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

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

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