Protein and metal cluster structure of the wheat metallothionein domain γ-Ec-1: the second part of the puzzle

Metallothioneins (MTs) are small cysteine-rich proteins coordinating various transition metal ions, including ZnII, CdII, and CuI. MTs are ubiquitously present in all phyla, indicating a successful molecular concept for metal ion binding in all organisms. The plant MT Ec-1 from Triticum aestivum, common bread wheat, is a ZnII-binding protein that comprises two domains and binds up to six metal ions. The structure of the C-terminal four metal ion binding βEdomain was recently described. Here we present the structure of the N-terminal second domain, γ-Ec-1, determined by NMR spectroscopy. The γ-Ec-1 domain enfolds an M 2 II Cys6 cluster and was characterized as part of the full-length Zn6Ec-1 protein as well as in the form of the separately expressed domain, both in the ZnII-containing isoform and the CdII-containing isoform. Extended X-ray absorption fine structure analysis of Zn2γ-Ec-1 clearly shows the presence of a ZnS4 coordination sphere with average Zn-S distances of 2.33Å. 113CdNMR experiments were used to identify the MII-Cys connectivity pattern, and revealed two putative metal cluster conformations. In addition, the general metal ion coordination abilities of γ-Ec-1 were probed with CdII binding experiments as well as by pH titrations of the ZnII and CdII forms, the latter suggesting an interaction of the γdomain and the βEdomain within the full-length protei

Localization and Spectroscopic Analysis of the Cu(I) Binding Site in Wheat Metallothionein Ec-1

The early cysteine-labeled metallothionein (MT) from Triticum aestivum (common wheat), denoted Ec-1, features two structurally well-defined domains, γ and βE, coordinating two and four Zn(II) ions, respectively. While the protein is currently assumed to function mainly in zinc homeostasis, a low amount of copper ions was also recently detected in a native Ec-1 sample. To evaluate the observed copper binding in more detail, the recombinant Zn6Ec-1 form was exposed to different amounts of Cu(I) ions and the resulting species characterized with spectroscopic methods. Data reveal that the first Cu(I) equivalent coordinates exclusively to the N-terminal γ-domain of the protein and replaces one Zn(II) ion. To analyze the ability of the γ-domain for coordination of monovalent metal ions in more detail, the γ-Ec-1 peptide fragment was incubated with increasing amounts of Cu(I) and the process monitored with UV–VIS, circular dichroism, and luminescence spectroscopy. Closely similar spectra are observed regardless if the apo- or the metal ion-loaded and, hence, pre-folded forms, were used for the titration experiments with Cu(I). The results indicate that low amounts of Cu(I) ions displace the two metal ions subsequently and stoichiometrically, despite the different coordination geometry requirements of Cu(I) and Zn(II)

Protein and metal cluster structure of the wheat metallothionein domain γ−Ec−1\mathrm{\gamma-E_c-1}: the second part of the puzzle

Metallothioneins (MTs) are small cysteine-rich proteins coordinating various transition metal ions, including Zn$^{II}$, Cd$^{II}$, and Cu$^{I}$. MTs are ubiquitously present in all phyla, indicating a successful molecular concept for metal ion binding in all organisms. The plant MT E$_c$-1 from Triticum aestivum, common bread wheat, is a Zn$^{II}$-binding protein that comprises two domains and binds up to six metal ions. The structure of the C-terminal four metal ion binding β$_E$ domain was recently described. Here we present the structure of the N-terminal second domain, γ-Ec-1, determined by NMR spectroscopy. The γ-E$_c$-1 domain enfolds an M$_{2}^{II}$Cys$_6$ cluster and was characterized as part of the full-length Zn$_6$E$_c$-1 protein as well as in the form of the separately expressed domain, both in the Zn$^{II}$-containing isoform and the Cd$^{II}$-containing isoform. Extended X-ray absorption fine structure analysis of Zn$_2$γ-E$_c$-1 clearly shows the presence of a ZnS$_4$ coordination sphere with average Zn–S distances of 2.33 Å. $^{113}$Cd NMR experiments were used to identify the M$^{II}$-Cys connectivity pattern, and revealed two putative metal cluster conformations. In addition, the general metal ion coordination abilities of γ-E$_c$-1 were probed with Cd$^{II}$ binding experiments as well as by pH titrations of the Zn$^{II}$ and Cd$^{II}$ forms, the latter suggesting an interaction of the γ domain and the β$_E$ domain within the full-length protein