Evidence of native metal-S(2-)-metallothionein complexes confirmed by the analysis of Cup1 divalent-metal-ion binding properties.
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.
Orihuela
Rubén
R
Departament de Química, Facultat de Ciències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.
Monteiro
Freddy
F
Pagani
Ayelén
A
Capdevila
Mercè
M
Atrian
Sílvia
S
D001483:Base Sequence; D002104:Cadmium / Q000032:analysis; D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D008668:Metallothionein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013440:Sulfides / Q000032:analysis / Q000737:chemistry*; D015032:Zinc / Q000032:analysis
C500769:CUP1-1 protein, S cerevisiae; D011994:Recombinant Proteins; D013440:Sulfides; D002104:Cadmium; D003300:Copper; D008668:Metallothionein; D015032:Zinc