Metallothionein

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Revision as of 04:37, 3 September 2008

Metallothionein (MT)was first identified in equine kidney cortex as a cadmium-binding protein responsible for the natural accumulation of cadmium in this tissue. It is a family of ubiquitous low molecular weight proteins containing 61-68 amino acids with an unusually high concentration of cysteine (30%). MT's have received their designation from their prominent metal and sulfur content which, varying with the metal species present, together may contribute to over 20% of their weight. The mammalian forms are characterized by a molecular weight of 6000-7000 Da, containing 60 to 68 amino acid residues, among them twenty are cys, and binding a total of 7 equiv. of bivalent metal ions. Aromatic amino acids are usually absent. All cysteine occur in the reduced form and are coordinated to the metal ions through mercaptide bonds, giving rise to spectroscopic features characteristic of metal-thiolate clusters.

MT's occur throughout the animal kingdom and are also found in higher plants, eukaryotic microorganisms, and in some prokaryotes. In animals, the genetically polymorphous proteins are most abundant in parenchymatous tissues, i.e. liver, kidney, pancreas, and intestines. There are wide variations in concentration in different species and tissues, reflecting effects of age, stage of development, dietary regimen, and other not yet fully identified factors. Although MT is a cytoplasmic protein, it can also accumulate in lysosomes, and during development it is observed in the nucleus.

MT's are genetically polymorphous protein families with subfamilies, subgroups and isoforms. The MT multigene family is composed of at least four isoforms. All are located on a single chromosome, i.e. chromosome 8 in mouse and chromosome 16 in human. In vertebrates all MT genes are divided into a 5' flanking region (5'UT), a 5' untranslated region (5'UTR), 3 coding exons separated by 2 introns and a 3' flanking end. The 5'UT contains regulatory elements, among them are one or more copies of the metal responsive element (MRE) which acts as a binding target for the transcription activating protein factor (MTF-1) regulating MT-gene expression. MT-I and -II exist in all tissues, are regulated in a coordinate fashion, and appear functionally equivalent. Other members of the MT gene family, however, show different patterns of expression: MT-III is found mainly in brain and MT-IV in stratified squamous epithelium.

Phylogenetic relationships of the various MT families have recently been established by different methods and approaches both from protein and gene MT sequences. The results of the analyses lead to the differentiation into a variety of phylogenetically related subfamilies and subgroups and have allowed to derive an evolutionary pedigree for the highly complex vertebrate family.

MT’s are thought to play roles both in the intracellular fixation of the essential trace elements zinc and copper, in controlling the concentrations of the free ions of these elements, in regulating their flow to their cellular destinations, in neutralizing the harmful influences of exposure to toxic elements such as cadmium and mercury and in the protection from of a variety of stress conditions. MT-I and -II can be induced easily by heavy metals, hormones, inflammation, acute stress, and many chemicals. In essence induction of MT has been proposed as an important adaptive mechanism in response to environmental stimuli. Induction of MT protects against metal toxicity, acts as a free radical scavenger protecting against oxidative damage, and protects against toxicity of alkylating anticancer drugs and other electrophiles.

MT also plays a major role in the regulation of the immune system because it binds zinc more strongly than the zinc-dependent hormone thymulin, which activates the thymus gland - the master gland of the immune system. Excess formation of MT can lead to inadequate free zinc to activate the thymulin, which results in an increasing incidence of infections. Some researchers suspect that the MT increase that occurs with aging causes atrophy of the thymus gland due to insufficient free zinc and is a reason for the increased incidence of infections and cancer as we age.

The biosynthesis of many MT’s is greatly enhanced both in vivo and in cultured cells by d10 transition metal ions and by certain hormones, cytokines, growth factors, tumor promoters and many other chemicals. This is now known to result from increase in the rate of metallothionein gene transcription mediated through the interaction between upstream regulatory sequences, DNA sequences and unidentified cellular cofactors. It appears to play a fundamental role in the metabolism of copper and zinc ions under various physiological conditions including its ability to donate metal ions to apo-zinc enzymes. Metallothionein may play an important role in sequestering toxic ions for instance Cd2+, Hg2+, Au+ and Pt+ , thus are important in heavy metal detoxification.

Structural Studies on Metallothionein

The structural properties of metallothioneins have been probed by several experimental techniques, including NMR spectroscopy, and X-ray absorption spectroscopy (XAS) as well as theoretical techniques.

Metallothioneins have a dumbell-like shape and unlike most metalloproteins which bind metal ions through finger or twist structures, metallothionein (MT) interacts with metals through its two domains – α and β, each a metal binding cluster. In the β domain consisting of residues 1-30, three metal ions are coordinated by nine cysteines. The alpha domain (residues 31-61) binds four metal ions to eleven cysteines.

The cysteines are deprotonated and coordinate the metals in a tetrahedral fashion. All cys are involved in the binding of the metals. MT’s contain almost no regular secondary structure elements. The best characterized mammalian metallothioneins contain a single polypeptide chain with 7 bound metal ions (either Zn2+ or Cd2+).

The structure of the rat liver metallothionein has been solved both by Nuclear Magnetic Resonance (NMR) and x-ray crystallography. The x-ray crystal structure of rat liver Zn2Cd5-MT refined at 2.0 Å resolution of R value 1.76 and NMR solution structures of rabbit liver Cd7-MTII and rat liver Cd7-MTII and human liver Cd7-MTII show that metallothionein contains two structurally independent α (C- terminal) and β (N-terminal) domains, which are linked in the protein via two amino acids. These investigations established the cysteine ligand to metal coordination pattern for the three-metal and four metal clusters, described hydrogen bonding within each domain and showed that all three proteins adopt a similar conformation, i.e. the seven metal ions are present in clusters of four and three metals bound to bridging and terminal cysteine thiolate ligands, with metal-to-thiolate ratios of M4S11 and M3S9 for the alpha and beta domains respectively. The NMR structure contains 7 Cd ions, whereas the x-ray structure has 4 Cd ions in the alpha domain, and a CdZn2 composition in the β domain. When both Zn and Cd are present, Cd binds preferentially to the α domain, whereas Zn is found preferentially in the β domain. All 20-cysteine residues participate in metal binding, and each of the seven Zn and Cd ions is tetrahedrally coordinated to four cysteine thiolate sulfur atoms.

Now, referring to the insilico studies regarding this protein we have the work of Fowle and Stillman. They have used molecular modeling (MM2) techniques to compare the structures of the metal thiolate binding site in Zn(II)-, Cd(II)-, and Hg(II)- metallothionein. Their calculations show the three-dimensional arrangement of all 62 amino acids in the rabbit liver isoform 2 peptide, the connectivities between each of the 20 cysteinyl thiolates, and of the bridging and terminal thiolates in the two cluster structures and thus gave an insight into the local environment of each metal, including the presence of crevices in both domains that suggest the likely spatial route for metal exchange. Reports of modelling metallothionein also include calculations of the metal thiolate core by Ab initio methods, MM/MD (Molecular Mechanics/Molecular Dynamics) of the complete protein.