicular GLUL as the first testicular protein shown to be affected by nickel exposure.

Proteomic Analysis of Rice Leaves Shows the Different Regulations to Osmotic Stress and Stress Signals

Following the idea of partial root-zone drying (PRD) in crop cultivation, the morphological and physiological responses to partial root osmotic stress (PROS) and whole root osmotic stress (WROS) were investigated in rice. WROS caused stress symptoms like leaf rolling and membrane leakage. PROS stimulated stress signals, but did not cause severe leaf damage. By proteomic analysis, a total of 58 proteins showed differential expression after one or both treatments, and functional classification of these proteins suggests that stress signals regulate photosynthesis, carbohydrate and energy metabolism. Two other proteins (anthranilate synthase and submergence-induced nickel-binding protein) were upregulated only in the PROS plants, indicating their important roles in stress resistance. Additionally, more enzymes were involved in stress defense, redox homeostasis, lignin and ethylene synthesis in WROS leaves, suggesting a more comprehensive regulatory mechanism induced by osmotic stress. This study provides new insights into the complex molecular networks within plant leaves involved in the adaptation to osmotic stress and stress signals.

Host-Dependent Expression of Rhizobium leguminosarum bv. viciae Hydrogenase Is Controlled at Transcriptional and Post-Transcriptional Levels in Legume Nodules

The legume host affects the expression of Rhizobium leguminosarum hydrogenase activity in root nodules. High levels of symbiotic hydrogenase activity were detected in R. leguminosarum bacteroids from different hosts, with the exception of lentil (Lens culinaris). Transcription analysis showed that the NifA-regulated R. leguminosarum hydrogenase structural gene promoter (P(1)) is poorly induced in lentil root nodules. Replacement of the P(1) promoter by the FnrN-dependent promoter of the fixN gene restored transcription of hup genes in lentil bacteroids, but not hydrogenase activity. In the P(fixN)-hupSL strain, additional copies of the hup gene cluster and nickel supplementation to lentil plants increased bacteroid hydrogenase activity. However, the level of activity in lentil still was significantly lower than in pea bacteroids, indicating that an additional factor is impairing hydrogenase expression inside lentil nodules. Immunological analysis revealed that lentil bacteroids contain reduced levels of both hydrogenase structural subunit HupL and nickel-binding protein HypB. Altogether, results indicate that hydrogenase expression is affected by the legume host at the level of both transcription of hydrogenase structural genes and biosynthesis or stability of nickel-related proteins HypB and HupL, and suggest the existence of a plant-dependent mechanism that affects hydrogenase activity during the symbiosis by limiting nickel availability to the bacteroid.

Interaction between the Helicobacter pylori accessory proteins HypA and UreE is needed for urease maturation.

Several accessory proteins are required for the maturation of two nickel-containing enzymes in the gastric pathogen Helicobacter pylori. These two enzymes are hydrogenase and urease. Among the accessory/maturation proteins, the nickel-binding HypA protein has been previously shown to be required for the full activity of both the hydrogenase and the urease enzymes, while another nickel-binding protein, UreE, is known to be solely involved in the urease maturation process. In this study, UreE was shown to be required under all nickel levels for full activation of the apourease. By use of cross-linking studies, an interaction between purified HypA and UreE proteins was identified, leading to the formation of a 34 kDa heterodimer complex. The cross-linked adduct was detected by immunoblotting with either anti-HypA or anti-UreE antiserum. By using a two-plasmid system in Escherichia coli, the highest urease activity was achieved under low nickel conditions only when the UreE protein was expressed along with the wild-type HypA protein, but not with its nickel-binding-deficient variant HypA H2A. Addition of only 1 microM NiCl(2) into minimal medium abolished the need for HypA to activate the urease. Although various attempts to show direct nickel transfer from HypA to UreE failed, these results suggest that interactions between the nickel-binding accessory proteins HypA and UreE are required to allow nickel transfer from HypA eventually to the apourease in H. pylori.

NikA Binds Heme: A New Role for anEscherichia coliPeriplasmic Nickel-Binding Protein

NikA is a periplasmic binding protein involved in nickel uptake in Escherichia coli. NikA was identified as a heme-binding protein in the periplasm of anaerobically grown cells overexpressing CydDC, an ABC transporter that exports reductant to the periplasm. CydDC-overexpressing cells accumulate a heme biosynthesis-derived pigment, P-574. For further biochemical and spectroscopic analysis, unliganded NikA was overexpressed and purified. NikA was found to comigrate with both hemin and protoporphyrin IX during gel filtration. Furthermore, tryptophan fluorescence quenching titrations demonstrated that both hemin and protoporphyrin IX bind to NikA with similar affinity. The binding affinity of NikA for these pigments (Kd approximately 0.5 microM) was unaltered in the presence and absence of saturating concentrations of nickel, suggesting that these tetrapyrroles bind to NikA in a manner independent of nickel. To test the hypothesis that NikA is required for periplasmic heme protein assembly, the effects of a nikA mutation (nikA::Tn5, Km(R) insertion) on accumulation of P-574 by CydDC-overexpressing cells was assessed. This mutation significantly lowered P-574 levels, implying that NikA may be involved in P-574 production. Thus, in the reducing environment of the periplasm, NikA may serve as a heme chaperone as well as a periplasmic nickel-binding protein. The docking of heme onto NikA was modeled using the published crystal structure; many of the predicted complexes exhibit a heme-binding cleft remote from the nickel-binding site, which is consistent with the independent binding of nickel and heme. This work has implications for the incorporation of heme into b- and c-type cytochromes.

Nickel binding to NikA: an additional binding site reconciles spectroscopy, calorimetry and crystallography

Intracellular nickel is required by Escherichia coli as a cofactor for a number of enzymes and is necessary for anaerobic respiration. However, high concentrations of nickel are toxic, so both import and export systems have evolved to control the cellular level of the metal. The nik operon in E. coli encodes a nickel-uptake system that includes the periplasmic nickel-binding protein NikA. The crystal structures of wild-type NikA both bound to nickel and in the apo form have been solved previously. The liganded structure appeared to show an unusual interaction between the nickel and the protein in which no direct bonds are formed. The highly unusual nickel coordination suggested by the crystal structure contrasted strongly with earlier X-ray spectroscopic studies. The known nickel-binding site has been probed by extensive mutagenesis and isothermal titration calorimetry and it has been found that even large numbers of disruptive mutations appear to have little effect on the nickel affinity. The crystal structure of a binding-site mutant with nickel bound has been solved and it is found that nickel is bound to two histidine residues at a position distant from the previously characterized binding site. This novel site immediately resolves the conflict between the crystal structures and other biophysical analyses. The physiological relevance of the two binding sites is discussed.