The βE-Domain of Wheat Ec-1 Metallothionein: A Metal-Binding Domain with a Distinctive Structure

Metallothioneins (MTs) are ubiquitous cysteine-rich proteins with a high affinity for divalent metal ions such as Zn-II, Cu-I and Cd-II that are involved in metal ion homeostasis and detoxification, as well as protection against reactive oxygen species. Here we show the NMR solution structure of the beta(E)-domain of the early cysteine-labeled protein (E-c-1) from wheat (beta(E)-E-c-1), which represents the first three-dimensional structure of a plant MT. The beta(E)-domain comprises the 51 C-terminal residues of E-c-1 and exhibits a distinctive unprecedented structure with two separate metal-biricling centers, a mononuclear Zn-II binding site constituted by two cysteine and two highly conserved histidine residues as found in certain zinc-finger motifs, and a cluster formed by three Zn-II ions coordinated by nine Cys residues that resembles the cluster in the beta-domain of vertebrate MTs. Cys-metal ion connectivities were determined by exhaustive structure calculations for all 7560 possible configurations of the three-metal cluster. Backbone dynamics investigated by N-15 relaxation experiments support the results of the structure determination in that beta(E)-E-c-1 is a rigidly folded polypeptide. To further investigate the influence of metal ion binding on the stability of the structure, we replaced Zn-II with Cd-II ions and examined the effects of metal ion release on incubation with a metal. ion chelator. (C) 2009 Elsevier Ltd. All rights reserved.