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Non-coordinative metal selectivity bias in human metallothioneins metal-thiolate clusters.

ABSTRACT: Mammalian metallothioneins (MT-1 through MT-4) are a class of metal binding proteins containing two metal-thiolate clusters formed through the preferential coordination of d10 metals, Cu(i) and Zn(ii), by 20 conserved cysteine residues located in two protein domains. MT metalation (homometallic or heterometallic Zn(ii)/Cu(i) species) appears to be isoform specific and controlling zinc and copper concentrations to perform specific and distinct biological functions. Structural and functional relationships, and in vivo metalation studies, identified evolutionary features defining the metal-selectivity nature for MTs. Metallothionein-3 (MT-3) has been shown to possess the most pronounced Cu-thionein character forming Cu(i)-containing species more favorably than metallothionein-2 (MT-2), which possesses the strongest Zn-thionein character. In this work, we identify isoform-specific determinants which control metal binding selectivity bias in different MTs isoforms. By studying the reactivity of Zn7MT-2, Zn7MT-3 and Zn7MT-3 mutants towards Cu(ii) to form Cu(i)4Zn4MTs, we have identified isoform-specific key non-coordinating residues governing folding/outer sphere control of metal selectivity bias in MTs metal clusters. By mutating selected residues and motifs in MT-3 to the corresponding MT-2 amino acids, we dissected key roles in modulating cluster dynamic and metal exchange rates, in increasing the Cu(i)-affinity in MT-3 N-terminal ?-domain and/or modulating the higher stability of the Zn(ii)-thiolate cluster in MT-2 ?-domain. We thus engineered MT-3 variants in which the copper-thionein character is converted into a zinc-thionein. These results provide new insights into the molecular determinants governing metal selectivity in metal-thiolate clusters.

SUBMITTER: Calvo JS

PROVIDER: S-EPMC6450656 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Non-coordinative metal selectivity bias in human metallothioneins metal-thiolate clusters.

Calvo Jenifer S JS Lopez Victor M VM Meloni Gabriele G

Metallomics : integrated biometal science 20181201 12

Mammalian metallothioneins (MT-1 through MT-4) are a class of metal binding proteins containing two metal-thiolate clusters formed through the preferential coordination of d10 metals, Cu(i) and Zn(ii), by 20 conserved cysteine residues located in two protein domains. MT metalation (homometallic or heterometallic Zn(ii)/Cu(i) species) appears to be isoform specific and controlling zinc and copper concentrations to perform specific and distinct biological functions. Structural and functional relat ...[more]

PMID: 30420986

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Project description:BackgroundThe degree of metal binding specificity in metalloproteins such as metallothioneins (MTs) can be crucial for their functional accuracy. Unlike most other animal species, pulmonate molluscs possess homometallic MT isoforms loaded with Cu(+) or Cd(2+). They have, so far, been obtained as native metal-MT complexes from snail tissues, where they are involved in the metabolism of the metal ion species bound to the respective isoform. However, it has not as yet been discerned if their specific metal occupation is the result of a rigid control of metal availability, or isoform expression programming in the hosting tissues or of structural differences of the respective peptides determining the coordinative options for the different metal ions. In this study, the Roman snail (Helix pomatia) Cu-loaded and Cd-loaded isoforms (HpCuMT and HpCdMT) were used as model molecules in order to elucidate the biochemical and evolutionary mechanisms permitting pulmonate MTs to achieve specificity for their cognate metal ion.ResultsHpCuMT and HpCdMT were recombinantly synthesized in the presence of Cd(2+), Zn(2+) or Cu(2+) and corresponding metal complexes analysed by electrospray mass spectrometry and circular dichroism (CD) and ultra violet-visible (UV-Vis) spectrophotometry. Both MT isoforms were only able to form unique, homometallic and stable complexes (Cd(6)-HpCdMT and Cu(12)-HpCuMT) with their cognate metal ions. Yeast complementation assays demonstrated that the two isoforms assumed metal-specific functions, in agreement with their binding preferences, in heterologous eukaryotic environments. In the snail organism, the functional metal specificity of HpCdMT and HpCuMT was contributed by metal-specific transcription programming and cell-specific expression. Sequence elucidation and phylogenetic analysis of MT isoforms from a number of snail species revealed that they possess an unspecific and two metal-specific MT isoforms, whose metal specificity was achieved exclusively by evolutionary modulation of non-cysteine amino acid positions.ConclusionThe Roman snail HpCdMT and HpCuMT isoforms can thus be regarded as prototypes of isoform families that evolved genuine metal-specificity within pulmonate molluscs. Diversification into these isoforms may have been initiated by gene duplication, followed by speciation and selection towards opposite needs for protecting copper-dominated metabolic pathways from nonessential cadmium. The mechanisms enabling these proteins to be metal-specific could also be relevant for other metalloproteins.

Dataset Information

Non-coordinative metal selectivity bias in human metallothioneins metal-thiolate clusters.

Publications

Non-coordinative metal selectivity bias in human metallothioneins metal-thiolate clusters.

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