Binding of Ni2+ to a histidine- and glutamine-rich protein, Hpn-like

Hpn-like (Hpnl) protein, encoded by the hpnl gene in Helicobacter pylori and featuring a histidine-rich and two glutamine-rich motifs, can render nickel tolerance to H. pylori when the external nickel level reaches toxic limits. We found that the recombinant Hpnl exists as an oligomer in the native state and binds to two molar equivalents of nickel ions per monomer with a dissociation constant of 3.8 μM. Nickel could be released from Hpnl either at acidic pH (pH_1/2 4.6) or in the presence of chelate ligands, such as EDTA (t _1/2 = 220, 355, and 716 min at pH 6.0, 7.0, and 7.5, respectively). Our combined spectroscopic data show that nickel ion coordinates to a nitrogen of a histidine residue possibly with a coordination number of four (square-planar geometry) or five. The growth of Escherichia coli cells with or without the hpnl gene implied a protective role of Hpnl under higher concentrations of external nickel ions. Hpnl may serve a role in binding/storage or detoxification of excess nickel ions. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Copper and zinc binding properties of the N-terminal histidine-rich sequence of Haemophilus ducreyi Cu,Zn superoxide dismutase

The Cu,Zn superoxide dismutase (Cu,ZnSOD) isolated from Haemophilus ducreyi possesses a His-rich N-terminal metal binding domain, which has been previously proposed to play a copper(II) chaperoning role. To analyze the metal binding ability and selectivity of the histidine-rich domain we have carried out thermodynamic and solution structural analysis of the copper(II) and zinc(II) complexes of a peptide corresponding to the first 11 amino acids of the enzyme (H₂N-HGDHMHNHDTK-OH, L). This peptide has highly versatile metal binding ability and provides one and three high affinity binding sites for zinc(II) and copper(II), respectively. In equimolar solutions the MHL complexes are dominant in the neutral pH-range with protonated lysine ε-amino group. As a consequence of its multidentate nature, L binds zinc and copper with extraordinary high affinity (K_D,Zn = 1.6 × 10⁻⁹ M and K_D,Cu = 5.0 × 10⁻¹² M at pH 7.4) and appears as the strongest zinc(II) and copper(II) chelator between the His-rich peptides so far investigated. These K_D values support the already proposed role of the N-terminal His-rich region of H. ducreyi Cu,ZnSOD in copper recruitment under metal starvation, and indicate a similar function in the zinc(II) uptake, too. The kinetics of copper(II) transfer from L to the active site of Cu-free N-deleted H. ducreyi Cu,ZnSOD showed significant pH and copper-to-peptide ratio dependence, indicating specific structural requirements during the metal ion transfer to the active site. Interestingly, the complex CuHL has significant superoxide dismutase like activity, which may suggest multifunctional role of the copper(II)-bound N-terminal His-rich domain of H. ducreyi Cu,ZnSOD.     

Short-chain oligopeptides with copper(II) binding properties: The impact of specific structural modifications on the copper(II) coordination abilities

A series of linear tetrapeptides containing two histidyl residues in position 2 and 4, namely DHGH, DHGdH, KHGH, KHGdH, Ac-DHGH-NH₂, Ac-DHGdH-NH₂, Ac-KHGH-NH₂, and Ac-KHGdH-NH₂, were synthesized and characterised. Their copper(II) binding properties were investigated in depth through a variety of physicochemical methods. Potentiometric titrations were first carried out to establish the stoichiometry and the stability of the resulting copper(II)-peptide complexes. The copper(II) chromophores that are formed in the various cases in dependence of pH were subsequently characterised by extensive spectroscopic analysis (UV-Vis, EPR, CD) in strict correlation with potentiometric data. The effects of the nature of the first amino acid (Lys versus Asp) and of N-terminal amino group protection on copper(II) binding were specifically addressed. On turn, the careful comparison of the copper(II) coordination abilities of the linear peptides with those of their cyclic analogs provided insight into the effects of cyclization on the overall metal binding properties.     

Oligomerization of the Cu(II) and Ni(II) tetraazaannulene complexes: A pulse radiolysis study of the associated reaction mechanism

The oligomerization of [Cu^II(H_x(tmdnTAA))]^x+ (x = 0, 1, 2 and (tmdnTAA))²⁻ is 2,4,9,11-tetramethyl-dinaphto[14]-2,4,6,9,11,13-hexaeneN₄) was initiated in homogeneous solution via the reaction of this Cu(II) complex with pulse radiolytically generated radicals. The reaction produces Cu(III) intermediates which are rapidly converted to Cu(II) ligand-radical species. In contrast to the mechanism proposed for the electrochemical oligomerization, where the local concentration of radicals is probably high, the reaction kinetics in homogeneous solution is propagated by a process where the Cu(II) ligand-radical precursors react with [Cu^II(H_x(tmdnTAA))]^x+.     

Vibrational circular dichroism and IR absorption of DNA complexes with Cu2+ ions

Vibrational circular dichroism (VCD) spectroscopy and simultaneous IR absorption measurements are applied to study the interaction of natural calf thymus DNA with Cu2+ ions at room temperature in a Cu2+ concentration range of 0-0.4M (a Cu2+/phosphate molar ratio [Cu]/[P] of 0-0.7). In some important instances, VCD provides more detailed insights than previous IR investigations whereas in several others it leads to the same interpretations. The Cu2+ ions bind to phosphate groups at a low metal concentration. Upon increasing the ion concentration, chelates are formed in which Cu2+ binds to the N7 of guanine (G) and a phosphate group. Detectable only by VCD, significant distortion of most guanine-cytosine (GC) base pairs occurs at a [Cu]/[P] ratio of 0.5 with only a minor affect on adenine-thymine (AT) base pairs, which favors a "sandwich" complex in which a Cu2+ ion is inserted between two adjacent guanines in a GpG sequence. The AT base pairs become significantly distorted when the metal concentration is increased to 0.7 [Cu]/[P]. A number of GC base pairs, which are possibly involved in sandwich complexes, remain stacked and paired even at 0.7 [Cu]/[P], preventing complete strand separation. The DNA secondary structure changes considerably from the standard B-form geometry at a [Cu]/[P] ratio of 0.4 and higher. A further transition to some intermediate conformation that is inconsistent with either the A- or Z-form or a completely denatured state is suggested in agreement with other works. In general, VCD proves to be a reliable indicator of the 3-dimensional structure of the DNA-metal ion complexes, which reveals structural details that cannot be deduced from the IR absorption spectra alone.     

Cu 2+ 对体外培养的日本沼虾血细胞的影响

以日本沼虾为实验材科研究血细胞的体外培养,发现添加15％胎牛血清(FBS)、100IU／m1青霉素、100μg/ml链霉素,渗透压为472mmol/L的199培养基有利于血细胞的生长,在此基础上测定了Cu2 对传代血细胞的影响,发现培养基中添加2μg/L Cu2 时血细胞生长较好,总磷含量、碱性磷酸酶活性均表现较高水平。     

Cu2+和Zn2+对普通小麦幼苗生长的影响

Zn^2＋在植物体中参与生长素的合成和某些酶系统的活动;Cu^2＋在催化氧化还原反应中起作用,是植物体内多种氧化酶的组成部分,与光合作用密切相关,在脂肪代谢、蛋白质分解中有一定的作用。当植物体内Cu^2＋和Zn^2＋含量超过一定浓度时对细胞有较大的毒害,危害植物的生长和发育,并可经食物链富集危害人的健康。本文研究了相同浓度的Cu^2＋和Zn^2＋对普通小麦幼苗生理生化特征的影响,以期为防止金属离子污染和培育抗性品种提供参考数据。     

New perspectives on S100 proteins: a multi-functional Ca 2+ -, Zn 2+ - and Cu 2+ -binding protein family

S100 proteins (16 members) show a very divergent pattern of cell- and tissue-specific expression, of subcel-lular localizations and relocations, of post-translational modifications, and of affinities for Ca 2+ , Zn 2+ , and Cu 2+ , consistent with their pleiotropic intra- and extracellular functions. Up to 40 target proteins are reported to interact with S100 proteins and for S100A1 alone 15 target proteins are presently known. Therefore it is not surprising that many functional roles have been proposed and that several human disorders such as cancer, neurodegenerative diseases, cardiomyopathies, inflammations, diabetes, and allergies are associated with an altered expression of S100 proteins. It is not unlikely that their biological activity in some cases is regulated by Zn 2+ and Cu 2+ , rather than by Ca 2+ Despite the numerous putative functions of S100 proteins, their three-dimensional structures of, e.g., S100B, S100A6, and S100A7 are surprisingly similar. They contain a compact dimerization domain whose conformation is rather insensitive to Ca 2+ binding and two lateral a-helices III and III, which project outward of each subunit when Ca 2+ is bound. Target docking depends on the two hydrophobic patches in front of the paired EF-hand generated by the binding of Ca 2+. The selec-tivity in target binding is assured by the central linker between the two EF-hands and the C-terminal tail. It appears that the S100-binding domain in some target proteins contains a basic amphiphilic a-helix and that the mode of interaction and activation bears structural similarity to that of calmodulin.© Kluwer Academic Publishers     

Study on synthesis, structure, and DNA-binding of Ni, Zn complexes with 2-phenylquinoline-4-carboylhydrazide

2-Phenylquinoline-4-carboylhydrazide (HL), and its novel nickel(II), zinc(II) complexes [M(HL)₂(L)]·2H₂O·NO₃ (M = Ni (1), M = Zn (2)), have been synthesized and characterized by elemental analysis, molar conductivity, and IR spectra. The crystal structure of [Ni(HL)₂(L)]·2H₂O·NO₃ obtained from ethanol solution was determined by X-ray diffraction analysis, crystallized in the rhombohedral system, space group , Z = 18, a = 31.913(3) Å, b = 31.913(3) Å, c = 27.709(2) Å, α = 90°, β = 90°, γ = 120°, R₁ = 0.0647. The interactions of the complexes and the ligand with calf thymus DNA had been investigated using UV-Vis spectra, fluorescent spectra, CD (circular dichroism) spectra, CV (cyclic voltammetry) and viscosity measurements. These compounds were tested against MFC (mouse forestomach carcinoma) cell lines. The complex 1 showed significant cytotoxic activity against MFC cell lines. The cleavage reaction on plasmid DNA has been monitored by agarose gel electrophoresis. Results suggest that the two complexes bound to DNA via a groove binding mode and the complexes can cleave pBR322 DNA.     

Copper-Ligand Interactions and Physiological free Radical Processes. Part 2. Influence of Cu2+ Ions on Cu+-Driven Oh Generation and Comparison with Their Effects on Fe2+-Driven Oh Production

In our search to establish a reference ·OH production system with respect to which the reactivity of copper(II) complexes could then be tested, the influence of free Cu²⁺ ions on the Cu⁺/H₂O₂ reaction has been investigated.

This influence depends on the C_Cu²⁺/C_Cu⁺ ratio. At low Cu²⁺ concentrations, ·OH damage to various detector molecules decreases with increasing Cu²⁺ concentrations until C_Cu²⁺/C_Cu⁺ reaches unity. Above this value, ·OH damage increases sharply until C_Cu²⁺/C_Cu⁺ becomes equal to 5 with salicylate and 2 with deoxyribose, ratios for which the protective effect of Cu²⁺ cancels. Finally, at higher concentrations, Cu²⁺ ions logically add their own ·OH production to that normally expected from Cu⁺ ions. The possible origin of this unprecedented alternate effect has been discussed. The possible influence of Cu⁺ ions on the generation of ·OH radicals by water gamma radiolysis has also been tested and, as already established for Cu²⁺ in a previous work, shown to be nonexistent. This definitely confirms that either form of ionised copper cannot scavenge ·OH radicals in the absence of a Iigand.     

Synthesis, characterisation of a complex of Cu(II) with a multidentate macrocyclic ligand and its interactions with anions

A macrocyclic ligand possessing a donor set of {N₃S₂} synthesised via Cs⁺-templation, 4-(pyridin-2-ylmethyl)-1,7-dithia-4,10-diazacyclododecane (L) and its Cu(II) complex, [CuL(NCMe)]²⁺ (6), are described. This Cu(II) complex interacts with a range of anions, F⁻, Cl⁻, Br⁻, I⁻, HCOO⁻, AcO⁻, CO₃²⁻, NO₃⁻, C₂O₄²⁻, H₂PO₄⁻, SCN⁻, CN⁻, BF₄⁻. Of the investigated anions, I⁻, SCN⁻, and CN⁻, show strong interaction with the Cu(II) centre as indicated by their spectral variations. The iodide particularly demonstrates distinct change in colour. This change originates from a newly appeared band at 471 nm upon iodide binding, which arises from the ligand (I⁻) to Cu(II) charge transfer (LMCT) in the iodide-substituted Cu(II) complex, [CuLI]⁺ (7). All organic compounds are characterised by NMR spectroscopy and/or microanalysis. The identities of the two Cu(II) complexes are confirmed by using microanalysis and the complex 6 is crystallographically analysed.     

Zn2+-linked dimerization of UreG from Helicobacter pylori, a chaperone involved in nickel trafficking and urease activation

The biosynthesis of the active metal-bound form of the nickel-dependent enzyme urease involves the formation of a lysine-carbamate functional group concomitantly with the delivery of two Ni(2+) ions into the precast active site of the apoenzyme and with GTP hydrolysis. In the urease system, this role is performed by UreG, an accessory protein belonging to the group of homologous P-loop GTPases, often required to complete the biosynthesis of nickel-enzymes. This study is focused on UreG from Helicobacter pylori (HpUreG), a bacterium responsible for gastric ulcers and cancer, infecting large part of the human population, and for which urease is a fundamental virulence factor. The soluble HpUreG was expressed in E. coli and purified to homogeneity. On-line size exclusion chromatography and light scattering indicated that apo-HpUreG exists as a monomer in solution. Circular dichroism, which demonstrated the presence of a well-defined secondary structure, and NMR spectroscopy, which revealed a large number of residues that appear structured on the basis of their backbone amide proton chemical shift dispersion, indicated that, at variance with other UreG proteins so far characterized, this protein is significantly folded in solution. The amino acid sequence of HpUreG is 29% identical to that of HypB from Methanocaldococcus jannaschii, a dimeric zinc-binding GTPase involved in the in vivo assembly of [Ni,Fe]-hydrogenase. A homology-based molecular model of HpUreG was calculated, which allowed us to identify structural and functional features of the protein. Isothermal titration microcalorimetry demonstrated that HpUreG specifically binds 0.5 equivalents of Zn(2+) per monomer (K(d) = 0.33 +/- 0.03 microM), whereas it has 20-fold lower affinity for Ni(2+) (K(d) = 10 +/- 1 microM). Zinc ion binding (but not Ni(2+) binding) causes protein dimerization, as confirmed using light scattering measurements. The structural rearrangement occurring upon Zn(2+)-binding and consequent dimerization was evaluated using circular dichroism and fluorescence spectroscopy. Fully conserved histidine and cysteine residues were identified and their role in zinc binding was verified by site-directed mutagenesis and microcalorimetry. The results are analyzed and discussed with respect to analogous examples of GTPases in nickel metabolism.     

N-terminal iron-mediated self-cleavage of human frataxin: regulation of iron binding and complex formation with target proteins

Frataxin is an iron-binding mitochondrial matrix protein that has been shown to mediate iron delivery during iron–sulfur cluster and heme biosynthesis. Mitochondrial processing peptidase (MPP) yields a form of human frataxin corresponding to residues 56–210. However, structural and functional studies have focused on a core structure that results from an ill-defined cleavage event at the N-terminus. Herein we show that the N-terminus of MPP-processed frataxin shows a unique high-affinity iron site and that this iron center appears to mediate a self-cleavage reaction. Moreover, the N-terminus appears to block previously defined iron-binding sites located on the carboxylate-rich surface defined by the helix (α1) and the β-sheet (β1), most likely through electrostatic contact with the carboxylate-rich surface on the core protein, as well as inhibiting iron-promoted binding of the iron–sulfur cluster assembly scaffold partner protein, ISU. The physiological significance of iron-mediated release of the N-terminal residues from this anionic surface is discussed.     

Helicobacter pylori interactions with host serum and extracellular matrix proteins: potential role in the infectious process.

Helicobacter pylori, a gram-negative spiral-shaped bacterium, specifically colonizes the stomachs of humans. Once established in this harsh ecological niche, it remains there virtually for the entire life of the host. To date, numerous virulence factors responsible for gastric colonization, survival, and tissue damage have been described for this bacterium. Nevertheless, a critical feature of H. pylori is its ability to establish a long-lasting infection. In fact, although good humoral (against many bacterial proteins) and cellular responses are observed, most infected persons are unable to eradicate the infection. A large body of evidence has shown that the interaction between H. pylori and the host is very complex. In addition to the effect of virulence factors on colonization and persistence, binding of specialized bacterial proteins, known as receptins, to certain host molecules (ligands) could explain the success of H. pylori as a chronically persisting pathogen. Some of the reported interactions are of high affinity, as revealed by their calculated dissociation constant. This review examines the binding of host proteins (serum and extracellular matrix proteins) to H. pylori and considers the significance of these interactions in the infectious process. A more thorough understanding of the kinetics of these receptin interactions could provide a new approach to preventing deeper tissue invasion in H. pylori infections and could represent an alternative to antibiotic treatment.     

Effects of Mg2+ and the beta gamma-subunit complex on the interactions of guanine nucleotides with G proteins

Mg2+ interacts with the alpha subunits of guanine nucleotide-binding regulatory proteins (G proteins) in the presence of guanosine-5'-[gamma-thio]triphosphate (GTP-gamma S) to form a highly fluorescent complex from which nucleotide dissociates very slowly. The apparent Kd for interaction of G alpha X GTP gamma S with Mg2+ is approximately 5 nM, similar to the Km for G protein GTPase activity X G beta gamma increases the rate of dissociation of GTP gamma S from G alpha X GTP gamma S or G alpha X GTP gamma S X Mg2+ at low concentrations of Mg2+. When the concentration of Mg2+ exceeds 1 mM, G beta gamma dissociates from G beta gamma X G alpha X GTP gamma S X Mg2+. Compared with the dramatic effect of Mg2+ on binding of GTP gamma S to G alpha, the metal has relatively little effect on the binding of GDP. However, G beta gamma increases the affinity of G alpha for GDP by more than 100-fold. High concentrations of Mg2+ promote the dissociation of GDP from G beta gamma X G alpha X GDP, apparently without causing subunit dissociation. The steady-state rate of GTP hydrolysis is strictly correlated with the rate of dissociation of GDP from G alpha under all conditions examined. Thus, there are at least two sites for interaction of Mg2+ with G protein-nucleotide complexes. Furthermore, binding of G beta gamma and GTP gamma S to G alpha is negatively cooperative, while the binding interaction between G beta gamma and GDP is strongly positive.     

Selective binding of Ni2+ and Cu2+ metal ions with naphthazarin esters isolated from Arnebia euchroma

Naphthazarin esters (C1–C4) isolated from the roots of Arnebia euchroma are found as skilled dual chemosensors for Ni²⁺ and Cu²⁺ among Pb²⁺, Na²⁺, K²⁺, Hg²⁺, Mg²⁺, and Ca²⁺ metal ions. C1–C4 esters exhibited a red shift of 54 nm with Ni²⁺ and 30 nm with Cu²⁺ metal ions in absorption. There is a formation of red-shifted bands between 517 and 613 nm in the absorption spectrum of C1–C4 sensors on binding with Ni²⁺ and Cu²⁺ ions. The addition of Ni²⁺ and Cu²⁺ ions to sensors C1–C4 stimulates a remarkable color change from reddish pink to purple and light blue, respectively. These color changes can be identified with the naked eye. The significant downfield shifts of CO and OH peaks in nuclear magnetic resonance (NMR) spectrum confirm the chelation as binding mechanism. With ultraviolet–visble and NMR studies, it is found that C1–C4 esters possessed notable selectivity and sensitivity toward Ni²⁺ and Cu²⁺ over other metal ions.     

Cu~(2+)胁迫条件对涡虫体内过氧化氢酶活性的影响

实验以0 .2 5、0 .5、1.0、2 .0mg·kg- 1 4种质量浓度的CuSO4 溶液培养东亚三角头涡虫(Dugesiajaponia) 4 8h后,提取其体内蛋白质并测定其过氧化氢酶(CAT)的活性。测定结果:涡虫体内蛋白的CAT酶活性依次为12 .5 9、2 3.74、18.37、18.72ū·mg- 1 。以自来水(实验室常规培养方法)为对照,其相应的酶活性为18.78ū·ｍｇ- 1 。实验结果表明:低浓度下Cu2 + 刺激CAT酶活性增加,而在高浓度下,由于涡虫耐受力的存在而使涡虫的CAT酶活性维持在正常水平(与对照组差异不大)。当Cu2 + 浓度达到4mg·kg- 1 时所培养的涡虫不到2 4h全部死亡。     

Cd~(2+)和Ni~(2+)对亚洲玉米螟求偶行为的影响

【目的】探索农田重金属Cd~(2+)和Ni~(2+)污染对植食性昆虫求偶行为潜在的影响。【方法】在人工饲料中添加不同类型的重金属,设置成Cd~(2+)、Ni~(2+)、Cd~(2+)-Ni~(2+)和空白对照(CK)4个处理,重金属浓度均为5 mg/kg。实验室条件下饲养亚洲玉米螟Ostrinia furnacalis(Guenée)初孵幼虫,系统观察其各代存活雌蛾求偶行为。【结果】重金属Cd~(2+)和Ni~(2+)对亚洲玉米螟3代存活雌蛾求偶百分率、求偶高峰期出现的时间及求偶持续时间都产生了影响。其中第2代求偶百分率与对照组差距最大,第3代受到的影响最小,甚至表现出促进作用;各重金属处理的求偶高峰期出现的时间仅在第2代与对照组保持一致外,第1代、第3代均发生改变;存活雌蛾求偶平均持续时间随着重金属饲养亚洲玉米螟代数的增加而逐代减少。与对照组各代各暗期最长求偶持续时间为4.89 h相比,重金属处理的雌蛾平均求偶持续时间最大值出现在Ni~(2+)处理第1代的第3个暗期,值为5.56 h;最小值为Cd~(2+)处理的第2代的第2、第3个暗期,值均为3.08 h。【结论】重金属Cd~(2+)和Ni~(2+)对亚洲玉米螟存活雌蛾的求偶行为产生了影响。     

The His-His sequence of the antimicrobial peptide demegen P-113 makes it very attractive ligand for Cu2+

Histatins are a family of histidine-rich, cationic peptides up to 38 amino acids long. As other antimicrobial peptides histatins exhibit in vitro activity against both bacteria and yeasts. A 12 amino acid amidated fragment of histatin 5, designated P-113 or demegen, has been identified as the smallest fragment retaining antimicrobial activity comparable to the parent compound. Demegen, AKRHHGYKRKFH, has three His and a N-terminal group known to participate in copper ion coordination. In this study potentiometric and spectroscopic (UV-vis, CD, EPR, NMR) measurements were used to evaluate the stability constants, stoichiometry and structures of Cu(2+) complexes with demegen P-113 and its analogues in aqueous solution. The main aim of this work was to understand the role of two adjacent histidine residues in metal ion binding. The comparison with results for modified ligands showed that two histydyl residues are basic for complex formation in the 4.5-7 pH range.