Metal co-ordination in rat liver metallothionein-2 prepared with or without reconstitution of the metal clusters, and comparison with rabbit liver metallothionein-2

doi:10.1016/0022-2836(87)90042-8

Journal of Molecular Biology

Volume 196, Issue 3, 5 August 1987, Pages 711-719

https://doi.org/10.1016/0022-2836(87)90042-8 Get rights and content

Abstract

Possible origins of the different metal co-ordination topologies in the recently determined structures of rat metallothionein-2 (MT2) in single crystals and rabbit MT2 in solution were investigated. A complete structure determination for rat MT2 in solution by nuclear magnetic resonance (n.m.r.) showed that the differences in the spatial structures cannot be attributed to the different primary structures of the two species. Comparison of [¹¹³Cd₇]MT2 obtained by reconstitution of the apoprotein in vitro with preparations using a different procedure showed, moreover, that the metal co-ordination observed in solution by n.m.r. is not an artefact of the protein reconstitution. Solutions of high-pressure liquid chromatographically homogeneous biosynthetic preparations of [¹¹³Cd, Zn]MT2 were obtained from rat liver following injection of ¹¹³Cd into rats in vivo, without further metal exchange after protein isolation. They contain a mixture of several forms of MT2 with different relative metal compositions, giving rise to an increased number of ¹¹³Cd resonances. For the components of the four-metal cluster, the major one of these different forms exhibits patterns in the two-dimensional [¹H, ¹¹³Cd]-correlated spectra that are indistinguishable from those of [¹¹³Cd₇]MT2, thereby implying identity of cluster co-ordination and topology. These results are discussed with regard to continued investigations into the differences between the solution structure and crystal structure of MT2.

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To make matters more complicated still, each of the in vitro overall approaches that either partly or completely failed with Sp1 has succeeded for other proteins. For instance, examples of successful cadmium substitution proceeding via addition of Cd(II) to apo-protein include metallothionein [197] and more recently Bud31 [49], while examples of substitution via treatment of zinc-bound protein with Cd-EDTA include CNOT4 and p44 [72]. Table 3 collects some examples of protocols that have been used to replace native structural zincs in proteins with 113Cd in published NMR studies.
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Mammalian Zn, Cd-MT2 complexes were the first to be solved using the 1980s commonly accepted methodology of X-ray diffraction [181]. However, simultaneously metal-MT2 complexes were among the first protein structures being solved by NMR [182–185]. In fact, after a discrepancy between the structures resulting from the two methodological approaches, the initially proposed crystal three-dimensional structure of rat MT2 had to be refined, so that a second crystal model, essentially coincident with the NMR-solved structure, was thereafter published [186].
Metallothioneins (MTs) are a particular type of small metalloprotein that possess a significant amount of Cys residues, which confer upon them an extraordinary capacity for heavy metal binding. Now, more than 50 years after their discovery and after the publication of thousands of papers, it is generally agreed that they are involved in different biological functions depending on the organisms and/or isoforms considered.
This review attempts to critically review the state-of-the-art of these unusual metal-binding proteins. Special attention is devoted to their chemical and structural characterization and reactivity, including a detailed overview of the most prominent techniques used for purification and quantification of MTs, as well as a section with a comparative description of all the three-dimensional structures so far known. Equally important are the biological aspects of MTs, which have also been extensively analyzed. Thus, their in vivo origin, localization and induction pathways, as well as their proposed biological significance is discussed. Finally, the most recent applications of MTs in the Biomedicine and Biotechnology fields and the future goals that, in our opinion, investigators should aim for in MT research are mentioned in the final sections.
A comparative study on interactions of cisplatin and ruthenium arene anticancer complexes with metallothionein using MALDI-TOF-MS
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The matrix used in these experiments was 20 mg/mL 2,5-dihydroxybenzoic acid (DHB), in a solution containing 1:1 acetonitrile and 1% H3PO4. The protein concentration of metallothionein stock solution was determined by measuring the absorbance of the dilution in 0.1% trifluroacetic acid (TFA) (pH 1.6) at 220 nm (the extinct coefficient of rabbit apo-MT at 220 nm is 48200 mol−1 cm−1 [39]). A aliquot of aqueous solution of ruthenium arene complex 1 (4 mM), 2 (4 mM) or cisplatin (1 mM) was mixed with MT-I or II in 10 mM Tris–HCl solution, of which the pH were adjusted using 0.1% TFA to 7.4 or 3, making the final molar ratio of metal compounds to MT (10 μM) be 1:2, 1:5 or 1:10, and the resulting mixture was incubated at 310 K for 48, 72 or 96 h prior to MALDI-TOF-MS analysis.
Metallothioneins (MTs) are a group of low molecular weight (6–7 KDa) proteins featured by high cysteine content which allows the proteins to bind to a diverse range of metals. MTs, of which the gene transcription can be induced by a variety of stimuli including hormones, cytokines and metal ions, have important biological functions such as storage, trafficking and homeostasis of metal ions, detoxification of heavy metals, resistance to ionizing radiation, scavenging hydroxyl radical, etc. Importantly, it has been reported that MTs play a role in oncogenesis and cancer prognosis, and are implicated in resistance of cancer cells to anticancer metallodrug cisplatin. In this work, we present a comparative study on interactions between MTs and cisplatin and ruthenium arene anticancer complexes using MALDI-TOF-MS. The results show that cisplatin coordinates to MT-I and MT-II at either pH 3.0 or 7.4, and exhibits a higher affinity to MT-II than to MT-I. The MT–Pt complexes increase significantly in content with decrease in pH values of solutions, indicative that cisplatin competes with zinc for coordination to cysteine residues on MTs and interferes with the binding of zinc to the proteins. While the ruthenium arene anticancer complexes [(η⁶-arene)Ru(en)Cl]PF₆ (arene = benzene or biphenyl, en = ethylenediamine) hardly bind to MTs at acidic pH and coordinate to MTs at a much lower level than cisplatin at neutral pH, which may account for the less toxicity and lack of cross-resistance to cisplatin for this class of ruthenium anticancer complexes. With the MT–Pt complexes as model protein complexes, DDT, the widely used unfolding thiol-containing agent in proteomic research was shown to reduce the coordination of platinum to MTs significantly, implying that DDT used as unfolding reagent at high concentration may cause dissociation of bound metallodrug and should be applied with care.
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Metallothioneins (MTs) constitute a family of proteins characterized by a high heavy metal [Zn(II), Cu(I)] content and also by an unusual cysteine abundance. Mammalian MTs are comprised of four major isoforms designated MT-1 trough MT-4. MT-1 and MT-2 are expressed in most tissues including the brain, whereas MT-3 (also called growth inhibitory factor) and MT-4 are expressed predominantly in the central nervous system and in keratinizing epithelia, respectively. All MT isoforms have been implicated in disparate physiological functions, such as zinc and copper metabolism, protection against reactive oxygen species, or adaptation to stress. In the case of MT-3, an additional involvement of this isoform in neuromodulatory events and in the pathogenesis of Alzheimer’s disease has also been suggested. It is essential to gain insight into how MTs are regulated in the brain in order to characterize MT functions, both in normal brain physiology, as well as in pathophysiological states. The focus of this review concerns the biology of the MT family in the context of their expression and functional roles in the central nervous system.
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The cadmium-binding properties of the C-terminal hexapeptide of mouse metallothionein I, Lys-Cys-Thr-Cys-Cys-Ala, were studied by circular dichroism spectroscopy (CD), differential pulse polarography (DPP) and $^{113} Cd-nuclear$ magnetic resonance (NMR).
The structure of the multiple cadmium binding sites could not be determined by $^{113} Cd-NMR$ because of the insolubility of the Cd–peptide samples at the high concentrations required for NMR. Therefore, alternative approaches were used: CD and DPP. The data were analyzed using a multivariate curve resolution (MCR) approach, based on factor analysis techniques, which allows the identification of the signal corresponding to different metal ions bound in different chemical environments. The CD study confirmed that the binding of Cd²⁺ induces important conformational changes in the structure of the peptidic complex, including the formation of a binuclear cluster. The DPP results obtained at various Cd²⁺-to-peptide concentration ratios and pH values, under conditions where electrode adsorption is low, if not negligible, indicated the formation of different Cd²⁺–peptide complexes, and a scheme for the electrochemical reduction of the complexed Cd²⁺ ions is proposed.
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Journal of Molecular Biology

Metal co-ordination in rat liver metallothionein-2 prepared with or without reconstitution of the metal clusters, and comparison with rabbit liver metallothionein-2

Abstract

J. Mol. Biol

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

J. Amer. Chem. Soc

Science