Evidence of Native Metal–S²⁻–Metallothionein Complexes Confirmed by the Analysis of Cup1 Divalent‐Metal‐Ion Binding Properties

Abstract

It has previously been shown that recombinant synthesis, under metal-supplemented conditions, of diverse metallothioneins (MTs) results in the recovery of a subpopulation of S(2-)-containing complexes in addition to the S(2-)-devoid canonical metal-MT species. Further significance of this finding has remained veiled by the possibility of it being a mere consequence of synthesis in a heterologous bacterial system. Herein, we present definitive evidence that S(2-) ligands are also constituents of native metal-MT complexes. Because, although practically universal, the highest S(2-) content is incorporated by copper-thioneins when coordinating divalent metal ions, we adapted the Saccharomyces cerevisiae Cup1 protein, which is the most paradigmatic copper-thionein, as an experimental model. Most significantly, native Cd-Cup1 complexes were purified and fully spectroscopically and spectrometrically characterized from the 301N mutant yeast strain, which allows Cup1 synthesis even in the absence of copper. These results undoubtedly revealed the presence of a Cd-S(2-)-Cup1 species in native preparations, which were only recovered when carefully avoiding the use of ion-exchange chromatography in the purification protocol. Furthermore, complete analysis of recombinant (Escherichia coli) Zn-Cup1, Cd-Cup1, and Cu-Cup1 and those complexes that result from Zn/Cd and Zn/Cu replacements in vitro and acidification/renaturalization processes yielded a comprehensive and comparative overview of the metal-binding abilities of Cup1. Overall, we consider the main conclusions of this study to go beyond the mere study of the particular Cup1 MT, so that they should be considered to delineate a new point of view on the interaction between copper-thioneins and divalent metal ions, still an unexplored aspect in MT research.

Metal Dealing at the Origin of the Chordata Phylum: The Metallothionein System and Metal Overload Response in Amphioxus

“…250 nm, and only a minor absorption at (-) 275 nm. The presence of S 2− -containing complexes in the preparation of both Cd-BfMT1 and Cd-BfMT2 preparations prompted us to analyzed whether their CD fingerprints evolved over time, a phenomenon that we have previously reported for preparations of the yeast Cd-Cup1, containing a significant proportion of S 2− -containing complexes [45]. The main changes are observed for the Cd-BfMT1 CD fingerprint, in which the initial broad signal evolves to form two new narrower signals ca.…”

“…Finally, the two highly dissimilar yeast MTs (Cup1 and Crs5) are of Cu-thionein character, but the extreme specificity for copper binding of Cup1 [45] is changed to a more subtle preference for Crs5 [30], compatible with a mixed metal metabolism role. A fairly comprehensive picture of MT differentiation in different taxa seems to emerge from these considerations.…”

Shaping mechanisms of metal specificity in a family of metazoan metallothioneins: evolutionary differentiation of mollusc metallothioneins

“…Significantly, the total equivalence between the features of the metal-MT complexes synthesized in these two hosts (bacteria and yeast) has recently been demonstrated for both cadmium and copper, using the Cup1 MT as a model system [35]. …”

Physiological relevance and contribution to metal balance of specific and non-specific Metallothionein isoforms in the garden snail, Cantareus aspersus

“…Furthermore, some of the Cd-species include S 2-ligands (Fig. 5c), which is a trait indicative of Cu-thionein character (Capdevila et al 2005;Bofill et al 2009;Orihuela et al 2010). Concordantly, this isoform exhibits intermediate features regarding its induction pattern and synthesis.…”