Dynamic structure of metallothionein

Vašák, Milan; Berger, Christopher L.; Kägi, Jeremias H. R.

doi:10.1016/0014-5793(84)80230-6

Cited by 22 publications

(4 citation statements)

References 15 publications

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“…Cd7-MT elutes from the Sephadex G-50 gel filtration column as a single chromatographic peak (Figure la) with an apparent molecular weight of 10700. A similar apparent molecular weight has also been found in a number of previous chromatographic studies (Kági et al, 1974;VaSák et al, 1984;Kági & Kojima, 1987). In the equilibrium ultracentrifugation experiments the same sample yielded a linear plot of In c against r2 (Figure 2A) from which a molecular weight of 6900 was calculated.…”

Section: Resultssupporting

confidence: 81%

Nonoxidative cadmium-dependent dimerization of cadmium-metallothionein from rabbit liver

Palumaa¹,

Mackay²,

Vašák³

1992

Biochemistry

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The effect of free Cd(II) ions on monomeric Cd7-metallothionein-2 (MT) from rabbit liver has been studied. Slow, concentration-dependent dimerization of this protein was observed by gel filtration chromatographic studies. The dimeric MT form, isolated by gel filtration, contains approximately two additional and more weakly bound Cd(II) ions per monomer. The incubation of MT dimers with complexing agents EDTA and 2-mercaptoethanol leads to the dissociation of dimers to monomers. The results of circular dichroism (CD) and electronic absorption studies indicate that the slow dimerization process is preceded by an initial rapid Cd-induced rearrangement of the monomeric Cd7-MT structure. The 113Cd NMR spectrum of the MT dimer revealed only four 113Cd resonances at chemical shift positions similar to those observed for the Cd4 cluster of the well-characterized monomeric 113Cd7-MT. This result suggests that on dimer formation major structural changes occur in the original three-metal cluster domain of Cd7-MT.

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Section: Resultssupporting

confidence: 81%

Nonoxidative cadmium-dependent dimerization of cadmium-metallothionein from rabbit liver

Palumaa¹,

Mackay²,

Vašák³

1992

Biochemistry

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“…The Stokes radius decreased from 17.8 to 15 Á when the ionic strength was increased from 0.015 to 0.5 M, indicating the large changes in the molecular shape of this protein. CD spectra obtained under similar conditions showed changes in the CD band at 227 nm, possibly attributable to a conformational transition of the polypeptide chain (Vasák et al, 1984).…”

Section: Discussionmentioning

confidence: 82%

“…present; (c) there are large differential temperature and ionic strength effects; (d) most resonances appear to be composed of several overlapping multiplets, many of which are doublets and triplets, i.e., Cd ions coupled to one or two other Cd ions, respectively, but with little clear indication of quartets or of pairs of triplets. Second, the results from other spectroscopic and structural studies should be considered, too: (a) tetrahedral-tetrathiolate metal coordination (Vasák, 1980); (b) the participation of all 20 cysteine residues in metal binding via mercaptide linkages (Kági & Vallee, 1961;Weser et al, 1973); (c) the capability of the COOH-terminal part of the protein to bind four Cd2+ ions (Winge & Miklossy, 1982); (d) the marked change of the shape of the MT molecule on raising the ionic strength (Vasák et al, 1984).…”

Section: Discussionmentioning

confidence: 99%

Cadmium-113 NMR studies of reconstituted seven-cadmium metallothionein: evidence for structural flexibility

Vašák¹,

Hawkes²,

Nicholson³

et al. 1985

Biochemistry

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A reproducible method for the reconstitution of rabbit liver metallothionein (MT) containing seven cadmium atoms per mole of protein is described. This protein was studied in detail by 113Cd NMR at 88-, 55-, and 44-MHz frequencies, including the effects of pH, temperature, and ionic strength on the spectra. Our results differ significantly from previous reports of 113Cd NMR on similar samples. Thus, the spectra of both chromatographically distinguishable isoforms MT1 and MT2 were not identical, and neither could be interpreted in terms of a unique static model with the seven Cd ions forming two independent clusters of four and three Cd ions. Large differential shifts of 113Cd resonances were observed with changes in temperature over the range 277-320 K and ionic strength (0.02-0.5 M). At low temperature a slow structural change (half-life of several minutes) was detected. The structure was more rigid at high ionic strength. The frequency dependence and two-dimensional J-resolved spectra revealed that 113Cd resonances were composed of several overlapping peaks, complicating the interpretation of fine structure in one-dimensional spectra. A new flexible model of the Cd cluster in metallothionein is proposed. This model incorporates dynamic thiolate exchange reactions and involves several configurational substates of the protein. The possible relationship of such flexibility to the function of metallothionein is discussed.

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“…The different modification numbers of cysteines in the [32][33][34][35][36][37][38][39][40][41][42][43] fragment (Table 1) and co-existence of several variously Zn(II)-depleted forms probably indicate high internal dynamics of Zn-S bonds, with their constant breakage and re-formation, as has been proposed in earlier studies. [57][58][59]66 Another plausible explanation relates to the stability of particular metal binding sites. The fourth Zn(II) equivalent may be partially distributed amongst a-and b-domains, as was proposed in the literature, 63 which could explain the observed protection of certain b-domain regions from alkylation (Table 1).…”

Section: Zn(ii) Binding To the A-domain Of Mt2 Is Sequential Under Cementioning

confidence: 99%

Crosstalk of the structural and zinc buffering properties of mammalian metallothionein-2

et al. 2018

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a Metallothioneins (MTs), small cysteine-rich proteins, present in four major isoforms, are key proteins involved in zinc and copper homeostasis in mammals. To date, only one X-ray crystal structure of a MT has been solved. It demonstrates seven bivalent metal ions bound in two structurally independent domains with M 4 S 11 (a) and M 3 S 9 (b) clusters. Recent discoveries indicate that Zn(II) ions are bound with MT2 with the range from nano-to picomolar affinity, which determines its cellular zinc buffering properties that are demonstrated by the presence of partially Zn(II)-depleted MT2 species. These forms serve as Zn(II) donors or acceptors and are formed under varying cellular free Zn(II) concentrations. Due to the lack of appropriate methods, knowledge regarding the structure of partially-depleted metallothionein is lacking. Here, we describe the Zn(II) binding mechanism in human MT2 with high resolution with respect to particular Zn(II) binding sites, and provide structural insights into Zn(II)-depleted MT species. The results were obtained by the labelling of metal-free cysteine residues with iodoacetamide and subsequent top-down electrospray ionization analysis, MALDI MS, bottom-up nanoLC-MALDI-MS/MS approaches and molecular dynamics (MD) simulations. The results show that the a-domain is formed sequentially in the first stages, followed by the formation of the b-domain, although both processes overlap, which is in contrast to the widely investigated cadmium MT. Independent ZnS 4 cores are characteristic for early stages of domain formation and are clustered in later stages. However, Zn-S network rearrangement in the b-domain upon applying the seventh Zn(II) ion explains its lower affinity. Detailed analysis showed that the weakest Zn(II) ion associates with the b-domain by coordination to Cys21, which was also found to dissociate first in the presence of the apo-form of sorbitol dehydrogenase. We found that Zn(II) binding to the isolated b-domain differs significantly from the whole protein, which explains its previously observed different Zn(II)-binding properties. MD results obtained for Zn(II) binding to the whole protein and isolated b-domain are highly convergent with mass spectrometry data. This study provides a comprehensive overview of the crosstalk of structural and zinc buffering related-to-thermodynamics properties of partially metal-saturated mammalian MT2 and sheds more light on other MT proteins and zinc homeostasis. Significance to metallomicsThe growing interest in metallomics within metal-dependent biological systems provides an opportunity to explore the structural and buffering properties of metallothioneins (MTs). The wide relevance of MTs is obvious due to their multiple biological functions such as participation in zinc and copper homeostasis. However, the MT metalloproteome is still not fully explored and is rather complicated in vivo or in vitro because of its highly dynamic structure, which lacks secondary structural elements and metamorphic behaviour. We have undertaken compreh...

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Dynamic structure of metallothionein

Cited by 22 publications

References 15 publications

Nonoxidative cadmium-dependent dimerization of cadmium-metallothionein from rabbit liver

Nonoxidative cadmium-dependent dimerization of cadmium-metallothionein from rabbit liver

Cadmium-113 NMR studies of reconstituted seven-cadmium metallothionein: evidence for structural flexibility

Crosstalk of the structural and zinc buffering properties of mammalian metallothionein-2

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