Characterization of the cadmium complex of peptide 49–61: a putative nucleation center for cadmium-induced folding in rabbit liver metallothionein IIA

Muñoz, Amalia; Laib, Frank; Petering, David H.; Shaw, C. Frank

doi:10.1007/s007750050335

Cited by 23 publications

(19 citation statements)

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“…It has been hypothesized that the C-terminal end of the apoMT structure is the reactive part of the molecule [32]. Between residues 50 and 61, there are four cysteinyl residues that comprise the only four consecutive ones which coordinate to a single Cd 2+ or Zn 2+ in either metal-thiolate cluster of the folded MT structure [62].…”

Section: Discussionmentioning

confidence: 99%

“…Between residues 50 and 61, there are four cysteinyl residues that comprise the only four consecutive ones which coordinate to a single Cd 2+ or Zn 2+ in either metal-thiolate cluster of the folded MT structure [62]. We have speculated that binding of metal ions to these particular sulfhydryl groups may serve as a nucleation site in the assembly of intact clusters [32]. That is, once the Cd 1 -MT species has formed, Cd 2+ would be expected to redistribute among thiolate ligands with non-rate-limiting kinetics, resulting in the construction of intact Cd-thiolate clusters.…”

Section: Discussionmentioning

confidence: 99%

“…However, little is known about the sites, such as metalloproteins, to which Cd 2+ binds and the properties of the reactions of apo-or Zn-MT with such sites. Studies of the reaction of apoMT and one of its component peptide sequences with Cd-carbonic anhydrase showed that both were remarkably effective in competing for protein-bound Cd 2+ [31,32]. There is evidence that, in vitro, Cd 2+ can substitute for Zn 2+ in zinc-finger structures and that such substitution can change the activities of the finger domains [22,28,33].…”

Section: Introductionmentioning

confidence: 99%

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Interprotein metal exchange between transcription factor IIIa and apo-metallothionein

Huang

Shaw

Petering

2004

Journal of Inorganic Biochemistry

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Zn 2+ and Cd 2+ ion exchange between transcription factor IIIA (TFIIIA) and apo-metallothionein (MT) were studied using a combination of methods including chromatography, ultrafiltration and UV spectroscopy. Under near stoichiometric conditions, apoMT was able to remove most if not all of the zinc ions from TFIIIA, whether or not the TFIIIA was bound to the 5S DNA internal control region (ICR), and concomitantly inhibit its DNA-binding activity as indicated by an electrophoretic mobility shift assay. The kinetics of the two processes were similar. The rate of the metal exchange reaction increased with the concentrations of both reactants. A second-order rate constant of 30 ± 10 M −1 s −1 was calculated. Similar observations were made for the reaction between apoMT and Cd-substituted TFIIIA, which proceeded without observable intermediates according to a spectrophotometric analysis. A very slow metal ion exchange occurred between Cd-TFIIIA and Zn-MT, but not between Cd-MT and Zn-TFIIIA. Comparative studies on the reaction of TFIIIA with a small competing ligand, ethylenedinitrilo-tetraacetic acid (EDTA), were also conducted. Although EDTA reacts with free Zn-TFIIIA, under similar conditions it failed to compete for Zn 2+ bound as Zn-TFIIIA-ICR.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Interprotein metal exchange between transcription factor IIIa and apo-metallothionein

Huang

Shaw

Petering

2004

Journal of Inorganic Biochemistry

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“…It was synthesized and purified as previously described [41]. The integrity of the peptides was confirmed by amino acid analysis, MALDI-MS, and SDS-PAGE.…”

Section: Synthesis Of Finger 3 Of Tfiiiamentioning

confidence: 99%

Zn-, Cd-, and Pb-transcription factor IIIA: properties, DNA binding, and comparison with TFIIIA-finger 3 metal complexes

Huang

Krepkiy

et al. 2004

Journal of Inorganic Biochemistry

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Properties of the metal ion binding sites of Zn-transcription factor IIIA (TFIIIA) were investigated to understand the potential of this type of zinc finger to undergo reactions that remove Zn 2+ from the protein. Zn-TFIIIA was purified from E. coli containing the cloned sequence for Xenopus laevis oocyte TFIIIA and its stoichiometry of bound Zn 2+ was shown to depend on the details of the isolation process. The average dissociation constant of Zn 2+ in Zn-TFIIIIA was 10 −7 . The dissociation constant for Zn-F3, the third finger from the N-terminus of TFIIIA, was 1.0 × 10 −8 . The reactivity of Zn-TFIIIA with a series of metal binding ligands, including 2-carboxy-2′-hydroxy-5′-sulfoformazylbenzene (zincon), 4-(2-pyridylazo)-resorcinol (PAR), and 3-ethoxy-2-oxo-butyraldehyde-bis-(N 4 -dimethylthiosemicarbazone) (H 2 KTSM 2 ) revealed similar kinetics. The reactivity of PAR with Zn-TFIIIA declined substantially when the protein was bound to the internal control region (ICR) of the 5S ribosomal DNA. Both Cd 2+ and Pb 2+ disrupt TFIIIA binding to its cognate DNA sequence. The Pb 2+ dissociation constant of Pb-F3 was measured as 2.5 × 10 −8 . According to NMR spectroscopy, F3 does not fold into a regular conformation in the presence of Pb 2+ .

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“…The proposed bond re-formation is caused by steric hindrance in the C 33 CSCC 37 motif, which could involve only three cysteine residues for coordination of the same Zn(II) ion. 56,60 The second metal ion is coordinated by Cys50, Cys60 and possibly Cys36/37.…”

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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Characterization of the cadmium complex of peptide 49–61: a putative nucleation center for cadmium-induced folding in rabbit liver metallothionein IIA

Cited by 23 publications

References 0 publications

Interprotein metal exchange between transcription factor IIIa and apo-metallothionein

Interprotein metal exchange between transcription factor IIIa and apo-metallothionein

Zn-, Cd-, and Pb-transcription factor IIIA: properties, DNA binding, and comparison with TFIIIA-finger 3 metal complexes

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

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