The Solution Structure and Dynamics of Cd-Metallothionein from Helix pomatia Reveal Optimization for Binding Cd over Zn

Metallothioneins (MTs) are cysteine-rich polypeptides that are naturally found coordinated to monovalent and/ or divalent transition metal ions. Three metallothionein isoforms from the Roman snail Helix pomatia are known. They differ in their physiological metal load and in their specificity for transition metal ions such as Cd2+ (HpCdMT isoform) and Cu+ (HpCuMT isoform) or in the absence of a defined metal specificity (HpCd/CuMT isoform). We have determined the solution structure of the Cd-specific isoform (HpCdMT) by nuclear magnetic resonance spectroscopy using recombinant isotopically labeled protein loaded with Zn2+ or Cd2+. Both structures display two-domain architectures, where each domain comprises a characteristic three-metal cluster similar to that observed in the β-domains of vertebrate MTs. The polypeptide backbone is well-structured over the entire sequence, including the interdomain linker. Interestingly, the two domains display mutual contacts, as observed before for the metallothionein of the snail Littorina littorea, to which both N- and C-terminal domains are highly similar. Increasing the length of the linker motionally decouples both domains and removes mutual contacts between them without having a strong effect on the stability of the individual domains. The structures of Cd6- and Zn6-HpCdMT are nearly identical. However, 15N relaxation, in particular 15N R2 rates, is accelerated for many residues of Zn6-HpCdMT but not for Cd6-HpCdMT, revealing the presence of conformational exchange effects. We suggest that this snail MT isoform is evolutionarily optimized for binding Cd rather than Zn

Drosophila MTN: a metazoan copper-thionein related to fungal forms

AbstractTwo Drosophila metallothioneins (MT) have been reported: MTN, a 40 residue peptide including 10 Cys, and MTO, a 43 residue peptide including 12 Cys. However, neither functional nor evolutionary analyses for either of the Drosophila MT are available. Here, heterologous expression of Mtn in Escherichia coli is reported. The metal binding abilities of the Cu- and Zn-MTN complexes conformed in vivo, as well as the features of the Cd- and Cu-aggregates produced by metal replacement in vitro, have been determined by atomic emission spectrometry, circular dichroism and electrospray ionization mass spectrometry. Primary structure relationships with other MT have been examined. The results indicate a close resemblance of MTN to fungal copper-thioneins