Metallothioneins: Structure and Functions

All metallothioneins (MTs) possess a highly conserved amino acid sequence and present only a few structural changes even when isolated from different animal species. In mammals, a single MT molecule is made up of 61–68 amino acids, depending on the isoform (the MT-1, MT-2, and MT-4 isoforms consist of 61–62 amino acids, whereas the MT-3 isoform comprises 68 amino acids), and the protein sequence is composed of up to 20 cysteine (Cys) residues (Vasak 2005; Vasak and Meloni 2011). Furthermore, in mammals, no aromatic amino acids are found in the MT molecules. Protein sequencing has revealed that the MT molecule is a single polypeptide chain, in which the Cys residues are organized in the sequences Cys-X-Cys, Cys-X-X-Cys, and Cys-Cys, where “X” denotes an amino acid other than Cys (Kojima et al. 1976; Huang and Yoshida 1977). The Cys residues are the metal-binding domains of the MT molecule, in which they are juxtaposed with lysine (Lys) and arginine (Arg) amino acid residues and arranged in two thiol-rich sites designated domains α and β (Fig. 2.1). The two metal-binding domains are separated by a non-cysteine-containing sequence often designated as the spacer or linker (Zangger et al. 2001; Babula et al. 2012). The α-domain consists of amino acids 31–68 and is located on the C-terminal edge, whereas the N-terminal β-domain contains amino acids 1–30 (Zangger et al. 2001; Dziegiel 2004). It has been demonstrated that the α-domain is capable of binding up to four, and the β-domain up to three, bivalent metal ions such as zinc, cadmium, mercury, or lead (Coyle et al. 2002b; Duncan et al. 2006). The part of the protein with no bound metal ions is termed apo-metallothionein (apo-MT) or thionein (Coyle et al. 2002b). Metallothioneins are also capable of reacting with up to 12 univalent metal ions (Palmiter 1998; Coyle et al. 2002b). Zinc ions, which naturally occur in the organism, are regarded as the main binding partner of apo-MT. However, other nonessential metal ions occurring pathologically in the organism—such as lead, copper, cadmium, mercury, platinum, chromate, bismuth, and silver—often possess higher affinity to the apo-MT-binding sites (Nordberg and Nordberg 2000; Ngu and Stillman 2009; Ngu et al. 2010b; Gumulec et al. 2011; Babula et al. 2012). So far, only iron ions (Fe2+) have been identified to possess lower affinity to the metal-binding sites of the apo-MT domains (Foster and Robinson 2011). Interestingly, only a small proportion of MT molecules was found bound to zinc ions in various organisms. In rat tissues, apo-MT has been shown to constitute up to 54 % of the total amount of MT, whereas higher apo-MT levels were detected in rat cancer cells (Yang et al. 2001). Recent studies have also identified small amounts of sulfide ligands bound to recombinant MT-1 and MT-4 proteins overexpressed in Escherichia coli (Capdevila et al. 2005; Tio et al. 2006). Nevertheless, studies analyzing MT proteins in the cytoplasm of mammalian cells have failed to detect sulfide ligands bound to their molecules (Mounicou et al. 2010).