Structural features specific to plant metallothioneins

Abstract

The metallothionein (MT) superfamily combines a large variety of small cysteine-rich proteins from nearly all phyla of life that have the ability to coordinate various transition metal ions, including Zn(II), Cd(II), and Cu(I). The members of the plant MT family are characterized by great sequence diversity, requiring further subdivision into four subfamilies. Very peculiar and not well understood is the presence of rather long cysteine-free amino acid linkers between the cysteine-rich regions. In light of the distinct differences in sequence to MTs from other families, it seems obvious to assume that these differences will also be manifested on the structural level. This was already impressively demonstrated with the elucidation of the three-dimensional structure of the wheat E(c)-1 MT, which revealed two metal cluster arrangements previously unprecedented for any MT. However, as this structure is so far the only one available for the plant MT family, other sources of information are in high demand. In this review the focus is thus set on any structural features known, deduced, or assumed for the plant MT proteins. This includes the determination of secondary structural elements by circular dichroism, IR, and Raman spectroscopy, the analysis of the influence of the long linker regions, and the evaluation of the spatial arrangement of the sequence separated cysteine-rich regions with the aid of, e.g., limited proteolytic digestion. In addition, special attention is paid to the contents of divalent metal ions as the metal ion to cysteine ratios are important for predicting and understanding possible metal-thiolate cluster structures. Very peculiar and not well understood is the presence of rather long Cys-free amino acid linkers between the Cys-rich regions. In light of the distinct differences in sequence to MTs from other families it seems obvious to assume that these differences will be also manifested on the structural level. This was already impressively demonstrated with the elucidation of the three-dimensional structure of the wheat E c -1 MT, which revealed two metal cluster arrangements previously unprecedented for any MT. However, as this structure is so far the only one available for the plant MT family other sources of information are of high demand.In this review the focus is thus set on any structural features known, deduced or assumed for the plant MT proteins. This includes the determination of secondary structural elements by CD, IR, and Raman spectroscopy, the analysis of the influence of the long linker regions, as well as the evaluation of the spatial arrangement of the sequence separated Cys-rich regions with the aid of e.g. limited proteolytic digestion. In addition, special attention is paid to the contents of divalent metal ions as the metal ion-to-Cys ratios are important for predicting and

The response of the different soybean metallothionein isoforms to cadmium intoxication

“…Total RNA was isolated with TRIZOL® reagent (Invitrogen) following the manufacturer's protocol and treated with DNase I (Fermentas). The cDNA was synthesized using an oligo(dT 18 ) primer from 1 μg of total RNA with the reverse transcriptase RevertAid™ (Fermentas) and RNaseOUT™ Recombinant RNase Inhibitor (Invitrogen). Quantitative real-time PCR reactions were performed in a 20-μL reaction volume with 0.5 μM gene-specific primers (listed in Table 1), 2 μL of 1/100 diluted cDNA as a template, and SYBR Green I (Invitrogen) as detection reagent.…”

“…As opposed to the large amount of knowledge of animal MTs, the structural and functional properties of plant MTs are little known (for recent plant MT reviews cf. [17,18]). The distribution of cysteine residues, as well as the length of the spacer region, served to further classify plant MTs into four types [19,20].…”

NMR Spectroscopy in Bioinorganic Chemistry

“…Beside some common characteristics shared by all members of the MT superfamily, plant MTs feature a number of unique properties such as higher sequence diversity, occurrence of aromatic amino acids including histidine, and long Cysfree amino acid stretches as mentioned above. [1][2][3] Not surprisingly, this variety is also reflected on the structural level as the first structure elucidation of a plant MT, i.e.T riticum aestivum (common bread wheat) Zn 6 E c -1, revealed two metal ion arrangements that were both unprecedented for MTs at that time. [9,10] The C-terminal β E -domain exposes a mononuclear binding site, M II Cys 2 His 2 , intertwined with the residues forming a M 3 II Cys 9 cluster (Fig.…”

“…[1][2][3][4][5] In addition, classical biochemical assays like in-line probing analyses or Tb 3+ cleavage assays are applied for the study of RNA, while application of mass spectrometry, MCD, and EXAFS forms an integral part of our research on plant MTs. Nuclear magnetic resonance (NMR) spectroscopy is a crucial technique that we use not only to obtain structural information on our molecules at an atomic level [6][7][8][9][10][11] but, even more important, to elucidate in detail their metal ion binding.…”

Genetic Engineering of Plants for Heavy Metal Removal from Soil

“…In plants, MTs are suggested to be involved in metal tolerance or homeostasis, detoxification, and distribution as they are able to bind metal ions by the formation of mercaptide bonds with the numerous cysteine residues. Recent reports show that MTs are also involved in the scavenging of reactive oxygen species (Hassinen et al 2011;Freisinger 2011;Leszczyszyn et al 2013). Each MT exhibits preferences for a special metal ion due to coordination residues other than cysteine and differences in folding and stability in dependence on the bound metal (Leszczyszyn et al 2007).…”