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.