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 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 beta(E) domain was recently described. Here we present the structure of the N-terminal second domain, gamma-E-c-1, determined by NMR spectroscopy. The gamma-E-c-1 domain enfolds an M (2) (II) Cys(6) 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 Zn-II-containing isoform and the Cd-II-containing isoform. Extended X-ray absorption fine structure analysis of Zn-2 gamma-E-c-1 clearly shows the presence of a ZnS4 coordination sphere with average Zn-S distances of 2.33 . Cd-113 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 gamma-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 gamma domain and the beta(E) domain within the full-length protein.