Structural basis for metal ion coordination and the catalytic mechanism of sphingomyelinases D

Sphingomyelinases D (SMases D) from Loxosceles spider venom are the principal toxins responsible for the manifestation of dermonecrosis, intravascular hemolysis, and acute renal failure, which can result in death. These enzymes catalyze the hydrolysis of sphingomyelin, resulting in the formation of ceramide 1-phosphate and choline or the hydrolysis of lysophosphatidyl choline, generating the lipid mediator lysophosphatidic acid. This report represents the first crystal structure of a member of the sphingomyelinase D family from Loxosceles laeta (SMase I), which has been determined at 1.75-angstrom resolution using the quick cryo-soaking technique and phases obtained from a single iodine derivative and data collected from a conventional rotating anode x-ray source. SMase I folds as an (alpha/beta)(8) barrel, the interfacial and catalytic sites encompass hydrophobic loops and a negatively charged surface. Substrate binding and/or the transition state are stabilized by a Mg2+ ion, which is coordinated by Glu(32), Asp(34), Asp(91), and solvent molecules. In the proposed acid base catalytic mechanism, His(12) and His(47) play key roles and are supported by a network of hydrogen bonds between Asp(34), Asp(52), Trp(230), Asp(233), and Asn(252).

A new approach for determining stability constants of metal ion complexes in aquatic systems based on flow injection-ion exchange-flame atomic absorption spectrometry coupling

A method based an ion exchange(IE)-atomic absorption spectrometry(AAS) coupled by flow techniques, allowing the determination of formation constants of, at least, the first species of complex systems, in aqueous solution, was developed.The IE-AAS coupling reduces significantly the number of experimental steps in comparison with IE batch methods, resulting in an important increase in analytical rate. The method is simple both from experimental and computational points of view, making possible its utilization by workers without special expertise in the field of complex equilibria in solution. on the other hand, taking into account mainly the amount of hollow cathode lamps available to date, the developed procedure may be applied, within certain limitations, to the study of many systems whose features prevent the use of traditional approaches.

Hydroxythiazole-Based Fluorescent Probes for Fluoride Ion Detection

This work describes the synthesis of five O-silyloxy-1,3-thiazoles and their use as fast-response turn-on probes for fluoride ion detection in polar aprotic solvents and in aqueous cetyltrimethylammonium bromide micellar medium. The fluoride-triggered deprotection of these silyl ethers results in ca. 180-nm shifts in the fluorescence emission wavelengths. All compounds are suitable for the detection of fluoride ions with a detection limit in DMSO of 107 mol?L1; derivatives containing a 2-pyridyl moiety in the thiazole system are more efficient than those with a 3- or 4-pyridyl moiety. Typical anionic interferents, such as acetate or chloride, are not detected by O-silyloxy-1,3-thiazoles, making these compounds very specific for fluoride.

Effect of denture cleansers on metal ion release and surface roughness of denture base materials

Chemical disinfectants are usually associated with mechanical methods to remove stains and reduce biofilm formation. This study evaluated the effect of disinfectants on release of metal ions and surface roughness of commercially pure titanium, metal alloys, and heat-polymerized acrylic resin, simulating 180 immersion trials. Disk-shaped specimens were fabricated with commercially pure titanium (Tritan), nickel-chromium-molybdenum-titanium (Vi-Star), nickel-chromium (Fit Cast-SB Plus), and nickel-chromium-beryllium (Fit Cast-V) alloys. Each cast disk was invested in the flasks, incorporating the metal disk to the heat-polymerized acrylic resin. The specimens (n=5) were immersed in these solutions: sodium hypochlorite 0.05%, Periogard, Cepacol, Corega Tabs, Medical Interporous, and Polident. Deionized water was used as a control. The quantitative analysis of metal ion release was performed using inductively coupled plasma mass spectrometry (ELAN DRC II). A surface analyzer (Surftest SJ-201P) was used to measure the surface roughness (µm). Data were recorded before and after the immersions and evaluated by two-way ANOVA and Tukey's test (α=0.05). The nickel release proved most significant with the Vi-Star and Fit Cast-V alloys after immersion in Medical Interporous. There was a significant difference in surface roughness of the resin (p=0.011) after immersion. Cepacol caused significantly higher resin roughness. The immersion products had no influence on metal roughness (p=0.388). It could be concluded that the tested alloys can be considered safe for removable denture fabrication, but disinfectant solutions as Cepacol and Medical Interporous tablet for daily denture immersion should be used with caution because it caused greater resin surface roughness and greater ion release, respectively.

Surface modification by metal ion implantation forming metallic nanoparticles in insulating matrix.

There is special interest in the incorporation of metallic nanoparticles in a surrounding dielectric matrix for obtaining composites with desirable characteristics such as for surface plasmon resonance, which can be used in photonics and sensing, and controlled surface electrical conductivity. We investigated nanocomposites produced through metallic ion implantation in insulating substrate, where the implanted metal self-assembles into nanoparticles. During the implantation, the excess of metal atom concentration above the solubility limit leads to nucleation and growth of metal nanoparticles, driven by the temperature and temperature gradients within the implanted sample including the beam-induced thermal characteristics. The nanoparticles nucleate near the maximum of the implantation depth profile (projected range), that can be estimated by computer simulation using the TRIDYN. This is a Monte Carlo simulation program based on the TRIM (Transport and Range of Ions in Matter) code that takes into account compositional changes in the substrate due to two factors: previously implanted dopant atoms, and sputtering of the substrate surface. Our study suggests that the nanoparticles form a bidimentional array buried few nanometers below the substrate surface. More specifically we have studied Au/PMMA (polymethylmethacrylate), Pt/PMMA, Ti/alumina and Au/alumina systems. Transmission electron microscopy of the implanted samples showed the metallic nanoparticles formed in the insulating matrix. The nanocomposites were characterized by measuring the resistivity of the composite layer as function of the dose implanted. These experimental results were compared with a model based on percolation theory, in which electron transport through the composite is explained by conduction through a random resistor network formed by the metallic nanoparticles. Excellent agreement was found between the experimental results and the predictions of the theory. It was possible to conclude, in all cases, that the conductivity process is due only to percolation (when the conducting elements are in geometric contact) and that the contribution from tunneling conduction is negligible.

Divalent metal-ion transporter DMT1 mediates both H+ -coupled Fe2+ transport and uncoupled fluxes

The H(+) -coupled divalent metal-ion transporter DMT1 serves as both the primary entry point for iron into the body (intestinal brush-border uptake) and the route by which transferrin-associated iron is mobilized from endosomes to cytosol in erythroid precursors and other cells. Elucidating the molecular mechanisms of DMT1 will therefore increase our understanding of iron metabolism and the etiology of iron overload disorders. We expressed wild type and mutant DMT1 in Xenopus oocytes and monitored metal-ion uptake, currents and intracellular pH. DMT1 was activated in the presence of an inwardly directed H(+) electrochemical gradient. At low extracellular pH (pH(o)), H(+) binding preceded binding of Fe(2+) and its simultaneous translocation. However, DMT1 did not behave like a typical ion-coupled transporter at higher pH(o), and at pH(o) 7.4 we observed Fe(2+) transport that was not associated with H(+) influx. His(272) --> Ala substitution uncoupled the Fe(2+) and H(+) fluxes. At low pH(o), H272A mediated H(+) uniport that was inhibited by Fe(2+). Meanwhile H272A-mediated Fe(2+) transport was independent of pH(o). Our data indicate (i) that H(+) coupling in DMT1 serves to increase affinity for Fe(2+) and provide a thermodynamic driving force for Fe(2+) transport and (ii) that His-272 is critical in transducing the effects of H(+) coupling. Notably, our data also indicate that DMT1 can mediate facilitative Fe(2+) transport in the absence of a H(+) gradient. Since plasma membrane expression of DMT1 is upregulated in liver of hemochromatosis patients, this H(+) -uncoupled facilitative Fe(2+) transport via DMT1 can account for the uptake of nontransferrin-bound plasma iron characteristic of iron overload disorders.

Functional properties of multiple isoforms of human divalent metal-ion transporter 1 (DMT1)

DMT1 (divalent metal-ion transporter 1) is a widely expressed metal-ion transporter that is vital for intestinal iron absorption and iron utilization by most cell types throughout the body, including erythroid precursors. Mutations in DMT1 cause severe microcytic anaemia in animal models. Four DMT1 isoforms that differ in their N- and C-termini arise from mRNA transcripts that vary both at their 5'-ends (starting in exon 1A or exon 1B) and at their 3'-ends giving rise to mRNAs containing (+) or lacking (-) the 3'-IRE (iron-responsive element) and resulting in altered C-terminal coding sequences. To determine whether these variations result in functional differences between isoforms, we explored the functional properties of each isoform using the voltage clamp and radiotracer assays in cRNA-injected Xenopus oocytes. 1A/IRE+-DMT1 mediated Fe2+-evoked currents that were saturable (K(0.5)(Fe) approximately 1-2 microM), temperature-dependent (Q10 approximately 2), H+-dependent (K(0.5)(H) approximately 1 muM) and voltage-dependent. 1A/IRE+-DMT1 exhibited the provisional substrate profile (ranked on currents) Cd2+, Co2+, Fe2+, Mn2+>Ni2+, V3+>>Pb2+. Zn2+ also evoked large currents; however, the zinc-evoked current was accounted for by H+ and Cl- conductances and was not associated with significant Zn2+ transport. 1B/IRE+-DMT1 exhibited the same substrate profile, Fe2+ affinity and dependence on the H+ electrochemical gradient. Each isoform mediated 55Fe2+ uptake and Fe2+-evoked currents at low extracellular pH. Whereas iron transport activity varied markedly between the four isoforms, the activity for each correlated with the density of anti-DMT1 immunostaining in the plasma membrane, and the turnover rate of the Fe2+ transport cycle did not differ between isoforms. Therefore all four isoforms of human DMT1 function as metal-ion transporters of equivalent efficiency. Our results reveal that the N- and C-terminal sequence variations among the DMT1 isoforms do not alter DMT1 functional properties. We therefore propose that these variations serve as tissue-specific signals or cues to direct DMT1 to the appropriate subcellular compartments (e.g. in erythroid cells) or the plasma membrane (e.g. in intestine).

Metal-ion-dependent biological properties of a chelator-derived somatostatin analogue for tumour targeting

Somatostatin-based radioligands have been shown to have sensitive imaging properties for neuroendocrine tumours and their metastases. The potential of [(55)Co(dotatoc)] (dotatoc =4,7,10-tricarboxymethyl-1,4,7,10-tetraazacyclododecane-1-ylacetyl-D-Phe-(Cys-Tyr-D-Trp-Lys-Thr-Cys)-threoninol (disulfide bond)) as a new radiopharmaceutical agent for PET has been evaluated. (57)Co was used as a surrogate of the positron emitter (55)Co and the pharmacokinetics of [(57)Co(dotatoc)] were investigated by using two nude mouse models. The somatostatin receptor subtype (sst1-sst5) affinity profile of [(nat)Co(dotatoc)] on membranes transfected with human somatostatin receptor subtypes was assessed by using autoradiographic methods. These studies revealed that [(57)Co(dotatoc)] is an sst2-specific radiopeptide which presents the highest affinity ever found for the sst2 receptor subtype. The rate of internalisation into the AR4-2J cell line also was the highest found for any somatostatin-based radiopeptide. Biodistribution studies, performed in nude mice bearing an AR4-2J tumour or a transfected HEK-sst2 cell-based tumour, showed high and specific uptake in the tumour and in other sst-receptor-expressing tissues, which reflects the high receptor binding affinity and the high rate of internalisation. The pharmacologic differences between [(57)Co(dotatoc)] and [(67)Ga(dotatoc)] are discussed in terms of the structural parameters found for the chelate models [Co(II)(dota)](2-) and [Ga(III)(dota)](-) whose X-ray structures have been determined. Both chelates show six-fold coordination in pseudo-octahedral arrangements.

Conversion of agonist site to metal-ion chelator site in the β2-adrenergic receptor

Previously metal-ion sites have been used as structural and functional probes in seven transmembrane receptors (7TM), but as yet all the engineered sites have been inactivating. Based on presumed agonist interaction points in transmembrane III (TM-III) and -VII of the β2-adrenergic receptor, in this paper we construct an activating metal-ion site between the amine-binding Asp-113 in TM-III—or a His residue introduced at this position—and a Cys residue substituted for Asn-312 in TM-VII. No increase in constitutive activity was observed in the mutant receptors. Signal transduction was activated in the mutant receptors not by normal catecholamine ligands but instead either by free zinc ions or by zinc or copper ions in complex with small hydrophobic metal-ion chelators. Chelation of the metal ions by small hydrophobic chelators such as phenanthroline or bipyridine protected the cells from the toxic effect of, for example Cu2+, and in several cases increased the affinity of the ions for the agonistic site. Wash-out experiments and structure–activity analysis indicated, that the high-affinity chelators and the metal ions bind and activate the mutant receptor as metal ion guided ligand complexes. Because of the well-understood binding geometry of the small metal ions, an important distance constraint has here been imposed between TM-III and -VII in the active, signaling conformation of 7TM receptors. It is suggested that atoxic metal-ion chelator complexes could possibly in the future be used as generic, pharmacologic tools to switch 7TM receptors with engineered metal-ion sites on or off at will.

Metal ion-mediated substrate-assisted catalysis in type II restriction endonucleases

The 2.15-Å resolution cocrystal structure of EcoRV endonuclease mutant T93A complexed with DNA and Ca2+ ions reveals two divalent metals bound in one of the active sites. One of these metals is ligated through an inner-sphere water molecule to the phosphate group located 3′ to the scissile phosphate. A second inner-sphere water on this metal is positioned approximately in-line for attack on the scissile phosphate. This structure corroborates the observation that the pro-SP phosphoryl oxygen on the adjacent 3′ phosphate cannot be modified without severe loss of catalytic efficiency. The structural equivalence of key groups, conserved in the active sites of EcoRV, EcoRI, PvuII, and BamHI endonucleases, suggests that ligation of a catalytic divalent metal ion to this phosphate may occur in many type II restriction enzymes. Together with previous cocrystal structures, these data allow construction of a detailed model for the pretransition state configuration in EcoRV. This model features three divalent metal ions per active site and invokes assistance in the bond-making step by a conserved lysine, which stabilizes the attacking hydroxide ion nucleophile.

Explanation by the double-metal-ion mechanism of catalysis for the differential metal ion effects on the cleavage rates of 5′-oxy and 5′-thio substrates by a hammerhead ribozyme

In a previous examination using natural all-RNA substrates that contained either a 5′-oxy or 5′-thio leaving group at the cleavage site, we demonstrated that (i) the attack by the 2′-oxygen at C17 on the phosphorus atom is the rate-limiting step only for the substrate that contains a 5′-thio group (R11S) and (ii) the departure of the 5′ leaving group is the rate-limiting step for the natural all-RNA substrate (R11O) in both nonenzymatic and hammerhead ribozyme-catalyzed reactions; the energy diagrams for these reactions were provided in our previous publication. In this report we found that the rate of cleavage of R11O by a hammerhead ribozyme was enhanced 14-fold when Mg2+ ions were replaced by Mn2+ ions, whereas the rate of cleavage of R11S was enhanced only 2.2-fold when Mg2+ ions were replaced by Mn2+ ions. This result appears to be exactly the opposite of that predicted from the direct coordination of the metal ion with the leaving 5′-oxygen, because a switch in metal ion specificity was not observed with the 5′-thio substrate. However, our quantitative analyses based on the previously provided energy diagram indicate that this result is in accord with the double-metal-ion mechanism of catalysis.

Two-dimensional protein crystallization via metal-ion coordination by naturally occurring surface histidines.

A powerful and potentially general approach to the targeting and crystallization of proteins on lipid interfaces through coordination of surface histidine residues to lipid-chelated divalent metal ions is presented. This approach, which should be applicable to the crystallization of a wide range of naturally occurring or engineered proteins, is illustrated here by the crystallization of streptavidin on a monolayer of an iminodiacetate-Cu(II) lipid spread at the air-water interface. This method allows control of the protein orientation at interfaces, which is significant for the facile production of highly ordered protein arrays and for electron density mapping in structural analysis of two-dimensional crystals. Binding of native streptavidin to the iminodiacetate-Cu lipids occurs via His-87, located on the protein surface near the biotin binding pocket. The two-dimensional streptavidin crystals show a previously undescribed microscopic shape that differs from that of crystals formed beneath biotinylated lipids.

A method for distance determination in proteins using a designed metal ion binding site and site-directed spin labeling: evaluation with T4 lysozyme.

The use of molecular genetics to introduce both a metal ion binding site and a nitroxide spin label into the same protein opens the use of paramagnetic metalnitroxyl interactions to estimate intramolecular distances in a wide variety of proteins. In this report, a His-Xaa3-His metal ion binding motif was introduced at the N terminus of the long interdomain helix of T4 lysozyme (Lys-65 --> His/Gln-69 --> His) of three mutants, each containing a single nitroxide-labeled cysteine residue at position 71, 76, or 80. The results show that Cu(II)-induced relaxation effects on the nitroxide can be quantitatively analyzed in terms of interspin distance in the range of 10-25 A using Redfield theory, as first suggested by Leigh [Leigh, J.S. (1970) J. Chem. Phys. 52, 2608-2612]. Of particular interest is the observation that distances can be determined both under rigid lattice conditions in frozen solution and in the presence of motion of the spins at room temperature under physiological conditions. The method should be particularly attractive for investigating structure in membrane proteins that are difficult to crystallize. In the accompanying paper, the technique is applied to a polytopic membrane protein, lactose permease.

Distance determination in proteins using designed metal ion binding sites and site-directed spin labeling: application to the lactose permease of Escherichia coli.

As shown in the accompanying paper, the magnetic dipolar interaction between site-directed metal-nitroxide pairs can be exploited to measure distances in T4 lysozyme, a protein of known structure. To evaluate this potentially powerful method for general use, particularly with membrane proteins that are difficult to crystallize, both a paramagnetic metal ion binding site and a nitroxide side chain were introduced at selected positions in the lactose permease of Escherichia coli, a paradigm for polytopic membrane proteins. Thus, three individual cysteine residues were introduced into putative helix IV of a lactose permease mutant devoid of native cysteine residues containing a high-affinity divalent metal ion binding site in the form of six contiguous histidine residues in the periplasmic loop between helices III and IV. In addition, the construct contained a biotin acceptor domain in the middle cytoplasmic loop to facilitate purification. After purification and spin labeling, electron paramagnetic resonance spectra were obtained with the purified proteins in the absence and presence of Cu(II). The results demonstrate that positions 103, 111, and 121 are 8, 14, and > 23 A from the metal binding site. These data are consistent with an alpha-helical conformation of transmembrane domain IV of the permease. Application of the technique to determine helix packing in lactose permease is discussed.

Structures of the apo- and the metal ion-activated forms of the diphtheria tox repressor from Corynebacterium diphtheriae.

The diphtheria tox repressor (DtxR) of Corynebacterium diphtheriae plays a critical role in the regulation of diphtheria toxin expression and the control of other iron-sensitive genes. The crystal structures of apo-DtxR and of the metal ion-activated form of the repressor have been solved and used to identify motifs involved in DNA and metal ion binding. Residues involved in binding of the activated repressor to the diphtheria tox operator, glutamine 43, arginine 47, and arginine 50, were located and confirmed by site-directed mutagenesis. Previous biochemical and genetic data can be explained in terms of these structures. Conformational differences between apo- and Ni-DtxR are discussed with regard to the mechanism of action of this repressor.