Evolution of Cd ²⁺ and Cu ⁺ binding in Helix pomatia metallothioneins

Abstract

Metallothioneins (MTs) are small proteins present in all kingdoms of life. Their high cysteine content enables them to bind metal ions, such as Zn ²⁺, Cd ²⁺, and Cu ⁺, providing means for detoxification and metal homeostasis. Three MT isoforms with distinct metal binding preferences are present in the Roman Snail Helix pomatia. Here, we use nuclear magnetic resonance (NMR) to follow the evolution of Cd ²⁺ and Cu ⁺ binding from the reconstructed ancestral Stylommatophora MT to the three H. pomatia MT (HpMT) isoforms. Information obtained from [ ¹⁵N, ¹H]-HSQC spectra and T ₂ relaxation times are combined to describe the conformational stability of the MT-metal complexes. A well-behaved MT-metal complex adopts a unique structure and does not undergo additional conformational exchange. The ancestor to all three HpMTs forms conformationally stable Cd ²⁺ complexes and closely resembles the Cd ²⁺-specific HpCdMT isoform, suggesting a role in Cd ²⁺ detoxification for the ancestral protein. All Cu ⁺-MT complexes, including the Cu ⁺-specific HpCuMT isoform, undergo a considerable amount of conformational exchange. The unspecific HpCd/CuMT and the Cu ⁺-specific HpCuMT isoforms form Cu ⁺ complexes with comparable characteristics. It is possible to follow how Cd ²⁺ and Cu ⁺ binding changed throughout evolution. Interestingly, Cu ⁺ binding improved independently in the lineages leading to the unspecific and the Cu ⁺-specific HpMT isoforms. C-terminal domains are generally less capable of coordinating the non-cognate metal ion than N-terminal domains, indicating a higher level of specialization of the C-domain. Our findings provide new insights into snail MT evolution, helping to understand the interplay between biological function and structural features toward a comprehensive understanding of metal preference.

Graphical Abstract

Graphical Abstract

Well-behaved metallothionein-metal complexes adopt unique structures that lack conformational exchange. The conformational stability of these complexes can be probed using NMR. We follow the evolution of metal binding in gastropod metallothioneins by investigating the conformational stability of Cd ²⁺ and Cu ⁺ complexes formed by extant and ancestral proteins.