Ian M. Armitage

Nitric oxide selectively releases metals from the amino-terminal domain of metallothioneins: potential role at inflammatory sites

Metallothioneins (MTs) and various other metal binding proteins release metals when exposed to nitric oxide (NO). We investigated the structural consequences of the interaction between MTs and NO by using 1H‐ and 113Cd‐NMR spectroscopy and found that only the three metals from the N‐terminal β‐domain were selectively released whereas the C‐terminal α‐domain remains intact. Since it has been proposed that the β‐domain is responsible for the postulated role of MTs in zinc homeostasis, whereas the tight binding of metals in the α‐domain appears to play a role in heavy metal detoxification, our results suggest a potential regulatory role of NO in zinc distribution. Specifically, we present a mechanism whereby MT counteracts the cytotoxic effects of NO at inflammatory sites.

3D solution structure of copper and silver-substituted yeast metallothioneins

3D solution structural calculations for yeast silver(I)‐substituted metallothionein (MT) and native copper(I) MT were completed using experimentally determined NOE and dihedral angle constraints, in conjunction with experimentally derived metal‐to‐Cys connectivities for AgMT which were assumed identical for CuMT. For the first 40 residues in both structures, the polypeptide backbone wraps around the metal cluster in two large parallel loops separated by a deep cleft containing the metal cluster. Minor differences between the two structures include differences in hydrogen bonds and the orientation of the N‐terminus with the overall protein volume conserved to within 6.5%.

NMR spectroscopic studies of I = 1/2 metal ions in biological systems

This article reviews the use of nuclear magnetic resonance methods of spin 1/2 metal nuclei to probe the metal binding site(s) in a variety of metalloproteins. The majority of the studies have involved native Zn(II) and Ca(II) metalloproteins where there has been isostructural substitution of these metal ions with the I = 1/2 (111/113)Cd(II) ion. Also included are recent studies that have utilized the 109Ag(I) ion to probe Cu(I) sites in yeast metallothionein and 199Hg(II) as a probe of the metal binding sites in mercury resistance proteins. Pertinent aspects for the optimal execution of these experiments along with the procedures for the metal substitution reactions are discussed together with the presentation of a 113Cd chemical shift correlation map with ligand type and coordination number. Specific examples of protein systems studied using the (111/113)Cd and 109Ag nuclei include the metallothionein superfamily of Zn(II)- and Cu(I)-binding proteins from mammalian, invertebrate, and yeast systems. In addition to the structural features revealed by these metal ion nuclear magnetic resonance studies, important new information is frequently provided about the dynamics at the active-site metal ion. In an effort for completeness, other less frequently used spin 1/2 metal nuclei are mentioned.

113Cd NMR as a probe of the active sites of metalloenzymes

113Cd NMR has been used to study the active site metal ion(s) of the 113Cd(II) derivatives of four Zn(II) metalloenzymes, carboxypeptidase A, carbonic anhydrases, alkaline phosphatase, and superoxide dismutase. The resonances of the enzyme-bound 113Cd(II) ions are extremely sensitive to ligand exchange, including solvent and inhibitor, and to changes in the metal ion coordination sphere. The nature of this behavior can be shown to parallel the known structural properties and proposed roles of the metal ion in the catalytic mechanisms. Models accounting for the exchange mechanisms which may be modulating the chemical shift, linewidth, and coupling are discussed.

Oxidative dimerization in metallothionein is a result of intermolecular disulphide bonds between cysteines in the α-domain

Upon storage under aerobic conditions metallothioneins (MTs) form a new species, which is characterized by a molecular mass approximately twice the size of monomeric MT and shifted (113/111)Cd- and (1)H-NMR resonances. The investigation of this oxidative dimerization process by NMR spectroscopy allowed us to structurally characterize this MT species that has been described to occur in vivo and might be synthesized under conditions of oxidative stress. The oxidative dimer was characterized by the formation of an intermolecular cysteine disulphide bond involving the alpha-domain, and a detailed analysis of chemical shift changes and intermolecular nuclear Overhauser effects points towards a disulphide bond involving Cys(36). In contrast to the metal-bridged (non-oxidative) dimerization, the metal-cysteine cluster structures in both MT domains remain intact and no conformational exchange or metal-metal exchange was observed. Also in contrast to the many recently reported oxidative processes which involve the beta-domain cysteine groups and result in the increased dynamics of the bound metal ions in this N-terminal domain, we found no evidence for any increased dynamics in the alpha-domain metals following this oxidation. Therefore these findings provide additional corroboration that metal binding in the C-terminal alpha-domain is rather tight, even under conditions of a changing cellular oxidation potential, compared with the more labile/dynamic nature of the metals in the N-terminal beta-domain cluster under similar conditions.

Characterization of the calcium-binding sites of calcineurin B

Calcineurin (CaN) is a calcium‐ and calmodulin‐dependent serine/threonine phosphatase whose inhibition by the immunosuppressant‐immunophilin complexes (cyclosporin‐cyclophilin and FK506‐FKBP) is considered key to the mechanism of immunosuppression. CaN is a heterodimer, consisting of a 59 kDa catalytic subunit (A) and a 19 kDa calcium‐binding regulatory subunit (B). The latter is postulated to harbor four calcium binding domains of the EF hand type. The titration of the CaN B apoprotein with the isomorphic Cd2+ was followed by 113Cd NMR and these data support one high‐affinity metal binding site and three lower‐affinity ones. Flow dialysis data with Ca2+ indicate one high affinity calcium binding site with K d ∼ 2.4 × 10−8 M and three other sites with K d ∼ 1.5 × 10−5 M. The chemical shifts of all four 113Cd resonances (−75, −93, −106 and −119 ppm) are in the same range as found in other 113Cd substituted calcium‐binding proteins, and are indicative of all‐oxygen coordination of pentagonal bipyramidal geometry.

Dynamics of interdomain and intermolecular interactions in mammalian metallothioneins

The structures of mammalian metallothioneins (MTs), as solved by X-ray crystallography and NMR spectroscopy, all show seven divalent metals bound in two separate domains. The marked differences in metal-mobilities found for the two domains has led to the proposal for a dual role for the two MT metal domains. The tight metal binding in the C-terminal alpha-domain supposedly constitutes the basis for the detoxification of excess heavy metals, while the more labile metals in the N-terminal beta-domain function in the homeostasis of the essential elements zinc and copper. In this overview, we compare the two types of dimers found for MTs and their influence on metal-mobilities. In the presence of excess metal, the N-terminal domain is responsible for the formation of metal-bridged dimers while under aerobic conditions, a specific intermolecular disulfide is formed between the C-terminal domains. Both forms of dimers not only involve different domains for their intermolecular protein interactions, they also exhibit radical differences in the reactive properties of their respective cluster bound metal ions. Since the metal exchange within each domain is also influenced by interdomain interactions, the relative orientation of the domains is also most likely important for MT functions. Thus far, the relative orientation of the two domains could only be obtained from the crystal structure. Here, we present evidence for increased mobility in the linker region as the reason for the lack of interdomain constraints in the solution NMR studies of mammalian MTs.

Nuclear magnetic resonance spectroscopy of skin: Predictive correlates for clinical application

The first application of phosphorous 31 (31P) and proton (1H) nuclear magnetic resonance (NMR) spectroscopy to the analysis of the metabolic profiles of skin flaps in a rat model and of human skin grafts is presented. Resonances of adenosine triphosphate (ATP), phosphocreatine (PCr), and inorganic phosphate (Pi) were identified in 31P nuclear magnetic resonance spectra. Resonances of phosphocreatine, creatine (Cr), and lactate (Lac) were identified in 1H nuclear magnetic resonance spectra. The most significant finding was the substantial presence of phosphocreatine as the major high-energy phosphometabolite in mammalian skin, a finding which heretofore has not been widely recognized. An energy shuttle between phosphocreatine and ATP is operative in skin to buffer the fall in ATP during ischemic (anaerobic) insult. Inability to replenish exhausted phosphocreatine reserves predictively correlates with eventual flap necrosis. We have defined and analyzed temporal fluxes in the phosphocreatine-creatine and phosphocreatine plus creatine-lactate ratios by proton nuclear magnetic resonance. Both are sensitive, accurate, and unambiguous early prognostic indices of eventual flap outcome. These findings support the concept that the fate of a flap may be established as early as 3 hours after elevation and have laid the groundwork for development and application of noninvasive in vivo nuclear magnetic resonance spectroscopy to the study of skin flaps in animals and humans.

113Cd nmr study of the metal cluster structure of human liver metallothionein

Cadmium-113 nuclear magnetic resonance (113Cd nmr) was used to elucidate the structural properties of the cadmium binding sites in human liver metallothionein. The isotopically labeled 113Cd-metallothionein was prepared by the in vitro exchange of the native metals (greater than 94% zinc) for 113CdCl2 during isolation. The two isoproteins, MT-1 and MT-2, showed 113Cd nmr resonances in the chemical shift range 610-670 ppm. The multiplet structure of the resonances is due to two bond scalar interactions between adjacent 113Cd ions linked by cysteine thiolate ligands. Homonuclear 113Cd decoupling experiments allowed the determination of the metal cluster structure, which, similar to the rabbit liver metallothionein, consists of a four- and a three-metal cluster designated cluster A and cluster B, respectively. Chemical shift similarities in the 113Cd nmr spectra of the human, rabbit and calf liver MT-1 and MT-2 are observed, especially for cluster A. Small variations in chemical shifts are explained in terms of differences in the primary structure between the two human isoproteins.

Inflammation and ER Stress Regulate Branched-Chain Amino Acid Uptake and Metabolism in Adipocytes

Inflammation plays a critical role in the pathology of obesity-linked insulin resistance and is mechanistically linked to the effects of macrophage-derived cytokines on adipocyte energy metabolism, particularly that of the mitochondrial branched-chain amino acid (BCAA) and tricarboxylic acid (TCA) pathways. To address the role of inflammation on energy metabolism in adipocytes, we used high fat-fed C57BL/6J mice and lean controls and measured the down-regulation of genes linked to BCAA and TCA cycle metabolism selectively in visceral but not in subcutaneous adipose tissue, brown fat, liver, or muscle. Using 3T3-L1 cells, TNFα, and other proinflammatory cytokine treatments reduced the expression of the genes linked to BCAA transport and oxidation. Consistent with this, [(14)C]-leucine uptake and conversion to triglycerides was markedly attenuated in TNFα-treated adipocytes, whereas the conversion to protein was relatively unaffected. Because inflammatory cytokines lead to the induction of endoplasmic reticulum stress, we evaluated the effects of tunicamycin or thapsigargin treatment of 3T3-L1 cells and measured a similar down-regulation in the BCAA/TCA cycle pathway. Moreover, transgenic mice overexpressing X-box binding protein 1 in adipocytes similarly down-regulated genes of BCAA and TCA metabolism in vivo. These results indicate that inflammation and endoplasmic reticulum stress attenuate lipogenesis in visceral adipose depots by down-regulating the BCAA/TCA metabolism pathway and are consistent with a model whereby the accumulation of serum BCAA in the obese insulin-resistant state is linked to adipose inflammation.