In this study, we present an NMR structure of the metallothionein (MT) from the snail Littorina littorea (LlMT) in complex with Cd . LlMT is capable of binding 9 Zn or 9 Cd ions. Sequence alignments with other snail MTs revealed that the protein is likely composed of three domains. The study revealed that the protein is divided into three individual domains, each of which folds into a single well-defined three-metal cluster. The central α2 and C-terminal β domains are positioned with a unique relative orientation. Two variants with longer and shorter linkers were investigated, which revealed that specific interdomain contacts only occurred with the wild-type linker. Moreover, a domain-swap mutant in which the highly similar α1 and α2 domains were exchanged was structurally almost identical. It is suggested that the expression of a three-domain MT confers an evolutionary advantage on Littorina littorea in terms of coping with Cd stress and adverse environmental conditions.

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 Cdspecific 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-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.

Die dreidimensionale Struktur des Metallothioneins (MT) der Großen Strandschnecke( Littorina littorea), LlMT, im Komplex mit Cd 2+ -Ionen wurdeN MR-spektroskopisch aufgeklärt. Das LlMT ist imstande,n eun Zn 2+ -o der neun Cd 2+ -Ionen zu binden. Sequenz-Alignments mit MTs anderer Gastropoden lassen darauf schließen, dass das Protein wahrscheinlich aus drei Domänen aufgebaut ist. Das Protein ist in drei Einzeldomänen unterteilt, von denen jede eine gut definierte Faltung in Form eines Drei-Metall-Clusters annimmt.Die mittlere a2-und die C-terminale b-Domäne sind zueinander in einer definierten relativen Orientierung positioniert. Zwei Varianten des LlMT,m it einem längeren und einem kürzeren Linker,wurden ebenfalls untersucht;die spezifischen Kontakte zwischen den Domänen traten nur im Fall des Wildtyp-Linkers auf.E ine weitere Mutante,b ei der die einander sehr ähnlichen a1-und a2-Domänen miteinander vertauscht wurden,z eigte eine fast identischeS truktur zum Wildtyp-MT.Die Expression eines Drei-Domänen-MT kçnnte Littorina littorea einen evolutionären Vorteil bei der Bewältigung von Cd 2+ -Stress und widrigen Umweltbedingungen verleihen.

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