X-ray diffraction study of the zinc(II) binding sites in yeast phenylalanine transfer RNA preferential binding of zinc to guanines in purine-purine sequences

An X-ray diffraction study of a zinc(II) complex of tRNA^Phe from yeast reveals the presence of five zin-binding sites on the tRNA molecule. Two of the cooperatively bound Mg²⁺ in the native tRNA structure are replaced by Zn²⁺. The remaining sites involve direct coordination of zinc to the N7 position of tRNA guanine bases, G15, G43 and G45. Thus, zinc displays a high specificity for binding to guanine bases in purine-purine sequences.     

Structural effects of Zn on cell membranes and molecular models

Zinc is an essential element for nutrition as well as for the proper development and function of brain cells, and its traces are present in a wide range of foods. It is a constituent of many enzyme systems and is an integral part of insulin and of the active site of intracellular enzymes. However, excessive accumulation of zinc or its release from the binding sites may become detrimental for neurons. With the aim to better understand the molecular mechanisms of the interaction of zinc ions with cell membranes, it was incubated with intact human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), cholinergic murine neuroblastoma cells, and molecular models of cell membranes. These consisted in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes present in the outer and inner monolayers of most plasmatic cell membranes, particularly that of human erythrocytes, respectively. The capacity of zinc ions to perturb the bilayer structures of DMPC and DMPE was assessed by X-ray diffraction, DMPC large unilamellar vesicles (LUV) and IUM were studied by fluorescence spectroscopy, intact human erythrocytes were observed with scanning electron microscopy (SEM), and neuroblastoma cell morphology was observed under inverted microscope. This study presents evidence that 0.1 mM Zn and higher concentrations affect cell membrane and molecular models.     

Interferonbeta-induced changes in metallothionein expression and subcellular distribution of zinc in HepG2 cells

We evaluated the changes of metallothionein induction and cellular zinc distribution in HepG2 cells by interferonbeta treatment. Immunohistochemical staining of metallothionein was observed in the cytoplasm and nuclei of hepatocytes; which was observed predominantly in the cells treated with interferon and zinc compared to those with zinc alone, interferon alone or the no-treated control. The cellular zinc level was higher in order of the interferon- and zinc-treated cells, the zinc-alone-treated cells, and the interferon-alone-treated cells. Flow cytometry showed that S-phase population increased in interferon-alone-treated cells and interferon- and zinc-treated cells, but not in zinc-alone-treated ones. Cellular elemental distribution was analyzed using in-air micro-particle induced X-ray emission. In zinc-alone-treated sample, X-ray spectra showed good consistency between the enhanced cellular zinc distribution and the phosphorous map. Localizations of bromine followed by interferon treatment were found accompanying a spatial correlation with the phosphorous map. The samples treated with interferon and zinc showed the marked accumulation of zinc and bromine. Discrete bromine accumulation sites were clearly visible with a strong spatial correlation followed by zinc accumulation. These findings suggest that interferonbeta in combination with zinc predominantly induces metallothionein expression in HepG2 cells. In addition, interferonbeta may promote the translocation of metallothionein-bound zinc from cytoplasm to S-phase nuclei.     

In situ X-ray absorption spectroelectrochemical study of hydroxocobalamin

 An in situ X-ray absorption spectroscopy (XAS) spectroelectrochemical study of aquocobalamin (system B_12a-B_12r-B_12s) has been carried out in aqueous solutions buffered at different pH values. To the best of our knowledge, this is the first structural study of aquocobalamin at room temperature under controlled oxidation conditions. Most of the previous work was in fact performed using frozen samples chemically treated to produce the species. The spectroelectrochemical approach offers several advantages: (1) the reduction products may be studied without poisoning the system with chemical reductive reagents and (2) any possible variation of the oxidation state owing to the electrons produced by the incident beam is avoided as the electrode, under potentiostatic control, acts as a scavenger. The spectroelectrochemical approach, together with more careful data analysis, has led to an improved interpretation of the XAS data. These conditions were not met in previous works where the oxidation state was not controlled and multiple scattering contributions were not taken into account. The general shape of the XAS spectra of the different species is not greatly affected by pH. A signature for the base-off square-planar coordination has been evidenced for the Co(II) compound at basic pH. A new signature for Co(I), indicating square-planar coordination, has been identified on the experimental spectra and simulated in theoretical X-ray absorption near-edge structure (XANES) studies. The flexibility of the electrochemical approach, that permits to unambiguously establish the formal oxidation state, has led to very reliable values for energy shift and peak intensity variations. The experimental XANES and extended X-ray absorption fine structure (EXAFS) spectra with a very good signal-to-noise ratio have been processed using the GNXAS package that takes into account multiple scattering contributions. EXAFS and XANES independent analysis result in the same structural model. The reduction from Co(III) to Co(II) produces the most significant structural changes: the cobalt coordination number decreases from six to five, and the edge position shifts by 2.4±0.3 eV. In addition, the XANES spectra are strongly modified. The reduction from Co(II) to Co(I) produces mainly electronic effects with no apparent change of the coordination number. A discussion of the limits and potentialities of EXAFS in this type of study has also been included. Received: 26 July 1999 / Accepted: 22 October 1999     

Characterization of the zinc sites in cobalamin-independent and cobalamin-dependent methionine synthase using zinc and selenium X-ray absorption spectroscopy

X-ray absorption spectroscopy has been used to investigate binding of selenohomocysteine to cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthase enzymes of Escherichia coli. We have shown previously [Peariso et al. (1998) J. Am. Chem. Soc. 120, 8410-8416] that the Zn sites in both enzymes show an increase in the number of sulfur ligands when homocysteine binds. The present data provide direct evidence that this change is due to coordination of the substrate to the Zn. Addition of L-selenohomocysteine to either MetE or the N-terminal fragment of MetH, MetH(2-649), causes changes in the zinc X-ray absorption near-edge structure that are remarkably similar to those observed following the addition of L-homocysteine. Zinc EXAFS spectra show that the addition of L-selenohomocysteine changes the coordination environment of the zinc in MetE from 2S + 2(N/O) to 2S + 1(N/O) + 1Se and in MetH(2-649) from 3S + 1(N/O) to 3S + 1Se. The Zn-S, Zn-Se, and Se-S bond distances determined from the zinc and selenium EXAFS data indicate that the zinc sites in substrate-bound MetE and MetH(2-649) both have an approximately tetrahedral geometry. The selenium edge energy for selenohomocysteine shifts to higher energy when binding to either methionine synthase enzyme, suggesting that there is a slight decrease in the effective charge of the selenium. Increases in the Zn-Cys bond distances upon selenohomocysteine binding together with identical magnitudes of the shifts to higher energy in the Se XANES spectra of MetE and MetH(2-649) suggest that the Lewis acidity of the Zn sites in these enzymes appears the same to the substrate and is electronically buffered by the Zn-Cys interaction.     

Zinc deficiency-induced iron accumulation, a consequence of alterations in iron regulatory protein-binding activity, iron transporters, and iron storage proteins

One consequence of zinc deficiency is an elevation in cell and tissue iron concentrations. To examine the mechanism(s) underlying this phenomenon, Swiss 3T3 cells were cultured in zinc-deficient (D, 0.5 microM zinc), zinc-supplemented (S, 50 microM zinc), or control (C, 4 microM zinc) media. After 24 h of culture, cells in the D group were characterized by a 50% decrease in intracellular zinc and a 35% increase in intracellular iron relative to cells in the S and C groups. The increase in cellular iron was associated with increased transferrin receptor 1 protein and mRNA levels and increased ferritin light chain expression. The divalent metal transporter 1(+)iron-responsive element isoform mRNA was decreased during zinc deficiency-induced iron accumulation. Examination of zinc-deficient cells revealed increased binding of iron regulatory protein 2 (IRP2) and decreased binding of IRP1 to a consensus iron-responsive element. The increased IRP2-binding activity in zinc-deficient cells coincided with an increased level of IRP2 protein. The accumulation of IRP2 protein was independent of zinc deficiency-induced intracellular nitric oxide production but was attenuated by the addition of the antioxidant N-acetylcysteine or ascorbate to the D medium. These data support the concept that zinc deficiency can result in alterations in iron transporter, storage, and regulatory proteins, which facilitate iron accumulation.     

ZnT-8, A Pancreatic Beta-Cell-Specific Zinc Transporter

The zinc content in the pancreatic beta cell is among the highest of the body. Zinc appears to be an important metal for insulin-secreting cells as insulin is stored inside secretory vesicles as a solid hexamer bound with two Zn²⁺ ions per hexamer. Zinc is also an important component of insulin secretion mechanisms and is likely to modulate the function of neighbouring cells via paracrine/autocrine interactions. Therefore beta cells undoubtedly need very efficient and specialized transporters to accumulate sufficient amounts of zinc in secretion vesicles. We report here the discovery and the characteristics of a new zinc transporter, ZnT-8, belonging to the CDF (Cation Diffusion Facilitator) family and expressed only in pancreatic beta cells. This transporter, localized in secretion vesicles membrane, facilitates the accumulation of zinc from the cytoplasm into intracellular insulin-containing vesicles and is a major component for providing zinc to insulin maturation and/or storage processes in insulin-secreting pancreatic beta cells. We discovered mammalian orthologs (rat, mouse, chimpanzee, and dog) and found these ZnT-8 proteins very similar (98% conserved amino acids) to human ZnT-8, indicating a high conservation during evolution.     

Metal poison inhibition of carbonic anhydrase

Carbonic anhydrase is inhibited by the “metal poison” cyanide. Several spectroscopic investigations of carbonic anhydrase where the natural zinc ion has been replaced by cobalt have further strengthened the view that cyanide and cyanate bind directly to the metal. We have determined the structure of human carbonic anhydrase II inhibited by cyanide and cyanate, respectively, by X-ray crystallography. It is shown that the inhibitors replace a molecule of water, which forms a hydrogen bond to the peptide nitrogen of Thr-199 in the native structure. The coordination of the zinc ion is hereby left unaltered compared to the native crystal structure, so that the zinc coordinates three histidines and one molecule of water or hydroxyl ion in a tetrahedral fashion. The binding site of the two inhibitors is identical to what earlier has been suggested to be the position of the substrate (CO₂) when attacked by the zinc bound hydroxyl ion. The peptide chain undergoes no significant alterations upon binding of either inhibitor. © 1993 Wiley-Liss, Inc.     

The role of Zn in the interplay among Langmuir-Blodgett multilayer and myelin basic protein: a quantitative analysis of XANES spectra

We have performed a quantitative analysis of the X-ray absorption near-edge structure (XANES) spectra at the Zinc K-edge of systems formed by phospholipid Langmuir-Blodgett multilayers (LBMLs) in the presence and in the absence of myelin basic protein (MBP) and in two hydration conditions. These spectra have been analysed by a new procedure called Minuit XANes (MXAN) which is able to perform a quantitative fit of XANES data in terms of structural parameters. By this method, we have been able to correlate the relevant differences between the spectra observed in the XANES range with the coordination changes due to reduction of the space around the Zinc when the level of hydration is lowered and/or the myelin basic protein is added. These spectral differences are peculiar of the XANES energy range, and are not present in the extended X-ray absorption fine structure (EXAFS) energy range where the analysis was previously performed. With this investigation, we give an unambiguous answer to the question of the role of zinc in such complexes by showing that the metal interacts with both the phospholipid heads of the substrate and the myelin basic protein.     

Metals on the move: zinc ions in cellular regulation and in the coordination dynamics of zinc proteins

Homeostatic control maintains essential transition metal ions at characteristic cellular concentrations to support their physiological functions and to avoid adverse effects. Zinc is especially widely used as a catalytic or structural cofactor in about 3000 human zinc proteins. In addition, the homeostatic control of zinc in eukaryotic cells permits functions of zinc(II) ions in regulation and in paracrine and intracrine signaling. Zinc ions are released from proteins through ligand-centered reactions in zinc/thiolate coordination environments, and from stores in cellular organelles, where zinc transporters participate in zinc loading and release. Muffling reactions allow zinc ions to serve as signaling ions (second messengers) in the cytosol that is buffered to picomolar zinc ion concentrations at steady-state. Muffling includes zinc ion binding to metallothioneins, cellular translocations of metallothioneins, delivery of zinc ions to transporter proteins, and zinc ion fluxes through cellular membranes with the result of removing the additional zinc ions from the cytosol and restoring the steady-state. Targets of regulatory zinc ions are proteins with sites for transient zinc binding, such as membrane receptors, enzymes, protein–protein interactions, and sensor proteins that control gene expression. The generation, transmission, targets, and termination of zinc ion signals involve proteins that use coordination dynamics in the inner and outer ligand spheres to control metal ion association and dissociation. These new findings establish critically important functions of zinc ions and zinc metalloproteins in cellular control.     

Complexation and detoxification of Zn and Cd in metal accumulating plants

Metal accumulating plants exposed to toxic levels of zinc (Zn) and cadmium (Cd) uptake metals through extracellular and intracellular complexation with inorganic and organic ligand formation. However, little is known about the nature and formation mechanism of these metal–ligand complexes. Though, Zn and Cd have many similar chemical properties, yet their complexation and compartmentalization in plants vary with plant species. In principal, the question arises what factors govern Zn and Cd partitioning in plants? What form of the metal is taken up by the root, and is further distributed and accumulated in both vegetative and reproductive tissues? Therefore, the aim of present study is to address several questions concerning the mechanisms of Zn and Cd coordination and compartmentalization in plants using X-ray absorption spectroscopy (XAS) technique. XAS allows direct determination of elemental oxidation states and coordination environments in different plant tissues. This review article briefly explains some other important techniques of XAS; EXAFS (extended X-ray absorption fine structure) and XANES (X-ray absorption near edge structure), which are employed for determining Zn and Cd complexation within the plant. Therefore, In present review, the predominant as well as the minor chemical forms of Zn and Cd present in particular plant tissue have been discussed which could give better insight towards metal accumulation and detoxification mechanisms operated in plants. This information could assist in employing suitable hyperaccumulator plants for metal phytoextraction and reclamation of metal contaminated sites.     

Synthesis and structural analysis of copper(II) cysteine complexes

This report describes the synthesis and structural analysis of stable copper(II) cysteine complexes. Pale pink copper(II) cysteine complexes were synthesized in mole ratios of 1:2, 1:4, and 1:6 of copper(II):cysteine in ethanol. Infrared spectroscopy and X-ray absorption spectroscopy confirmed that copper(II) binding occurred via the thiol ligand of cysteine. XANES analysis showed that the oxidation state of copper remained as copper(II) and the local atomic geometry was similar in all of the cysteine complexes. The EXAFS data indicate that the copper(II) cysteine complexes are forming ring type structures with sulfur ligands from the cysteines acting as bridging ligands. X-ray diffraction revealed that the copper(II) cysteine complexes formed monoclinic cells with maximum crystallinity found in the 1:4 copper(II):cysteine complex.     

Calcium binding sites in the vesicles of the carotid and aortic body chief cells

Summary Chief cells of the carotid and aortic body chemoreceptors possess numerous cytoplasmic dense-core vesicles which are known to contain primarily dopamine. Following fixation in solutions containing 50 mM CaCl₂, a 20–30 nm electron-dense particle (EDP) is often observed eccentrically located in many of the vesicles. Approximately 44 % of the carotid body and 16 % of the aortic body vesicles contain an EDP. The EDP probably represents the Ca^{+ +} binding site critical to the stimulus-secretion coupling events culminating in exocytosis of these vesicles. The presence of Ca^{+ +} in the cytoplasmic vesicles was verified by electron probe X-ray microanalysis.Supported by a Grant-in-Aid from the American Heart Association (77630) and by funds contributed in part by the Texas Affiliate. The authors wish to thank Ms. Teri Heitman for her excellent technical assistance     

Toward a molecular understanding of zinc metabolism

The absorption of zinc is increased when the dietary zinc supply is low. This is caused by increased intestinal transport and reduced secretion of endogenous zinc into the intestine. Kinetic analysis of zinc transport, based on data from either the isolated perfused intestine or brush border membrane vesicles, demonstrates uptake velocity is increased homeostatically by a carrier-mediated phase of transport in response to low dietary zinc. Zinc within intestinal cells binds to high molecular weight proteins and metallothionein. Expression of the metallothionein gene is altered by zinc status and the protein appears to have a function in intestinal cells. Zinc transport across the basolateral membrane is also carrier-mediated and may be ATP-driven. Newly absorbed zinc is transported via albumin, first to the liver and then is redistributed to other tissues, particularly muscle and bone which provide the greatest reserves. Plasma zinc levels remain relatively constant except during periods of dietary zinc depletion and acute responses to stress, infection or inflammation where they are depressed. Experiments with intact rats and isolated rat liver parenchymal cells have shown that hepatic zinc turnover is rapid. Stimulation of liver cells by glucocorticoids, glucagon, epinephrine, cAMP or interleukin-1-like factors alters uptake/exchange kinetics such that there is a net accumulation of cellular zinc. Metallothionein gene expression is enhanced by these hormonal signals, and a considerable portion of the newly accumulated zinc is accounted for as that associated with this zinc-binding protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino Acid Composition, Immunoreactivity, Sequence Analysis, and Function of Bovine Hippocampal Metallothionein Isoforms

Abstract: The high concentration of zinc in the hippocampal mossy fiber axon boutons is localized in the vesicles and is mobilized by exocytosis of the zinc-laden vesicles. Because "free" zinc in excess is a neurotoxic substance inhibiting an extensive number of sulfhydryl-containing enzymes and receptor sites, we hypothesized that low-molecular-weight zinc binding proteins must exist in the hippocampus to regulate the steady-state concentration of zinc. In this communication, we report that the bovine hippocampus synthesizes metallothionein (MT) isoforms that are similar, but not identical, to those of the rat brain MT isoforms and cross-react poorly with antibodies formed against the hepatic MT isoforms, suggesting that the immunologically dominant regions of hippocampal MT (residues 1–29) are not conserved. A comparative sequence analysis of bovine hippocampal MTs and bovine hepatic MT isoforms I and II revealed a 90% sequence identity, being mostly different in residues 1–29. The results of these studies suggest that the hippocampal MT isoforms, which are synthesized on a continuous basis, may play a role in regulating the transport, accumulation, and compartmentation of zinc in the hippocampus.     

ZnT-2, a mammalian protein that confers resistance to zinc by facilitating vesicular sequestration.

A cDNA encoding a second zinc transporter (ZnT-2) was isolated from a rat kidney cDNA expression library by complementation of a zinc-sensitive BHK cell line. The protein predicted from the open reading frame of ZnT-2 cDNA has 359 amino acids and initiates with a CTG codon. It resembles ZnT-1 (a plasma membrane protein that stimulates zinc efflux) in overall topology in that it has six membrane-spanning domains, a histidine-rich intracellular loop and a long C-terminal tail; however, the overall amino acid identity is only 26%. Unlike ZnT-1, which is in the plasma membrane and lowers cellular zinc by stimulating zinc efflux, ZnT-2 is localized on vesicles and allows the zinc-sensitive BHK cells to accumulate zinc to levels that are much higher than non-transformed cells can tolerate. Zinc was visualized within these vesicles with zinquin, a zinc-specific fluorescent probe. The intracellular compartment that accumulates zinc is acidic as revealed by staining with acridine orange or LysoTracker. Prolonged exposure of cells expressing ZnT-2 to zinc causes an accretion of intracellular vesicles. We suggest that ZnT-2 protects these cells from zinc toxicity by facilitating zinc transport into an endosomal/lysosomal compartment.     

Zinc transporter 2 (SLC30A2) can suppress the vesicular zinc defect of adaptor protein 3-depleted fibroblasts by promoting zinc accumulation in lysosomes

Zinc accumulation in the lumen of cytoplasmic vesicles is one of the mechanisms by which cells can store significant amounts of this essential but potentially toxic biometal. Previous studies had demonstrated reduced vesicular zinc levels in fibroblasts from mutant mice deficient in adaptor protein 3 (AP-3), a complex involved in protein trafficking to late endosomes and lysosomes. We have observed a similar phenotype in the human fibroblastoid cell line, M1, upon small interference RNA-mediated AP-3 knockdown. A survey of the expression and localization of zinc transporter (ZnT) family members identified ZnT2, ZnT3, and ZnT4 as likely mediators of vesicular zinc accumulation in M1 cells. Expression of green fluorescence protein (GFP)-tagged ZnT2 and ZnT3 promoted accumulation of vesicular zinc as visualized using the indicator zinquin. Moreover, GFP-ZnT2 overexpression elicited a significant accumulation of zinc within mature lysosomes, which in untransfected M1 cells contained little or no chelatable zinc, and restored the zinc storage capability of AP-3-deficient cells. These results suggest that ZnT2 can facilitate vesicular zinc accumulation independently of AP-3 function, and validate the M1 fibroblastoid line as a human cell culture system amenable to the study of vesicular zinc regulation using techniques compatible with functional genomic approaches.     

X-ray absorption spectroscopy studies of the adducts formed between cytotoxic gold compounds and two major serum proteins

Gold metallodrugs form a class of promising antiproliferative agents showing a high propensity to react with proteins. We exploit here X-ray absorption spectroscopy (XAS) methods [both X-ray absorption near-edge spectroscopy (XANES) and extended X-ray absorption fine structure (EXAFS)] to gain insight into the nature of the adducts formed between three representative gold(I, III) metallodrugs (i.e., auranofin, [Au(2,2′-bipyridine)(OH)₂](PF₆), Aubipy, and dinuclear [Au₂(6,6′-dimethyl-2,2′-bipyridine)₂(μ-O)₂](PF₆)₂, Auoxo6) and two major plasma proteins, namely, bovine serum albumin (BSA) and human serum apotransferrin (apoTf). The following metallodrug–protein systems were investigated in depth: auranofin/apoTf, Aubipy/BSA, and Auoxo6/apoTf. XANES spectra revealed that auranofin, upon protein binding, conserves its gold(I) oxidation state. Protein binding most probably takes place through release of the thiosugar ligand and its subsequent replacement by a thiol (or a thioether) from the protein. This hypothesis is independently supported by EXAFS results. In contrast, the reactions of Aubipy with serum albumin and of Auoxo6 with serum apoTf invariantly result in gold(III) to gold(I) reduction. Gold(III) reduction, clearly documented by XANES, is accompanied, in both cases, by release of the bipyridyl ligands; for Auoxo6 cleavage of the gold–gold dioxo bridge is also observed. Gold(III) reduction leads to formation of protein-bound gold(I) species, with deeply modified metal coordination environments, as evidenced by EXAFS. In these adducts, the gold(I) centers are probably anchored to the protein through nitrogen donors. In general, these two XAS methods, i.e., XANES and EXAFS, used here jointly, allowed us to gain independent structural information on metallodrug/protein systems; detailed insight into the gold oxidation state and the local environment of protein-bound metal atoms was achieved in the various cases.