Metal binding to brain-specific metallothionein-3 studied by electrospray ionization mass spectrometry.

Metallothionein-3 (MT-3) is a brain-specific isoform of metallothioneins, which is down-regulated in Alzheimer's disease (AD), inhibits the growth of neurons in vitro, and differs from common MTs also in gene regulation. To elucidate the differences in structure and function between MT-3 and common MTs, Zn2+ and Cd2+ binding to MT-3 and MT-1 were studied using electrospray ionization time of flight mass spectrometry (ESI TOF MS) at pH values between 7.5 and 2.7. The metal binding properties of MT-3 differ considerably from those of MT-1. After reconstitution with a metal excess, metallated MT-3 exists as a mixture of Zn7MT-3 (or Cd7MT-3, respectively) and several metalloforms with stoichiometries below and above seven. In contrast, MT-1 exists as a single Zn7MT-1 (or Cd7MT-1). Lowering of pH leads to a stepwise release of metals from metallated MT-3, first from extra sites, then from the 3-metal cluster and finally from the 4-metal cluster. At acidic pH values the 4-metal cluster of MT-3 is slightly more stable than that of MT-1. The results demonstrate higher structural plasticity, dynamics and metal binding capacity of MT-3 than of MT-1, which makes MT-3 suitable as a zinc buffer-transfer molecule in zinc-enriched neurons functioning at conditions of fluctuating zinc concentrations.     

Electrospray ionization mass spectrometry of zinc, cadmium, and copper metallothioneins: evidence for metal-binding cooperativity

Electrospray ionization (ESI) mass spectra of both well-characterized and novel metallothioneins (MTs) from various species were recorded to explore their metal-ion-binding modes and stoichiometries. The ESI mass spectra of the zinc- and cadmium-binding MTs showed a single main peak corresponding to metal-to-protein ratios of 4, 6, or 7. These findings combined with data obtained by other methods suggest that these MTs bind zinc or cadmium in a single predominant form and are consistent with the presence of three- and four-metal clusters. An unstable copper-specific MT isoform from Roman snails (Helix pomatia) could be isolated intact and was shown to preferentially bind 12 copper ions. To obtain additional information on the formation and relative stability of metal-thiolate clusters in MTs, a mass spectrometric titration study was conducted. One to seven molar equivalents of zinc or of cadmium were added to metal-free human MT-2 at neutral pH, and the resulting complexes were measured by ESI mass spectrometry. These experiments revealed that the formation of the four-metal cluster and of the thermodynamically less stable three-metal cluster is sequential and largely cooperative for both zinc and cadmium. Minor intermediate forms between metal-free MT, Me4MT, and fully reconstituted Me7MT were also observed. The addition of increasing amounts of cadmium to metal-free blue crab MT-I resulted in prominent peaks whose masses were consistent with apoMT, Cd3MT, and Cd6MT, reflecting the known structure of this MT with two Me3Cys9 centers. In a similar reconstitution experiment performed with Caenorhabditis elegans MT-II, a series of signals corresponding to apoMT and Cd3MT to Cd6MT species were observed.     

Brain-specific metallothionein-3 has higher metal-binding capacity than ubiquitous metallothioneins and binds metals noncooperatively

Zinc metabolism in the cells is largely regulated by ubiquitous small proteins, metallothioneins (MT). Metallothionein-3 is specifically expressed in the brain and is down regulated in Alzheimer's disease. We demonstrate by mass spectrometry that MT-3, in contrast to common MTs, binds Zn(2+) and Cd(2+) in a noncooperative manner and can also bind higher stoichiometries of metals than seven. MT-3 reconstituted with seven metals exists in a dynamic equilibrium of different metalloforms, where the prevalent metalloform is Me(7)MT-3, but metalloforms with 6, 8, and even 9 metals are also present. The results from pH and stability studies demonstrate that the heterogeneity of metalloforms originates from the N-terminal beta-cluster, whereas the C-terminal alpha-cluster of MT-3 binds four metal ions such as that of common MTs. Experiments with EDTA demonstrate that the beta-cluster of ZnMT-3 has a higher metal transfer potential than the beta-cluster of Zn(7)MT-2. Moreover, ZnMT-3 loses metals during ultrafiltration. MT-3, reconstituted with an excess of Zn(2+) or Cd(2+), exists as a dynamic mixture of metalloforms with higher than 7 metal stoichiometries (8-11). Such forms of ZnMT-3 are unstable and decompose partly already during a rapid gel filtration, whereas CdMT-3 forms are more stable. Extra metal ions may bind to the beta-cluster region as well as to the carboxylates of MT-3. The specific metal-binding properties of MT-3 could be functionally implemented for buffering of fluctuating concentrations of zinc in zincergic neurons and for transfer of zinc to synaptic vesicles.     

The metal-binding properties of the blue crab copper specific CuMT-2: a crustacean metallothionein with two cysteine triplets

Most crustacean metallothioneins (MTs) contain 18 Cys residues and bind six divalent metal ions. The copper-specific CuMT-2 (MTC) of the blue crab Callinectes sapidus with 21 Cys residues, of which six are organized in two uncommon Cys-Cys-Cys sequences, represents an exception. However, its metal-binding properties are unknown. By spectroscopic and spectrometric techniques we show that all 21 Cys residues of recombinant MTC participate in the binding of Cu(I), Zn(II), and Cd(II) ions, indicating that both Cys triplets act as ligands. The fully metallated M₈ ^II–MTC (M is Zn, Cd) form possesses high- and low-affinity metal binding sites, as evidenced by the formation of Zn₆–MTC and Cd₇–MTC species from M₈ ^II–MTC after treatment with Chelex 100. The NMR characterization of Cd₇–MTC suggests the presence of a two-domain structure, each domain containing one Cys triplet and encompassing either the three-metal or the four-metal thiolate cluster. Whereas the metal–Cys connectivities in the three-metal cluster located in the N-terminal domain (residues 1–31) reveal a Cd₃Cys₉ cyclohexane-like structure, the presence of dynamic processes in the C-terminal domain (residues 32–64) precluded the determination of the organization of the four-metal cluster. Absorption and circular dichroism features accompanying the stepwise binding of Cu(I) to MTC suggest that all 21 Cys are involved in the binding of eight to nine Cu(I) ions (Cu_8–9–MTC). The subsequent generation of Cu₁₂–MTC involves structural changes consistent with a decrease in the Cu(I) coordination number. Overall, the metal-binding properties of MTC reported here contribute to a better understanding of the role of Cys triplets in MTs.     

Organization and assembly of metal-thiolate clusters in epithelium-specific metallothionein-4

Mammalian metallothionein-4 (MT-4) was found to be specifically expressed in stratified squamous epithelia where it plays an essential but poorly defined role in regulating zinc or copper metabolism. Here we report on the organization, stability, and the pathway of metal-thiolate cluster assembly in MT-4 reconstituted with Cd(2+) and Co(2+) ions. Both the (113)Cd NMR studies of (113)Cd(7)MT-4 and the spectroscopic characterization of Co(7)MT-4 showed that, similar to the classical MT-1 and MT-2 proteins, metal ions are organized in two independent Cd(4)Cys(11) and Cd(3)Cys(9) clusters with each metal ion tetrahedrally coordinated by terminal and bridging cysteine ligands. Moreover, we have demonstrated that the cluster formation in Cd(7)MT-4 is cooperative and sequential, with the Cd(4)Cys(11) cluster being formed first, and that a distinct single-metal nucleation intermediate Cd(1)MT-4 is required in the cluster formation process. Conversely, the absorption and circular dichroism features of metal-thiolate clusters in Cd(7)MT-4 indicate that marked differences in the cluster geometry exist when compared with those in Cd(7)MT-1/2. The biological implication of our studies as to the role of MT-4 in zinc metabolism of stratified epithelia is discussed.     

Spectroscopic studies on metal distribution in Co(II)/Zn(II) mixed-metal clusters in rabbit liver metallothionein 2.

Metal selectivity of metal-thiolate clusters in rabbit liver metallothionein (MT) 2 has been studied by examining the metal distribution of two similarly sized divalent metal ions, cobalt and zinc, which have different thiolate affinity. The forms of mixed-metal cluster species in (Co/Zn)7-MT generated with different ratios of both metal ions offered to the metal-free protein were investigated using EPR, ultraviolet/visible absorption and MCD spectroscopy. The results demonstrated that the distribution of these metals between the two metal-thiolate clusters is not random. Thus, the EPR absorption intensities of the bound Co(II) ions in the Zn-cluster matrix increased linearly up to a ratio of Co(II)/Zn(II) equivalents of 3:4, with the final EPR intensity of three non-interacting Co(II)-binding sites. This EPR behaviour is consistent with a binding scheme in which one Co(II) ion occupies a metal-binding site within the three-metal cluster and the remaining two Co(II) ions occupy two distinctly separate sites in the four-metal cluster. With four or more Co(II) ions in the cluster matrix, magnetic coupling between adjacent, sulphur-bridged Co(II) ions was observed. In previous studies on mixed-metal clusters in MT formed with Co(II)/Cd(II), Zn(II)/Cd(II) and Cd(II)/Fe(II), changes in the respective cluster volumes were shown to be a significant factor dictating the widely differing metal distributions in these systems. Based on the results of the current study, it is suggested that both the sizes of the two metal ions and their relative affinities towards the cysteine-thiolate ligands are important in the formation of mixed-metal clusters in MT.     

Engineering of metallothionein-3 neuroinhibitory activity into the inactive isoform metallothionein-1

The third isoform of mammalian metallothioneins (MT-3), mainly expressed in brain and down-regulated in Alzheimer's disease, exhibits neuroinhibitory activity in vitro and a highly flexible structure that distinguishes it from the widely expressed MT-1/-2 isoforms. Previously, we showed that two conserved prolyl residues of MT-3 are crucial for both the bioactivity and cluster dynamics of this isoform. We have now used genetic engineering to introduce these residues into mouse MT-1. The S6P,S8P MT-1 mutant is inactive in neuronal survival assays. However, the additional introduction of the unique Thr5 insert of MT-3 resulted in a bioactive MT-1 form. Temperature-dependent and saturation transfer (113)Cd NMR experiments performed on the (113)Cd-reconstituted wild-type and mutant Cd(7)-MT-1 forms revealed that the gain of MT-3-like neuronal inhibitory activity is paralleled by an increase in conformational flexibility and intersite metal exchange in the N-terminal Cd(3)-thiolate cluster. The observed correlation suggests that structure/cluster dynamics are critical for the biological activity of MT-3. We propose that the interplay between the specific Pro-induced conformational requirements and those of the metal-thiolate bonds gives rise to an alternate and highly fluctuating cluster ensemble kinetically trapped by the presence of the (5)TCPCP(9) motif. The functional significance of such heterogeneous cluster ensemble is discussed.     

113Cd NMR studies on metal-thiolate cluster formation in rabbit Cd(II)-metallothionein: evidence for a pH dependence

The formation of two metal-thiolate clusters in rabbit liver metallothionein 2 (MT) has been examined by 113Cd NMR spectroscopy at pH 7.2 and 8.6. The chemical shifts of the 113Cd resonances developing in the course of apoMT titration with 113Cd(II) ions have been compared with those of fully metal occupied 113Cd7-MT. At pH 7.2 and at low metal occupancy (less than 4), a cooperative formation of the four-metal cluster (cluster A) occurs. Further addition of 113Cd(II) ions generates all the resonances of the three-metal cluster (cluster B) in succession, suggesting cooperative metal binding to this cluster also. In contrast, similar studies at pH 8.6, at low metal occupancy (less than 4), reveal a broad NMR signal centered at 688 ppm. This observation indicates that an entirely different protein structure exists. When exactly 4 equiv of 113Cd(II) are bound to apoMT, the 113Cd NMR spectrum changes to the characteristic spectrum of cluster A. Further addition of 113Cd(II) ions again leads to the cooperative formation of cluster B. These results stress the determining role of the cluster A domain on the overall protein fold. The observed pH dependence of the cluster formation in MT can be rationalized by the different degree of deprotonation of the cysteine residues (pKa approximately 8.9), i.e., by the difference in the Gibbs free energy required to bind Cd(II) ions to the thiolate ligands at both pH values.     

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.     

Role of metallothionein in cadmium traffic and toxicity in kidneys and other mammalian organs

Metallothioneins are cysteine-rich, small metal-binding proteins present in various mammalian tissues. Of the four common metallothioneins, MT-1 and MT-2 (MTs) are expressed in most tissues, MT-3 is predominantly present in brain, whereas MT-4 is restricted to the squamous epithelia. The expression of MT-1 and MT-2 in some organs exhibits sex, age, and strain differences, and inducibility with a variety of stimuli. In adult mammals, MTs have been localized largely in the cell cytoplasm, but also in lysosomes, mitochondria and nuclei. The major physiological functions of MTs include homeostasis of essential metals Zn and Cu, protection against cytotoxicity of Cd and other toxic metals, and scavenging free radicals generated in oxidative stress. The role of MTs in Cd-induced acute and chronic toxicity, particularly in liver and kidneys, is reviewed in more details. In acute toxicity, liver is the primary target, whereas in chronic toxicity, kidneys are major targets of Cd. The intracellular MTs bind Cd ions and form CdMT. In chronic intoxication, Cd stimulates de novo synthesis of MTs; it is assumed that toxicity in the cells starts when loading with Cd ions exceeds the buffering capacity of intracellular MTs. CdMT, released from the Cd-injured organs, or when applied parenterally for experimental purposes, reaches the kidneys via circulation, where it is filtered, endocytosed in the proximal tubule cells, and degraded in lysosomes. Liberated Cd can immediately affect the cell structures and functions. The resulting proteinuria and CdMT in the urine can be used as biomarkers of tubular injury.     

Fish and molluscan metallothioneins

Metallothioneins (MTs) are noncatalytic peptides involved in storage of essential ions, detoxification of nonessential metals, and scavenging of oxyradicals. They exhibit an unusual primary sequence and unique 3D arrangement. Whereas vertebrate MTs are characterized by the well-known dumbbell shape, with a beta domain that binds three bivalent metal ions and an alpha domain that binds four ions, molluscan MT structure is still poorly understood. For this reason we compared two MTs from aquatic organisms that differ markedly in primary structure: MT 10 from the invertebrate Mytilus galloprovincialis and MT A from Oncorhyncus mykiss. Both proteins were overexpressed in Escherichia coli as glutathione S-transferase fusion proteins, and the MT moiety was recovered after protease cleavage. The MTs were analyzed by gel electrophoresis and tested for their differential reactivity with alkylating and reducing agents. Although they show an identical cadmium content and a similar metal-binding ability, spectropolarimetric analysis disclosed significant differences in the Cd7-MT secondary conformation. These structural differences reflect the thermal stability and metal transport of the two proteins. When metal transfer from Cd7-MT to 4-(2-pyridylazo)resorcinol was measured, the mussel MT was more reactive than the fish protein. This confirms that the differences in the primary sequence of MT 10 give rise to peculiar secondary conformation, which in turn reflects its reactivity and stability. The functional differences between the two MTs are due to specific structural properties and may be related to the different lifestyles of the two organisms.     

(Cu,Zn)-metallothioneins from fetal bovine liver. Chemical and spectroscopic properties

Two metallothioneins (MTs) from bovine fetal liver were purified by a combination of gel filtration and ion-exchange chromatography. The primary structures of the isoproteins MT-1 and MT-2 were elucidated by peptide and amino acid sequence analysis. The amino-terminal part was deduced from automated Edman degradations of the pyridylethylated CNBr-cleaved derivatives. The remaining part of the sequence was established by a comparison of the carboxamidomethylated tryptic peptides to those from equine liver MT-1A and MT-2B. Peptides differing in either amino acid composition or retention time from high pressure liquid chromatography were further subjected to manual Edman degradations or carboxypeptidase Y digestion. The two isoproteins consist of 61 amino acids and show a sequence identity of 90%. When compared with the primary structures of other mammalian MTs, the 20 cysteinyl residues are totally conserved, in agreement with their function as metal ligands. The two isoproteins contain Cu and Zn at a ratio of 3:4. Spectroscopic data reveal absorption properties typical for both Cu- and Zn-thiolate transitions. The marked differences of MT-1 and MT-2 in the Cu-thiolate CD features can be attributed to the six amino acid substitutions occurring exclusively in the amino-terminal parts of the molecules. It is proposed that in bovine fetal MTs also the three copper ions are preferentially bound to the first 9 cysteinyl residues (cluster B) and the four zinc ions to the remaining 11 cysteinyl residues (cluster A) suggested previously by 113Cd NMR spectroscopy of calf liver MTs (Briggs, R. W., and Armitage, I. M. (1982) J. Biol. Chem. 257, 1259-1262).     

Metal co-ordination in rat liver metallothionein-2 prepared with or without reconstitution of the metal clusters, and comparison with rabbit liver metallothionein-2

Possible origins of the different metal co-ordination topologies in the recently determined structures of rat metallothionein-2 (MT2) in single crystals and rabbit MT2 in solution were investigated. A complete structure determination for rat MT2 in solution by nuclear magnetic resonance (n.m.r.) showed that the differences in the spatial structures cannot be attributed to the different primary structures of the two species. Comparison of [113Cd7]MT2 obtained by reconstitution of the apoprotein in vitro with preparations using a different procedure showed, moreover, that the metal co-ordination observed in solution by n.m.r. is not an artefact of the protein reconstitution. Solutions of high-pressure liquid chromatographically homogeneous biosynthetic preparations of [113Cd, Zn]MT2 were obtained from rat liver following injection of 113Cd into rats in vivo, without further metal exchange after protein isolation. They contain a mixture of several forms of MT2 with different relative metal compositions, giving rise to an increased number of 113Cd resonances. For the components of the four-metal cluster, the major one of these different forms exhibits patterns in the two-dimensional [1H, 113Cd]-correlated spectra that are indistinguishable from those of [113Cd7]MT2, thereby implying identity of cluster coordination and topology. These results are discussed with regard to continued investigations into the differences between the solution structure and crystal structure of MT2.     

A distinct Cu4-thiolate cluster of human metallothionein-3 is located in the N-terminal domain

Metallothionein-3 (MT-3), also known as neuronal growth inhibitory factor, is a metalloprotein expressed almost exclusively in the brain. Isolated MT-3 contains four Cu(I) and three Zn(II) ions organized in homometallic metal-thiolate clusters located in two independent protein domains. In this work a Cu(I) binding to metal-free MT-3 has been studied, aiming at the better understanding of the domain specificity for this metal ion. The cluster formation was followed by electronic absorption, circular dichroism, and by luminescence spectroscopy at room temperature and 77 K. The stepwise incorporation of Cu(I) into recombinant human apo-MT-3 revealed the cooperative formation of two Cu(4)S(9) clusters in succession, formed in both protein domains, i.e. Cu(4)- and Cu(8)-MT-3. Further binding of four Cu(I) caused an expansion of these Cu(I) cores, leading to fully metal-loaded Cu(12)-MT-3 containing Cu(6)S(9) and Cu(6)S(11) clusters in the beta- and alpha-domains of the protein, respectively. The location of the preferentially formed Cu(4) cluster in the protein was established by immunochemistry. Using domain-specific antibodies, in combination with limited tryptic digestion of a partially metal-occupied Cu(4)-MT-3, we could demonstrate that the Cu(4)S(9) cluster is located in the N-terminal beta-domain of the protein that contains a total of nine cysteine ligands.     

NMR structure of the sea urchin (Strongylocentrotus purpuratus) metallothionein MTA.

The three-dimensional structure of [(113)Cd7]-metallothionein-A (MTA) of the sea urchin Strongylocentrotus purpuratus was determined by homonuclear(1)H NMR experiments and heteronuclear [(1)H, (113)Cd]-correlation spectroscopy. MTA is composed of two globular domains, an N-terminal four-metal domain of the amino acid residues 1 to 36 and a Cd4Cys11cluster, and a C-terminal three-metal domain including the amino acid residues 37 to 65 and a Cd3Cys9cluster. The structure resembles the known mammalian and crustacean metallothioneins, but has a significantly different connectivity pattern of the Cys-metal co-ordination bonds and concomitantly contains novel local folds of some polypeptide backbone segments. These differences can be related to variations of the Cys sequence positions and thus emphasize the special role of the cysteine residues in defining the structure of metallothioneins, both on the level of the domain architecture and the topology of the metal-thiolate clusters.     

Molecular characterization and function analysis of MT-10 and MT-20 metallothionein isoforms from Mytilus galloprovincialis

Structure and function of molluscan metallothioneins (MTs) are still poorly understood. The sea mussel Mytilus galloprovincialis displays two MT isoforms which differ in both primary sequences and physiological functions. MT-10 is the constitutive isoform, whereas MT-20 is mainly induced by cadmium (Cd). Both MTs were produced as recombinant proteins and showed identical Cd content and similar Cd-binding properties. Conversely, circular dichroism disclosed marked differences in the secondary conformations of the two Cd(7)-MTs. The possible relapses of these structural differences on protein stability and function were assessed. MT-10 presented a higher thermal stability and a more compact structure than MT-20, as it was inferred by absorption and emission spectroscopy studies. Moreover, the kinetics of Cd-release clearly indicated that MT-10 is much more sensitive to oxidation than is MT-20. The observed differences between MT-10 and MT-20 are discussed in terms of the different physiological roles exerted by the two isoforms in mussel.     

Effect of the two conserved prolines of human growth inhibitory factor (metallothionein-3) on its biological activity and structure fluctuation: comparison with a mutant protein

Human neuronal growth inhibitory factor, a metalloprotein classified as metallothionein-3 (MT-3), impairs the survival and the neurite formation of cultured neurons. In these studies the double P7S/P9A mutant (mutMT-3) and single mutants P7S and P9A of human Zn(7)-MT-3 were generated, and their effects on the biological activity and the structure of the protein were examined. The biological results clearly established the necessity of both proline residues for the inhibitory activity, as even single mutants were found to be inactive. Using electronic absorption, circular dichroism (CD), magnetic CD (MCD), and (113)Cd NMR spectroscopy, the structural features of the metal-thiolate clusters in the double mutant Cd(7)-mutMT-3 were investigated and compared with those of wild-type Cd(7)-MT-3 [Faller, P., Hasler, D. W., Zerbe, O., Klauser, S., Winge, D. R., and Vasák, M. (1999) Biochemistry 38, 10158] and the well characterized Cd(7)-MT-2a from rabbit liver. Similarly to (113)Cd(7)-MT-3 the (113)Cd NMR spectrum of (113)Cd(7)-mutMT-3 at 298 K revealed four major and three minor resonances (approximately 20% of the major ones) between 590 and 680 ppm, originating from a Cd(4)S(11) cluster in the alpha-domain and a Cd(3)S(9) cluster in the beta-domain, respectively. Due to the presence of dynamic processes in the structure of MT-3 and mutMT-3, all resonances showed the absence of resolved homonuclear [(113)Cd-(113)Cd] couplings and large apparent line widths (between 140 and 350 Hz). However, whereas in (113)Cd(7)-mutMT-3 the temperature rise to 323 K resulted in a major recovery of the originally NMR nondetectable population of the Cd(3)S(9) cluster resonances, no such temperature effect was observed in (113)Cd(7)-MT-3. To account for the observed NMR features, a dynamic structural model for the beta-domain is proposed, which involves a folded and a partially unfolded state. It is suggested that in the partially unfolded state a slow cis/trans isomerization of Cys-Pro(7) or Cys-Pro(9) amide bonds in (113)Cd(7)-MT-3 takes place and that this process represents a rate-limiting step in a correct domain refolding. In addition, closely similar apparent stability constants of human MT-3, mutMT-3, and rabbit MT-2a with Cd(II) and Zn(II) ions were found. These results suggest that specific structural features dictated by the repetitive (Cys-Pro)(2) sequence in the beta-domain of MT-3 and not its altered metal binding affinity compared to MT-1/MT-2 isoforms are responsible for the biological activity of this protein.     

Characterization of Trypanosoma brucei brucei S-adenosyl-L-methionine decarboxylase and its inhibition by Berenil, pentamidine and methylglyoxal bis(guanylhydrazone).

Reaction of rat liver cadmium-metallothionein-II(Cd-MT-II) with p-hydroxymercuribenzoate(pHOHgBzO-) causes displacement of bound Cd. When pHOHgBzO- -induced displacement of 109Cd is observed after dialysis of the reaction mixture, the stoichiometry is consistent with stepwise displacement of tetraco-ordinate Cd atoms by non-random entry of reagent into the polynuclear clusters. 113Cd n.m.r. allows direct observation of the effects on bound Cd of stepwise titration of 113Cd-MT-II with pHOHgBzO-. The first equivalent reduces all resonances approximately equally. Subsequently differential reactivity of the protein thiolates towards the reagent gives rise to differential decreases in the 113Cd signal intensities. Resonances previously attributed to a three-metal cluster are lost before those arising from the four-metal cluster. These results are interpreted in terms of current models of the MT structure. They are distinct from the results of reaction of MT with 5,5'-dithiobis-(2-nitrobenzoic acid), which distinguishes between only two classes of thiolates, terminal and bridging. Such different patterns of reactivity of the protein thiolates may underlie a biological activity of this protein.     

Chemistry and biology of mammalian metallothioneins

Metallothioneins (MTs) are a class of ubiquitously occurring low molecular mass, cysteine- and metal-rich proteins containing sulfur-based metal clusters formed with Zn(II), Cd(II), and Cu(I) ions. In mammals, four distinct MT isoforms designated MT-1 through MT-4 exist. The first discovered MT-1/MT-2 are widely expressed isoforms, whose biosynthesis is inducible by a wide range of stimuli, including metals, drugs, and inflammatory mediators. In contrast, MT-3 and MT-4 are noninducible proteins, with their expression primarily confined to the central nervous system and certain squamous epithelia, respectively. MT-1 through MT-3 have been reported to be secreted, suggesting that they may play different biological roles in the intracellular and extracellular space. Recent reports established that these isoforms play an important protective role in brain injury and metal-linked neurodegenerative diseases. In the postgenomic era, it is becoming increasingly clear that MTs fulfill multiple functions, including the involvement in zinc and copper homeostasis, protection against heavy metal toxicity, and oxidative damage. All mammalian MTs are monomeric proteins, containing two metal–thiolate clusters. In this review, after a brief summary of the historical milestones of the MT-1/MT-2 research, the recent advances in the structure, chemistry, and biological function of MT-3 and MT-4 are discussed.     

Metal selectivity of clusters in rabbit liver metallothionein.

In mammalian metallothioneins the metals are organized in two adamantane-type clusters with three and four metal ions which are tetrahedrally coordinated by thiolate ligands. The metal selectivity of the metal-thiolate clusters in rabbit liver metallothionein has been studied by offering two ions, i.e. Co(II)/Cd(II), Zn(II)/Cd(II) or Co(II)/Zn(II), to the metal-free protein. The heterogeneous metal complexes thus formed were characterized by electronic absorption, magnetic circular dichroism. 113Cd-NMR and EPR spectroscopy. In the case of Co/Cd-metallothionein, homometallic cluster occupation occurs, with the Cd(II) ions bound exclusively to the four-metal cluster. In contrast, heterometallic clusters were formed for both Zn/Cd- and Co/Zn-metallothionein. Based on evidence from corresponding inorganic structures of adamantane metal-thiolate cages, it is suggested that the major factor governing the cluster type is the protein structure perturbation due to the cluster volume variations. Thus, while metal thiolate affinities are important in the folding process, size-match selectivity is the dominant factor in the metal-loaded protein.