The Solution Structure and Dynamics of Cd-Metallothionein from Helix pomatia Reveal Optimization for Binding Cd over Zn.

Metallothioneins (MTs) are cysteine-rich polypeptides that are naturally found coordinated to monovalent and/or divalent transition metal ions. Three metallothionein isoforms from the Roman snail Helix pomatia are known. They differ in their physiological metal load and in their specificity for transition metal ions such as Cd2+ (HpCdMT isoform) and Cu+ (HpCuMT isoform) or in the absence of a defined metal specificity (HpCd/CuMT isoform). We have determined the solution structure of the Cd-specific isoform (HpCdMT) by nuclear magnetic resonance spectroscopy using recombinant isotopically labeled protein loaded with Zn2+ or Cd2+. Both structures display two-domain architectures, where each domain comprises a characteristic three-metal cluster similar to that observed in the ß-domains of vertebrate MTs. The polypeptide backbone is well-structured over the entire sequence, including the interdomain linker. Interestingly, the two domains display mutual contacts, as observed before for the metallothionein of the snail Littorina littorea, to which both N- and C-terminal domains are highly similar. Increasing the length of the linker motionally decouples both domains and removes mutual contacts between them without having a strong effect on the stability of the individual domains. The structures of Cd6- and Zn6-HpCdMT are nearly identical. However, 15N relaxation, in particular 15N R2 rates, is accelerated for many residues of Zn6-HpCdMT but not for Cd6-HpCdMT, revealing the presence of conformational exchange effects. We suggest that this snail MT isoform is evolutionarily optimized for binding Cd rather than Zn.

Analysis of Metal-Binding Features of the Wild Type and Two Domain-Truncated Mutant Variants of Littorina littorea Metallothionein Reveals Its Cd-Specific Character.

After the resolution of the 3D structure of the Cd9-aggregate of the Littorina littorea metallothionein (MT), we report here a detailed analysis of the metal binding capabilities of the wild type MT, LlwtMT, and of two truncated mutants lacking either the N-terminal domain, Lltr2MT, or both the N-terminal domain, plus four extra flanking residues (SSVF), Lltr1MT. The recombinant synthesis and in vitro studies of these three proteins revealed that LlwtMT forms unique M9-LlwtMT complexes with Zn(II) and Cd(II), while yielding a complex mixture of heteronuclear Zn,Cu-LlwtMT species with Cu(I). As expected, the truncated mutants gave rise to unique M6-LltrMT complexes and Zn,Cu-LltrMT mixtures of lower stoichiometry with respect to LlwtMT, with the SSVF fragment having an influence on their metal binding performance. Our results also revealed a major specificity, and therefore a better metal-coordinating performance of the three proteins for Cd(II) than for Zn(II), although the analysis of the Zn(II)/Cd(II) displacement reaction clearly demonstrates a lack of any type of cooperativity in Cd(II) binding. Contrarily, the analysis of their Cu(I) binding abilities revealed that every LlMT domain is prone to build Cu4-aggregates, the whole MT working by modules analogously to, as previously described, certain fungal MTs, like those of C. neoformans and T. mesenterica. It is concluded that the Littorina littorea MT is a Cd-specific protein that (beyond its extended binding capacity through an additional Cd-binding domain) confers to Littorina littorea a particular adaptive advantage in its changeable marine habitat.

Biomphalaria glabrata Metallothionein: Lacking Metal Specificity of the Protein and Missing Gene Upregulation Suggest Metal Sequestration by Exchange Instead of through Selective Binding.

The wild-type metallothionein (MT) of the freshwater snail Biomphalaria glabrata and a natural allelic mutant of it in which a lysine residue was replaced by an asparagine residue, were recombinantly expressed and analyzed for their metal-binding features with respect to Cd2+, Zn2+ and Cu⁺, applying spectroscopic and mass-spectrometric methods. In addition, the upregulation of the Biomphalaria glabrataMT gene was assessed by quantitative real-time detection PCR. The two recombinant proteins revealed to be very similar in most of their metal binding features. They lacked a clear metal-binding preference for any of the three metal ions assayed-which, to this degree, is clearly unprecedented in the world of Gastropoda MTs. There were, however, slight differences in copper-binding abilities between the two allelic variants. Overall, the missing metal specificity of the two recombinant MTs goes hand in hand with lacking upregulation of the respective MT gene. This suggests that in vivo, the Biomphalaria glabrata MT may be more important for metal replacement reactions through a constitutively abundant form, rather than for metal sequestration by high binding specificity. There are indications that the MT of Biomphalaria glabrata may share its unspecific features with MTs from other freshwater snails of the Hygrophila family.

Structural Adaptation of a Protein to Increased Metal Stress: NMR Structure of a Marine Snail Metallothionein with an Additional Domain.

In this study, we present an NMR structure of the metallothionein (MT) from the snail Littorina littorea (LlMT) in complex with Cd2+ . LlMT is capable of binding 9 Zn2+ or 9 Cd2+ ions. Sequence alignments with other snail MTs revealed that the protein is likely composed of three domains. The study revealed that the protein is divided into three individual domains, each of which folds into a single well-defined three-metal cluster. The central α2 and C-terminal ß domains are positioned with a unique relative orientation. Two variants with longer and shorter linkers were investigated, which revealed that specific interdomain contacts only occurred with the wild-type linker. Moreover, a domain-swap mutant in which the highly similar α1 and α2 domains were exchanged was structurally almost identical. It is suggested that the expression of a three-domain MT confers an evolutionary advantage on Littorina littorea in terms of coping with Cd2+ stress and adverse environmental conditions.

Understanding the 7-Cys module amplification of C. neoformans metallothioneins: how high capacity Cu-binding polypeptides are built to neutralize host nutritional immunity.

Cryptococcus neoformans metallothioneins (MTs), CnMT1 and CnMT2, have been identified as essential infectivity and virulence factors of this pathogen. Both MTs are unusually long Cu-thioneins, exhibiting protein architecture and metal-binding abilities compatible with the hypothesis of resulting from three and five tandem repetitions of 7-Cys motives, respectively, each of them folding into Cu5-clusters. Through the study of the Zn(II)- and Cu(I)-binding capabilities of several CnMT1 truncated mutants, we show that a 7-Cys segment of CnMT1 folds into Cu5-species, of additive capacity when joined in tandem. All the obtained Cu-complexes share practically similar architectural features, if judging by their almost equivalent CD fingerprints, and they also share their capacity to restore copper tolerance in MT-devoid yeast cells. Besides the analysis of the modular composition of these long fungal MTs, we evaluate the features of the Cys-rich stretch spacer and flanking sequences that allow the construction of stable metal clusters by adjacent union of binding modules. Overall, our data support a mechanism by which some microbial MTs may have evolved to enlarge their original metal co-ordination capacity under the specific selective pressure of counteracting the Cu-based immunity mechanisms evolved by the infected hosts.

Cantareus aspersus metallothionein metal binding abilities: the unspecific CaCd/CuMT isoform provides hints about the metal preference determinants in metallothioneins.

In Proteomics, gene/protein families including both specialized and non-specialized paralogs are an invaluable tool to study the evolution of structure/function relationships in proteins. Metallothioneins (MTs) of the pulmonate gastropod molluscs (snails) offer one of the best materials to study the metal-binding specificity of proteins, because they consist of a polymorphic system that includes members with extremely distinct metal preferences but with a high protein sequence similarity. Cantareus aspersus was the first snail where three paralogous MTs were isolated: the highly specific cadmium (CaCdMT) and copper (CaCuMT) isoforms, and an unspecific CaCd/CuMT isoform, so called because it was natively isolated as a mixed Cd and Cu complex. In this work, we have thoroughly analyzed the Zn(2+)-, Cd(2+)- and Cu(+)-binding abilities of these three CaMTs by means of the spectroscopic and spectrometric characterization of the respective recombinant, as well as in vitro-substituted, metal-complexes. The comparison with the orthologous HpMTs and the study of the isoform-determinant residues allow correlating the protein sequence variability with the coordination capabilities of these MTs. Surprisingly, the CaCuMT isoform exhibits a stronger Cu-thionein character than the HpCuMT ortholog, and the CaCd/CuMT isoform could be defined as a non-optimized Cu-thionein, which has not attained any defined functional differentiation in the framework of the snail MT gene/protein family.

Rhenium and technetium tricarbonyl, {M(CO)3} (+) (M = Tc, Re), binding to mammalian metallothioneins: new insights into chemical and radiopharmaceutical implications.

This paper deals with the binding of the four mammalian metallothioneins (MTs) to the organometallic metal fragment {fac-M(CO)3}(+) (M = (99)Tc, Re), which is highly promising for the preparation of second-generation radiopharmaceuticals. The study of the transmetallation reaction between zinc and rhenium in Zn7-MT1 by means of UV-vis and CD spectroscopy demonstrated the incorporation of the {fac-Re(CO)3}(+) fragment to the MTs. This reaction should be performed at 70 °C to accelerate the reaction rate, a result that is consistent with the reported reactivity of the rhenium fragment. ESI-TOF MS demonstrated the formation of mixed-metal species as Zn6,{Re(CO)3}-MT, Zn6,{Re(CO)3}2-MT, and Zn5,{Re(CO)3}3-MT, as well as the different reactivity of the four MT isoforms. Hence, Zn-MT3 showed the highest reactivity, in agreement with its high Cu-thionein character, whereas Zn-MT2 exhibited the lowest reactivity, in line with its high Zn-thionein character. The reactivity of the Zn-loaded forms of MT1 and MT4 is intermediate between those of MT3 and MT2. The study of the binding of the {fac-(99)Tc(CO)3}(+) fragment to MTs showed a significant and very interesting different reactivity in relation to rhenium. The transmetallation reaction is much more effective with technetium than with rhenium and significant amounts of mixed Zn x ,{(99)Tc(CO)3} y -MT species were formed with the four MT isoforms whereas only MT3 rendered similar amounts of rhenium derivatives. The results obtained in this study support the possible use of technetium for labelling mammalian metallothioneins and also for possible radiopharmaceutical applications.

Chemically and biologically harmless versus harmful ferritin/copper-metallothionein couples.

The simultaneous measurement of the decrease of available Fe(II) ions and the increase of available Fe(III) ions allowed the analysis of the ferroxidase activity of two distinct apoferritins. Although recombinant human apoferritin (HuFtH) rapidly oxidizes Fe(II) to Fe(III) , this iron is not properly stored in the ferritin cavity, as otherwise occurs in horse-spleen H/L-apoferritin (HsFt; H=heavy subunit, L=light subunit). Iron storage in these apoferritins was also studied in the presence of two copper-loaded mammalian metallothioneins (MT2 and MT3), a scenario that occurs in different brain-cell types. For HuFtH, unstored Fe(III) ions trigger the oxidation of Cu-MT2 with concomitant Cu(I) release. In contrast, there is no reaction with Cu-MT2 in the case of HsFt. Similarly, Cu-MT3 does not react during either HuFtH or HsFt iron reconstitution. Significantly, the combination of ferritin and metallothionein isoforms reported in glia and neuronal cells are precisely those combinations that avoid a harmful release of Fe(II) and Cu(I) ions.

Does Variation of the Inter-Domain Linker Sequence Modulate the Metal Binding Behaviour of Helix pomatia Cd-Metallothionein?

Snail metallothioneins (MTs) constitute an ideal model to study structure/function relationships in these metal-binding polypeptides. Helix pomatia harbours three MT isoforms: the highly specific CdMT and CuMT, and an unspecific Cd/CuMT, which represent paralogous proteins with extremely different metal binding preferences while sharing high sequence similarity. Preceding work allowed assessing that, although, the Cys residues are responsible for metal ion coordination, metal specificity or preference is achieved by diversification of the amino acids interspersed between them. The metal-specific MT polypeptides fold into unique, energetically-optimized complexes of defined metal content, when binding their cognate metal ions, while they produce a mixture of complexes, none of them representing a clear energy minimum, with non-cognate metal ions. Another critical, and so far mostly unexplored, region is the stretch linking the individual MT domains, each of which represents an independent metal cluster. In this work, we have designed and analyzed two HpCdMT constructs with substituted linker segments, and determined their coordination behavior when exposed to both cognate and non-cognate metal ions. Results unequivocally show that neither length nor composition of the inter-domain linker alter the features of the Zn(II)- and Cd(II)-complexes, but surprisingly that they influence their ability to bind Cu(I), the non-cognate metal ion.

His-containing plant metallothioneins: comparative study of divalent metal-ion binding by plant MT3 and MT4 isoforms.

Metallothioneins (MTs) are a superfamily of Cys-rich, low-molecular weight metalloproteins that bind heavy metal ions. These cytosolic metallopeptides, which exist in most living organisms, are thought to be involved in metal homeostasis, metal detoxification, and oxidative stress protection. In this work, we characterise the Zn(II)- and Cd(II)-binding abilities of plant type 3 and type 4 MTs identified in soybean and sunflower, both of them being His-containing peptides. The recombinant metal-MT complexes synthesised in Zn(II) or Cd(II)-enriched Escherichia coli cultures have been analysed by ESI-MS, and CD, ICP-AES, and UV spectroscopies. His-to-Ala type 3 MT mutants have also been constructed and synthesised for the study of the role of His in divalent metal ion coordination. The results show comparable divalent metal-binding capacities for the MTs of type 3, and suggest, for the first time, the participation of their conserved C-term His residues in metal binding. Interesting features for the Zn(II)-binding abilities of type 4 MTs are also reported, as their variable His content may be considered crucial for their biological performance.

Cognate and noncognate metal ion coordination in metal-specific metallothioneins: the Helix pomatia system as a model.

The Helix pomatia metallothionein (MT) system, namely, its two highly specific forms, HpCdMT and HpCuMT, has offered once again an optimum model to study metal-protein specificity. The present work investigates the most unexplored aspect of the coordination behavior of MT polypeptides with respect to either cognate or noncognate metal ions, as opposed to the standard studies of cognate metal ion coordination. To this end, we analyzed the in vivo synthesis of the corresponding complexes with their noncognate metals, and we performed a detailed spectroscopic and spectrometric study of the Zn(2+)/Cd(2+) and Zn(2+)/Cu(+) in vitro replacement reactions on the initial Zn-HpMT species. An HpCuMTAla site-directed mutant, exhibiting differential Cu(+)-binding abilities in vivo, was also included in this study. We demonstrate that when an MT binds its cognate metal, it yields well-folded complexes of limited stoichiometry, representative of minimal-energy conformations. In contrast, the incorporation of noncognate metal ions is better attributed to an unspecific reaction of cysteinic thiolate groups with metal ions, which is dependent on their concentration in the surrounding milieu, where no minimal-energy structure is reached, and otherwise, the MT peptide acts as a multidentate ligand that will bind metal ions until its capacity has been saturated. Additionally, we suggest that previous binding of an MT polypeptide with its noncognate metal ion (e.g., binding of Zn(2+) to the HpCuMT isoform) may preclude the correct folding of the complex with its cognate metal ion.

Understanding the interaction of an antitumoral platinum(II) 7-azaindolate complex with proteins and DNA.

The reactivity of the [Pt(dmba)(aza-N1)(dmso)] complex 1, (a potential antitumoral drug with lower IC50 than cisplatin in several tumoral cell lines) with different proteins and oligonucleotides is investigated by means of mass spectrometry (ESI-TOF MS). The results obtained show a particular binding behaviour of this platinum(II) complex. The interaction of 1 with the assayed proteins apparently takes place by Pt-binding to the most accessible coordinating amino acids, presumably at the surface of the protein -this avoiding protein denaturation or degradation- with the subsequent release of one or two ligands of 1. The specific reactivity of 1 with distinct proteins allows to conclude that the substituted initial ligand (dmso or azaindolate) is indicative of the nature of the protein donor atom finally bound to the platinum(II) centre, i.e. N- or S-donor amino acid. Molecular modeling calculations suggest that the release of the azaindolate ligand is promoted by a proton transfer to the non-coordinating N present in the azaindolate ring, while the release of the dmso ligand is mainly favoured by the binding of a deprotonated Cys. The interaction of complex 1 with DNA takes always place through the release of the azaindolate ligand. Interestingly, the interaction of 1 with DNA only proceeds when the oligonucleotides are annealed forming a double strand. Complex 1 is also capable to displace ethidium bromide from DNA and it also weakly binds to DNA at the minor groove, as shown by Hoechst 33258 displacement experiments. Furthermore, complex 1 is also a good inhibitor of cathepsin B (an enzyme implicated in a number of cancer related events). Therefore, although compound 1 is definitely able to bind proteins that can hamper its arrival to the nuclear target, it should be taken into consideration as a putative anticancer drug due to its strong interaction with oligonucleotides and its effective inhibition of cat B.

Shaping mechanisms of metal specificity in a family of metazoan metallothioneins: evolutionary differentiation of mollusc metallothioneins.

BACKGROUND: The 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. RESULTS: HpCuMT 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. CONCLUSION: The 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.

Metallothionein protein evolution: a miniassay.

Metallothionein (MT) evolution is one of the most obscure yet fascinating aspects of the study of these atypical metal-binding peptides. The different members of the extremely heterogeneous MT protein superfamily probably evolved through a web of duplication, functional differentiation, and/or convergence events leading to the current scenario, which is particularly hard to interpret in terms of molecular evolution. Difficulties in drawing straight evolutionary relationships are reflected in the lack of definite MT classification criteria. Presently, MTs are categorized either according to a pure taxonomic clustering or depending on their metal binding preferences and specificities. Extremely well documented MT revisions were recently published. But beyond classic approaches, this review of MT protein evolution will bring together new aspects that have seldom been discussed before. Hence, the emergence of life on our planet, since metal ion utilization is accepted to be at the root of the emergence of living organisms, and global trends that underlie structural and functional MT diversification, will be presented. Major efforts are currently being devoted to identifying rules for function-constrained MT evolution that may be applied to different groups of organisms.

Zn- and Cu-thioneins: a functional classification for metallothioneins?

This report intends to provide the reader with a deeper insight in the chemical, and extensively biological, characteristics of the metallothionein (MT) system. We have devoted nearly 20 years to the study of MTs and this has allowed us to form what we believe is a more complete picture of this peculiar family of metalloproteins. At the beginning of the 1990s, the landscape of this field was quite different from the overall picture we have now. Many researchers have contributed to the readjustment of this part of scientific knowledge. In our case, we implemented a unified method for obtaining MTs, for characterizing their metal-binding features, and for applying a unified research rationale. All this has helped to enlarge the initial picture that was mainly dominated by mammalian MT1/MT2 and yeast Cup1, by introducing approximately 20 new MTs. It has also allowed some characteristics to be clarified and examined in more detail, such as the cooperativity or the coexistence of multiple species in the metal-substitution reactions, the availability of Ag(I) or Cd(II) for use as respective probes for the Cu(I) and Zn(II) binding sites, the participation of chloride or sulfide ligands in the metal coordination spheres, and the feasibility of using in vitro data as representative of in vivo scenarios. Overall, the results yield enough data to consider new criteria for a proposal of classification of MTs based on MT metal-binding features, which complements the previous classifications, and that can shed light on the still controversial physiological functions of this peculiar superfamily of metalloproteins.

Physiological relevance and contribution to metal balance of specific and non-specific Metallothionein isoforms in the garden snail, Cantareus aspersus.

Variable environmental availability of metal ions represents a constant challenge for most organisms, so that during evolution, they have optimised physiological and molecular mechanisms to cope with this particular requirement. Metallothioneins (MTs) are proteins that play a major role in metal homeostasis and as a reservoir. The MT gene/protein systems of terrestrial helicid snails are an invaluable model for the study of metal-binding features and MT isoform-specific functionality of these proteins. In the present study, we characterised three paralogous MT isogenes and their expressed products in the escargot (Cantareus aspersus). The metal-dependent transcriptional activation of the three isogenes was assessed using quantitative Real Time PCR. The metal-binding capacities of the three isoforms were studied by characterising the purified native complexes. All the data were analysed in relation to the trace element status of the animals after metal feeding. Two of the three C. aspersus MT (CaMT) isoforms appeared to be metal-specific, (CaCdMT and CaCuMT, for cadmium and copper respectively). A third isoform (CaCd/CuMT) was non-specific, since it was natively recovered as a mixed Cd/Cu complex. A specific role in Cd detoxification for CaCdMT was revealed, with a 80-90% contribution to the Cd balance in snails exposed to this metal. Conclusive data were also obtained for the CaCuMT isoform, which is involved in Cu homeostasis, sharing about 30-50% of the Cu balance of C. aspersus. No apparent metal-related physiological function was found for the third isoform (CaCd/CuMT), so its contribution to the metal balance of the escargot may be, if at all, of only marginal significance, but may enclose a major interest in evolutionary studies.

Lack of DNA helicase Pif1 disrupts zinc and iron homoeostasis in yeast.

The Saccharomyces cerevisiae gene PIF1 encodes a conserved eukaryotic DNA helicase required for both mitochondrial and nuclear DNA integrity. Our previous work revealed that a pif1Δ strain is tolerant to zinc overload. In the present study we demonstrate that this effect is independent of the Pif1 helicase activity and is only observed when the protein is absent from the mitochondria. pif1Δ cells accumulate abnormal amounts of mitochondrial zinc and iron. Transcriptional profiling reveals that pif1Δ cells under standard growth conditions overexpress aconitase-related genes. When exposed to zinc, pif1Δ cells show lower induction of genes encoding iron (siderophores) transporters and higher expression of genes related to oxidative stress responses than wild-type cells. Coincidently, pif1Δ mutants are less prone to zinc-induced oxidative stress and display a higher reduced/oxidized glutathione ratio. Strikingly, although pif1Δ cells contain normal amounts of the Aco1 (yeast aconitase) protein, they completely lack aconitase activity. Loss of Aco1 activity is also observed when the cell expresses a non-mitochondrially targeted form of Pif1. We postulate that lack of Pif1 forces aconitase to play its DNA protective role as a nucleoid protein and that this triggers a domino effect on iron homoeostasis resulting in increased zinc tolerance.

Evidence of native metal-S(2-)-metallothionein complexes confirmed by the analysis of Cup1 divalent-metal-ion binding properties.

It has previously been shown that recombinant synthesis, under metal-supplemented conditions, of diverse metallothioneins (MTs) results in the recovery of a subpopulation of S(2-)-containing complexes in addition to the S(2-)-devoid canonical metal-MT species. Further significance of this finding has remained veiled by the possibility of it being a mere consequence of synthesis in a heterologous bacterial system. Herein, we present definitive evidence that S(2-) ligands are also constituents of native metal-MT complexes. Because, although practically universal, the highest S(2-) content is incorporated by copper-thioneins when coordinating divalent metal ions, we adapted the Saccharomyces cerevisiae Cup1 protein, which is the most paradigmatic copper-thionein, as an experimental model. Most significantly, native Cd-Cup1 complexes were purified and fully spectroscopically and spectrometrically characterized from the 301N mutant yeast strain, which allows Cup1 synthesis even in the absence of copper. These results undoubtedly revealed the presence of a Cd-S(2-)-Cup1 species in native preparations, which were only recovered when carefully avoiding the use of ion-exchange chromatography in the purification protocol. Furthermore, complete analysis of recombinant (Escherichia coli) Zn-Cup1, Cd-Cup1, and Cu-Cup1 and those complexes that result from Zn/Cd and Zn/Cu replacements in vitro and acidification/renaturalization processes yielded a comprehensive and comparative overview of the metal-binding abilities of Cup1. Overall, we consider the main conclusions of this study to go beyond the mere study of the particular Cup1 MT, so that they should be considered to delineate a new point of view on the interaction between copper-thioneins and divalent metal ions, still an unexplored aspect in MT research.

The zn- or cu-thionein character of a metallothionein determines its metal load when synthesized in physiological (metal-unsupplemented) conditions.

The present work comprises the recombinant synthesis of four metallothioneins (MTs) in metal-unsupplemented cultures and the characterization of the recovered metal complexes by means of analytical and spectrometric techniques. The four MTs are two Drosophila (MtnA and MtnB), one yeast (Crs5), and one mouse (mMT1) metallothionein isoforms. These four MTs exhibit distinct metal binding preferences, from a clear Cu-thionein character to a definite Zn-thionein nature, respectively. Although in all cases, the only metal ion present in the purified complexes is Zn(2+), our results highlight an inherently different behaviour of those two types of MTs, in conditions that would mimic their synthesis in physiological environments. Therefore, intrinsically different roles can be hypothesized for the constitutively-produced MT peptides in the absence of any metal overload, depending on their Zn- or Cu-thionein character.

Metallomics reveals a persisting impact of cadmium on the evolution of metal-selective snail metallothioneins.

The tiny contribution of cadmium (Cd) to the composition of the earth's crust contrasts with its high biological significance, owing mainly to the competition of Cd with the essential zinc (Zn) for suitable metal binding sites in proteins. In this context it was speculated that in several animal lineages, the protein family of metallothioneins (MTs) has evolved to specifically detoxify Cd. Although the multi-functionality and heterometallic composition of MTs in most animal species does not support such an assumption, there are some exceptions to this role, particularly in animal lineages at the roots of animal evolution. In order to substantiate this hypothesis and to further understand MT evolution, we have studied MTs of different snails that exhibit clear Cd-binding preferences in a lineage-specific manner. By applying a metallomics approach including 74 MT sequences from 47 gastropod species, and by combining phylogenomic methods with molecular, biochemical, and spectroscopic techniques, we show that Cd selectivity of snail MTs has resulted from convergent evolution of metal-binding domains that significantly differ in their primary structure. We also demonstrate how their Cd selectivity and specificity has been optimized by the persistent impact of Cd through 430 million years of MT evolution, modifying them upon lineage-specific adaptation of snails to different habitats. Overall, our results support the role of Cd for MT evolution in snails, and provide an interesting example of a vestigial abiotic factor directly driving gene evolution. Finally, we discuss the potential implications of our findings for studies devoted to the understanding of mechanisms leading to metal specificity in proteins, which is important when designing metal-selective peptides.