Structural features specific to plant metallothioneins

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 ZnII, CdII, and CuI. 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 Ec-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 structure

An optimized intein-mediated protein ligation approach for the efficient cyclization of cysteine-rich proteins

Head-to-tail backbone cyclization of proteins is a widely used approach for the improvement of protein stability. One way to obtain cyclic proteins via recombinant expression makes use of engineered Intein tags, which are self-cleaving protein domains. In this approach, pH-induced self-cleavage of the N-terminal Intein tag generates an N-terminal cysteine residue at the target protein, which then attacks in an intramolecular reaction the C-terminal thioester formed by the second C-terminal Intein tag resulting in the release of the cyclic target protein. In the current work we aimed to produce a cyclic analog of the small γ-Ec-1 domain of the wheat metallothionein, which contains six cysteine residues. During the purification process we faced several challenges, among them premature cleavage of one or the other Intein tag resulting in decreasing yields and contamination with linear species. To improve efficiency of the system we applied a number of optimizations such as the introduction of a Tobacco etch virus cleavage site and an additional poly-histidine tag. Our efforts resulted in the production of a cyclic protein in moderate yields without any contamination with linear protein specie

Metal ion binding properties of Triticum aestivum Ec-1 metallothionein: evidence supporting two separate metal thiolate clusters

Metallothioneins are ubiquitous low molecular mass, cysteine-rich proteins with an extraordinary high metal ion content. In contrast to the situation for the vertebrate forms, information regarding the properties of members of the plant metallothionein family is still scarce. We present the first spectroscopic investigation aiming to elucidate the metal ion binding properties and metal thiolate cluster formation of the Tricium aestivum (common wheat) early cysteine-labeled plant metallothionein (Ec-1). For this, the protein was overexpressed recombinantly in Escherichia coli. Recombinant Ec-1 is able to bind a total of six divalent d 10 metal ions in a metal thiolate cluster arrangement. The pH stability of the zinc and cadmium clusters investigated is comparable to stabilities found for mammalian metallothioneins. Using cobalt(II) as a paramagnetic probe, we were able to show the onset of cluster formation taking place with the addition of a fourth metal ion equivalent to the apo protein. Limited proteolytic digestion experiments complemented with mass spectrometry and amino acid analysis provide clear evidence for the presence of two separate metal thiolate clusters. One cluster consists of four metal ions and is made up by a part of the protein containing 11 cysteine residues, comparable to the situation found in the mammalian counterparts. The second cluster features two metal ions coordinated by six cysteine residues. The occurrence of the latter cluster is unprecedented in the metallothionein superfamily so fa

Metal ion binding properties of Tricium aestivum Ec-1 metallothionein: evidence supporting two separate metal thiolate clusters

Metallothioneins are ubiquitous low molecular mass, cysteine-rich proteins with an extraordinary high metal ion content. In contrast to the situation for the vertebrate forms, information regarding the properties of members of the plant metallothionein family is still scarce. We present the first spectroscopic investigation aiming to elucidate the metal ion binding properties and metal thiolate cluster formation of the Tricium aestivum (common wheat) early cysteine-labeled plant metallothionein (Ec-1). For this, the protein was overexpressed recombinantly in Escherichia coli. Recombinant Ec-1 is able to bind a total of six divalent d 10 metal ions in a metal thiolate cluster arrangement. The pH stability of the zinc and cadmium clusters investigated is comparable to stabilities found for mammalian metallothioneins. Using cobalt(II) as a paramagnetic probe, we were able to show the onset of cluster formation taking place with the addition of a fourth metal ion equivalent to the apo protein. Limited proteolytic digestion experiments complemented with mass spectrometry and amino acid analysis provide clear evidence for the presence of two separate metal thiolate clusters. One cluster consists of four metal ions and is made up by a part of the protein containing 11 cysteine residues, comparable to the situation found in the mammalian counterparts. The second cluster features two metal ions coordinated by six cysteine residues. The occurrence of the latter cluster is unprecedented in the metallothionein superfamily so fa

The Bioinorganic Periodic Table

Life depends on metals. While carbon, in terms of abundance and versatility, is considered THE element of life, the vast variety and diversity of the chemistry taking place in living organisms could not be achieved without metal ions. More than twenty metals are found in the human body, most of them being essential, some beneficial, and for others it is still unknown what role they might fulfil in a living cell. Here we give a short introduction into the bioinorganic world of the periodic table, providing just a few examples of key metals for life and aiming to give a flavour to gain further insights into this exciting field of inorganic chemistry at the intersection to the life sciences

NMR Spectroscopy in Bioinorganic Chemistry

Multinuclear and multidimensional nuclear magnetic resonance (NMR) spectroscopy is applied in our groups to gain insights into the role of metal ions for the function and structure of large biomolecules. Specifically, NMR is used i) to investigate how metal ions bind to nucleic acids and thereby control the folding and structure of RNAs, ii) to characterize how metal ions are able to stabilize modified nucleic acids to be used as potential nanowires, and iii) to characterize the formation, structure, and role of the diverse metal clusters within plant metallothioneins. In this review we summarize the various NMR experiments applied and the information obtained, demonstrating the important and fascinating part NMR spectroscopy plays in the field of bioinorganic chemistry

Metal ion release from metallothioneins: proteolysis as an alternative to oxidation

Metallothioneins (MTs) are among others involved in the cellular regulation of essential ZnII and CuI ions. However, the high binding affinity of these proteins requires additional factors to promote metal ion release under physiological conditions. The mechanisms and efficiencies of these processes leave many open questions. We report here a comprehensive analysis of the ZnII-release properties of various MTs with special focus on members of the four main subfamilies of plant MTs. ZnII competition experiments with the metal ion chelator 4-(2-pyridylazo)resorcinol (PAR) in the presence of the cellular redox pair glutathione (GSH) / glutathione disulfide (GSSG) show that plant MTs from the subfamilies MT1, MT2, and MT3 are remarkably more affected by oxidative stress than those from the Ec subfamily and the well-characterized human MT2 form. In addition, we evaluated proteolytic digestion with trypsin and proteinase K as an alternative mechanism for selective promotion of metal ion release from MTs. Also here the observed percentage of liberated metal ions depends strongly on the MT form evaluated. Closer evaluation of the data additionally allowed deducing the thermodynamic and kinetic properties of the ZnII release processes. The CuI-form of chickpea MT2 was used to exemplify that both oxidation and proteolysis are also effective ways to increase the transfer of copper ions to other molecules. ZnII release experiments with the individual metal-binding domains of Ec-1 from wheat grain reveal distinct differences to the full-length protein. This triggers the question about the roles of the long cysteine-free peptide stretches typical for plant MTs