Mammalian Metallothioneins Research Articles

ESI-MS analysis of Cu(I) binding to apo and Zn7 human metallothionein 1A, 2, and 3 identifies the formation of a similar series of metallated species with no individual isoform optimization for Cu(I).

Metallothioneins (MTs) are cysteine-rich proteins involved in metal homeostasis, heavy metal detoxification, and protection against oxidative stress. Whether the four mammalian MT isoforms exhibit different metal binding properties is not clear. In this paper, the Cu(I) binding properties of the apo MT1A, apo MT2, and apo MT3 are compared and the relative Cu(I) binding affinities are reported. In all three isoforms, Cu4, Cu6, and Cu10 species form cooperatively, and MT1A and MT2 also form a Cu13 species. The Cu(I) binding properties of Zn7-MT1A, Zn7-MT2, and Zn7-MT3 are compared systematically using isotopically pure 63Cu(I) and 68Zn(II). The species formed in each MT isoform were detected through electrospray ionization-mass spectrometry and further characterized using room temperature phosphorescence spectroscopy. The mixed metal Cu, Zn species forming in MT1A, MT2, and MT3 have similar stoichiometries and their emission spectral properties indicate that analogous clusters form in the three isoforms. Three parallel metallation pathways have been proposed through analysis of the detailed Cu, Zn speciation in MT1A, MT2, and MT3. Pathway ① results in Cu5Zn5-MT and Cu9Zn3-MT. Pathway ② involves Cu6Zn4-MT and Cu10Zn2-MT. Pathway ③ includes Cu8Zn4-MT. Speciation analysis indicates that Pathway ② is the preferred pathway for MT2. This is also evident in the phosphorescence spectra with the 750nm emission from Cu6Zn4-MT being most prominent in MT2. We see no evidence for different MT isoforms being optimized or exhibiting preferences for certain metals. We discuss the probable stoichiometry for MTs in vivo based on the in vitro determined binding constants.

Supermetalation of Cd-MT3 beyond the two-domain model

The flexibility of mammalian metallothioneins (MTs) has contributed to the difficulty in obtaining structural information for this family of metalloproteins that bind divalent metals with its twenty cysteines. While the two-domain structure for Cd7MT is well-established as a Cd4S11 and Cd3S9, a third structure has been reported when 8 Cd(II) ions bind to MT1. Isoform 3 of the MT family, MT3, has been of interest to the research community since its isolation as a growth inhibitory factor isolated in brain tissue, and has since been noted as a prominent participant in the mediation of neurodegenerative diseases and regular brain development. The differences between MT3 and the other isoforms of MT include an additional hexapeptide insertion of acidic residues in the α domain as well as the introduction of two prolines in the β domain. It is unclear whether these changes impact the metalation properties of MT3. We report the formation of a Cd8MT3 species is characterized by electrospray ionization mass spectrometry and UV–visible absorption spectroscopy. We report that the spectroscopic properties of this supermetalated Cd8MT3 are similar to those of the supermetalated Cd8MT1, with a clear indication of changes in structure from “fully-metalated” Cd7MT3 to supermetalated Cd8MT3 from circular dichroism spectra and both 1D 113Cd and 2D 1H-113Cd HSQC NMR spectra. We conclude that the metalation properties are not impacted significantly due to the amino acid changes in MT3, and that the cysteinyl thiols are the key players in determining the capacity of metal-binding and the structure of metal-thiolate clusters.

The connection of α- and β-domains in mammalian metallothionein-2 differentiates Zn(II) binding affinities, affects folding, and determines zinc buffering properties.

Mammalian metallothioneins (MTs) are small Cys-rich proteins involved in Zn(II) and Cu(I) homeostasis. They bind seven Zn(II) ions in two distinct β- and α-domains, forming Zn3Cys9 and Zn4Cys11 clusters, respectively. After six decades of research, their role in cellular buffering of Zn(II) ions has begun to be understood recently. This is because of different affinities of bound ions and the proteins' coexistence in variously Zn(II)-loaded Zn4-7MT species in the cell. To date, it has remained unclear how these mechanisms of action occur and how the affinities are differentiated despite the Zn(S-Cys)4 coordination environment being the same. Here, we dissect the molecular basis of these phenomena by using several MT2 mutants, hybrid protein, and isolated domains. Through a combination of spectroscopic and stability studies, thiol(ate) reactivity, and steered molecular dynamics, we demonstrate that both protein folding and thermodynamics of Zn(II) ion (un)binding significantly differ between isolated domains and the whole protein. Close proximity reduces the degrees of freedom of separated domains, making them less dynamic. It is caused by the formation of intra- and interdomain electrostatic interactions. The energetic consequence of domains connection has a critical impact on the role of MTs in the cellular environment, where they function not only as a zinc sponge but also as a zinc buffering system keeping free Zn(II) in the right concentrations. Any change of that subtle system affects the folding mechanism, zinc site stabilities, and cellular zinc buffer components.

From methodological limitations to the function of metallothioneins - A guide to approaches for determining weak, moderate, and tight affinity zinc sites.

Mammalian metallothioneins (MTs) are small cysteine-rich proteins whose primary role is participation in zinc and copper homeostasis. Ever since their discovery, MTs have been investigated in terms of metal-binding affinity. The initial concept of seven Zn(II) ions (Zn7MT) bound with the same, undifferentiated low picomolar affinity in the α and β domains prevailed for many years and derived from spectroscopic studies. The application of fluorescent zinc probes has changed the perception of MTs, showing that they function in nano- to subnanomolar free zinc concentrations due to the presence of tight, moderate, and weak binding sites. The discovery of Zn(II)-depleted MTs in many tissues and determination of cellular free Zn(II) concentrations with differentiated zinc affinity sites revealed the critical importance of partially saturated Zn4-6MTs species in cellular zinc buffering in a wide pico- to nanomolar range of free Zn(II) concentrations. Until today, there is no clear agreement on the presence of differentiated or only tight zinc sites. Here, we present a series of spectroscopic, mass spectrometry-based, and enzymatic competition experiments which reveal how weak, moderate, or high affinity ligands interact with human MT2, with special attention to the determination of Zn(II) affinities. The results show that the simplification of the stability model is the major reason for determining significantly different stability data that obscured the actual MTs function. Therefore, we emphasize that different metal affinities are the single most reason for their presumed function, which changed over the years from tight binding and, thus, storage to one that is highly dynamic.

The Bioinorganic Chemistry of Mammalian Metallothioneins.

The functions, purposes, and roles of metallothioneins have been the subject of speculations since the discovery of the protein over 60 years ago. This article guides through the history of investigations and resolves multiple contentions by providing new interpretations of the structure-stability-function relationship. It challenges the dogma that the biologically relevant structure of the mammalian proteins is only the one determined by X-ray diffraction and NMR spectroscopy. The terms metallothionein and thionein are ambiguous and insufficient to understand biological function. The proteins need to be seen in their biological context, which limits and defines the chemistry possible. They exist in multiple forms with different degrees of metalation and types of metal ions. The homoleptic thiolate coordination of mammalian metallothioneins is important for their molecular mechanism. It endows the proteins with redox activity and a specific pH dependence of their metal affinities. The proteins, therefore, also exist in different redox states of the sulfur donor ligands. Their coordination dynamics allows a vast conformational landscape for interactions with other proteins and ligands. Many fundamental signal transduction pathways regulate the expression of the dozen of human metallothionein genes. Recent advances in understanding the control of cellular zinc and copper homeostasis are the foundation for suggesting that mammalian metallothioneins provide a highly dynamic, regulated, and uniquely biological metal buffer to control the availability, fluctuations, and signaling transients of the most competitive Zn(II) and Cu(I) ions in cellular space and time.

An Integrated Mass Spectrometry and Molecular Dynamics Simulations Approach Reveals the Spatial Organization Impact of Metal-Binding Sites on the Stability of Metal-Depleted Metallothionein-2 Species.

Mammalian metallothioneins (MTs) are a group of cysteine-rich proteins that bind metal ions in two α- and β-domains and represent a major cellular Zn(II)/Cu(I) buffering system in the cell. At cellular free Zn(II) concentrations (10–11–10–9 M), MTs do not exist in fully loaded forms with seven Zn(II)-bound ions (Zn7MTs). Instead, MTs exist as partially metal-depleted species (Zn4–6MT) because their Zn(II) binding affinities are on the nano- to picomolar range comparable to the concentrations of cellular Zn(II). The mode of action of MTs remains poorly understood, and thus, the aim of this study is to characterize the mechanism of Zn(II) (un)binding to MTs, the thermodynamic properties of the Zn1–6MT2 species, and their mechanostability properties. To this end, native mass spectrometry (MS) and label-free quantitative bottom-up and top-down MS in combination with steered molecular dynamics simulations, well-tempered metadynamics (WT-MetaD), and parallel-bias WT-MetaD (amounting to 3.5 μs) were integrated to unravel the chemical coordination of Zn(II) in all Zn1–6MT2 species and to explain the differences in binding affinities of Zn(II) ions to MTs. Differences are found to be the result of the degree of water participation in MT (un)folding and the hyper-reactive character of Cys21 and Cys29 residues. The thermodynamics properties of Zn(II) (un)binding to MT2 are found to differ from those of Cd(II), justifying their distinctive roles. The potential of this integrated strategy in the investigation of numerous unexplored metalloproteins is attested by the results highlighted in the present study.

Metallothionein dimerization evidenced by QD-based Förster resonance energy transfer and capillary electrophoresis

Herein, we report a new simple and easy-to-use approach for the characterization of protein oligomerization based on fluorescence resonance energy transfer (FRET) and capillary electrophoresis with LED-induced detection. The FRET pair consisted of quantum dots (QDs) used as an emission tunable donor (emission wavelength of 450 nm) and a cyanine dye (Cy3), providing optimal optical properties as an acceptor. Nonoxidative dimerization of mammalian metallothionein (MT) was investigated using the donor and acceptor covalently conjugated to MT. The main functions of MTs within an organism include the transport and storage of essential metal ions and detoxification of toxic ions. Upon storage under aerobic conditions, MTs form dimers (as well as higher oligomers), which may play an essential role as mediators in oxidoreduction signaling pathways. Due to metal bridging by Cd2+ ions between molecules of metallothionein, the QDs and Cy3 were close enough, enabling a FRET signal. The FRET efficiency was calculated to be in the range of 11–77%. The formation of MT dimers in the presence of Cd2+ ions was confirmed by MALDI-MS analyses. Finally, the process of oligomerization resulting in FRET was monitored by CE, and oligomerization of MT was confirmed.

Metallothionein Cd4S11 cluster formation dominates in the protection of carbonic anhydrase.

Metallothioneins (MTs) are ubiquitous proteins vital for essential metal homeostasis and heavy metal detoxification. The twenty-cysteinyl mammalian metallothioneins protect the proteome by sequestering heavy metals into thermodynamically stable metal thiolate structures when metalated with seven Cd2+. At physiological pH, the first metal (M) thiolate (SCys) structures formed involve M(SCys)4 terminal thiolates. With higher metal loading, M4(SCys)11 and M3(SCys)9 clusters form. As a regulator of the metallome, it is necessary to understand metal sequestration properties of MT in solution with other metalloproteins. We report that the association between apo-MT and apo-carbonic anhydrase (CA) enhances the formation of the protective mode of MT, in which Cd4(SCys)11-clusters form at much lower concentration levels than for the free apo-MT at physiological pH. Using stopped-flow kinetics and electrospray ionization mass spectrometry, we quantified this protective effect, determining that it is significant at pH 7.4, but the effect diminishes at pH 5.0. We report for the first time, the absolute stepwise binding constants of Cd2+ binding to human MT1a both in the presence and absence of CA through calibration by the known binding constant of Cd2+ to bovine CA. We report that this protein association affects the Cd2+ metalation rates of MT. The data support the physiological role of MTs as protectors of the metalloproteome from the toxic effects of Cd2+.

Kinetics of competitive Cd2+ binding pathways: the realistic structure of intrinsically disordered, partially metallated metallothioneins.

The 20-cysteine mammalian metallothioneins are considered to be central to the homeostatic control of the essential metals Zn(ii) and Cu(i) and, as part of their metal-loaded status, play a role in reversing oxidative stress. Native apo-MT does not adopt a well-known structural motif, being described as a random-coil or intrinsically-disordered. Conclusions reached from a combination of ESI-mass spectral charge states, As(iii) metallation of apo-MT at low pH, from molecular dynamic calculations and from metallation of the α-domain fragment, suggest that in fact the native apo-MT adopts a structure that is highly efficient towards metallation at physiological pH. The results in this paper show that the initial (M < 5) Cd(ii) metallation at physiological pH takes place to form structures based on isolated Cd(SCYS)4 units, beads. At pH 5, cysteine bridged Cd4(SCYS)11 clusters form. ESI-mass spectral profile of cysteine modification of apo-MT at physiological pH shows that it is folded, whereas in the presence of 3 M guandinium hydrochloride the apo-MT is unfolded. Stopped flow kinetic studies of the Cd(ii) metallation shows that the reaction is much slower for the unfolded vs. the folded apo-MT for formation of either beads or clusters. Metallation is also much slower for the formation of clusters than the formation of beads. These results are first to quantify the presence of structure in native apo-MT in terms of the critical metallation properties. The implications of this study suggest that oxidation of apo-MT due to ageing or other agent will negatively impact the metallation process for essential metals.

Pb(ii) binding to the brain specific mammalian metallothionein isoform MT3 and its isolated αMT3 and βMT3 domains.

The toxicity of lead, one of the most ubiquitous toxic metals, is well known. Some of its pathological effects are related to its preference for the sulfhydryl groups of proteins. Metallothioneins (MT) are a particular family of metalloproteins characterized by their high Cys content that, among other functions, are linked to the detoxification of heavy metals. In mammals, 4 MT isoforms have been found. The MT3 isoform, also called "neuronal growth inhibitory factor", is mainly synthesized in the brain and contains several structural differences that may contribute to important functional differences between it and other MT isoforms. The abilities of recombinant MT3 and its individual αMT3 and βMT3 fragments to bind Pb(ii) have been investigated here, under different pH conditions, by means of spectroscopy, mass spectrometry and isothermal titration calorimetry. The results obtained show that the binding of Pb(ii) to the intact MT3 protein is relatively unaffected by pH, while the individual domains interact with Pb(ii) in a pH-sensitive manner. The mass spectrometry data reveal the evolution with time of the initially formed Pb-MT complexes. In the case of the full length protein, Pb(ii) remains bound for a long period of time. With the isolated fragments, the lead is eventually released. The Pb-species formed depend on the amount of Pb(ii) present in solution. The thermodynamic data recorded, as measured by ITC, for the replacement of Zn(ii) by Pb(ii) in reactions with Zn-MT3, Zn-αMT3 and Zn-βMT3 are all similar, and in all cases, the displacement of Zn(ii) by Pb(ii) is thermodynamically favorable. Zn-Replete and Pb-replete MT3 have distinctive circular dichroism spectra, suggestive of structural differences with different metallation status.

Unravelling the mechanistic details of metal binding to mammalian metallothioneins from stoichiometric, kinetic, and binding affinity data.

Metallothioneins (MTs) are small, cysteine-rich proteins, found throughout Nature. Their ability to bind a number of different metals with a range of stoichiometric ratios means that this protein family is critically important for essential metal (Zn2+ and Cu+) homeostasis, metal storage, metal donation to nascent metalloenzymes as well as heavy metal detoxification. With its 20 cysteines, metallothionein is also considered to protect cells against oxidative stress. MT has been studied by a large number of researchers over the last 6 decades using a variety of spectroscopic techniques. The lack of distinguishing chromophores for the multitude of binding sites has made the evaluation of stoichiometric properties for different metals challenging. Initially, only 113Cd-NMR spectroscopy could provide strong evidence for the proposed cluster formation of Cd-MT. The extraordinary development of electrospray ionization mass spectrometry (ESI-MS), where all coexisting species in solution are observed, revolutionized MT research. Prior to the use of ESI-MS data, a range of "magic numbers" representing metal-to-MT molar ratios were reported from optical spectroscopic studies. The availability of ESI mass spectral data led to (i) the confirmation of cluster formation, (ii) a conceptual understanding of the cooperativity involved in multiple metal binding events, (iii) the presence of domain specificity between regions of the protein and (iv) mechanistic details involving both binding affinities and rate constants. The kinetic experiments identified the presence of multiple individual binding sites, each with a unique rate constant and an analogous binding affinity. The almost linear trend in rate constants as a function of bound As3+ provided a unique insight that became a critical step in the complete understanding of the mechanistic details of the metalation of MT. To fully define the biological function of this sulfur-rich protein it is necessary to determine kinetic rate constants and binding affinities for the essential metals. Recently, Zn2+ competition experiments between both of the isolated fragments (α and β) and the full-length protein (βα-MT 1a) as well as Zn2+ competition between βα-MT 1a and carbonic anhydrase were reported. From these data, the trend in binding affinities and the values of the Kf of the 7 bimolecular reactions involved in metalation were determined. From the analysis of ESI-MS data for Cu+ binding to βα-MT 1a at different pH-values, a trend in the 20 binding affinities for the complete metalation mechanism was reported. This review details a personal view of the historical development of the determination of stoichiometry for metal binding, the structure of the binding sites, the rates of the metalation reactions and the underlying binding affinities for each metalation step. We have attempted to summarize the experimental developments that led to the publication in May 2017 of the experimental determination of the 20 binding constants for the 20 sequential bimolecular reactions for Cu+ binding to the 20 Cys of apoMT as a function of pH that show the appearance and disappearance of clusters. We report both published data and in a series of tables an assembly of stoichiometries, and equilibrium constants for Zn2+ and Cu+ for many different metallothioneins.

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

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 β-domain and/or modulating the higher stability of the Zn(ii)-thiolate cluster in MT-2 β-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.

The Functions of Metamorphic Metallothioneins in Zinc and Copper Metabolism.

Recent discoveries in zinc biology provide a new platform for discussing the primary physiological functions of mammalian metallothioneins (MTs) and their exquisite zinc-dependent regulation. It is now understood that the control of cellular zinc homeostasis includes buffering of Zn2+ ions at picomolar concentrations, extensive subcellular re-distribution of Zn2+, the loading of exocytotic vesicles with zinc species, and the control of Zn2+ ion signalling. In parallel, characteristic features of human MTs became known: their graded affinities for Zn2+ and the redox activity of their thiolate coordination environments. Unlike the single species that structural models of mammalian MTs describe with a set of seven divalent or eight to twelve monovalent metal ions, MTs are metamorphic. In vivo, they exist as many species differing in redox state and load with different metal ions. The functions of mammalian MTs should no longer be considered elusive or enigmatic because it is now evident that the reactivity and coordination dynamics of MTs with Zn2+ and Cu+ match the biological requirements for controlling—binding and delivering—these cellular metal ions, thus completing a 60-year search for their functions. MT represents a unique biological principle for buffering the most competitive essential metal ions Zn2+ and Cu+. How this knowledge translates to the function of other families of MTs awaits further insights into the specifics of how their properties relate to zinc and copper metabolism in other organisms.

Mammalian Metallothionein-3: New Functional and Structural Insights.

Metallothionein-3 (MT-3), a member of the mammalian metallothionein (MT) family, is mainly expressed in the central nervous system (CNS). MT-3 possesses a unique neuronal growth inhibitory activity, and the levels of this intra- and extracellularly occurring metalloprotein are markedly diminished in the brain of patients affected by a number of metal-linked neurodegenerative disorders, including Alzheimer’s disease (AD). In these pathologies, the redox cycling of copper, accompanied by the production of reactive oxygen species (ROS), plays a key role in the neuronal toxicity. Although MT-3 shares the metal-thiolate clusters with the well-characterized MT-1 and MT-2, it shows distinct biological, structural and chemical properties. Owing to its anti-oxidant properties and modulator function not only for Zn, but also for Cu in the extra- and intracellular space, MT-3, but not MT-1/MT-2, protects neuronal cells from the toxicity of various Cu(II)-bound amyloids. In recent years, the roles of zinc dynamics and MT-3 function in neurodegeneration are slowly emerging. This short review focuses on the recent developments regarding the chemistry and biology of MT-3.

Chemistry of mammalian metallothioneins and their interaction with amyloidogenic peptides and proteins.

Cu and Zn ions are essential in most living beings. Their metabolism is critical for health and mis-metabolism can be lethal. In the last two decades, a large body of evidence has reported the role of copper, zinc and iron, and oxidative stress in several neurodegenerative diseases like Alzheimer's, Parkinson's, prion diseases, etc. To what extent this mis-metabolism is causative or a consequence of these diseases is still a matter of research. In this context metallothioneins (MTs) appear to play a central gate-keeper role in controlling aberrant metal-protein interactions. MTs are small proteins that can bind high amounts of Zn(ii) and Cu(i) ions in metal-cluster arrangements via their cysteine thiolates. Moreover, MTs are well known antioxidants. The present tutorial outlines the chemistry underlying the interconnection between copper(i/ii) and zinc(ii) coordination to amyloidogenic proteins and MTs, and their redox properties in generation and/or silencing reactive oxygen species (overproduced in oxidative stress) and other reactants. These studies have revealed the coordination chemistry involved in neurodegenerative diseases and the interactions between MTs and amyloidogenic protein metal-complexes (like amyloid-β, α-synuclein and prion-protein). Overall, the protective role of MTs in neurodegenerative processes is emerging, serving as a foundation for exploring MT chemistry as inspiration for therapeutic approaches.

Metallothionein regulates intracellular zinc signaling during CD4(+) T cell activation.

BackgroundThe ultra-low redox potential and zinc binding properties of the intracellular pool of mammalian metallothioneins (MT) suggest a role for MT in the transduction of redox signals into intracellular zinc signals. Increased expression of MT after exposure to heavy metals, oxidative stress, or inflammatory cytokines leads to an increased intracellular redox-mobilizable zinc pool that can affect downstream zinc-sensitive signaling pathways. CD4+ T helper cells are poised to be influenced by MT transduced zinc signaling because they produce intracellular reactive oxygen species following activation through the T cell receptor and are sensitive to small changes in intracellular [Zn2+].ResultsMT expression and intracellular [Zn2+] are both increased during primary activation and expansion of naïve CD4+ T cells into the Tr1 phenotype in vitro. When Tr1 cells from wildtype mice are compared with congenic mice lacking functional Mt1 and Mt2 genes, the expression of intracellular MT is associated with a greater increase in intracellular [Zn2+] immediately following exposure to reactive oxygen species or upon restimulation through the T cell receptor. The release of Zn2+ from MT is associated with a greater increase in p38 MAPK activation following restimulation and decreased p38 MAPK activation in MT knockout Tr1 cells can be rescued by increasing intracellular [Zn2+]. Additionally, IL-10 secretion is increased in MT knockout Tr1 cells compared with wildtype controls and this increase is prevented when the intracellular [Zn2+] is increased experimentally.ConclusionsDifferences in zinc signaling associated with MT expression appear to be a result of preferential oxidation of MT and concomitant release of Zn2+. Although zinc is released from many proteins following oxidation, release is greater when the cell contains an intracellular pool of MT. By expressing MT in response to certain environmental conditions, CD4+ T cells are able to more efficiently release intracellular zinc and regulate signaling pathways following stimulation. The link between MT expression and increased zinc signaling following activation represents an important immunomodulatory mechanism of MT and illuminates the complex role MT plays in shaping immune responses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-016-0151-2) contains supplementary material, which is available to authorized users.

Cadmium binding mechanisms of isolated domains of human MT isoform 1a: Non-cooperative terminal sites and cooperative cluster sites

A number of biological functions have been ascribed to mammalian metallothioneins (MTs) including zinc and copper homeostatic regulation, redox activity and detoxification of heavy metals like cadmium and mercury. It is unclear how these small, fluxional, cysteine rich proteins manage to play each of these vital roles. Using a combination of cadmium and pH titrations of the isolated domains of human MT isoform 1a monitored by electrospray ionization mass spectrometry and circular dichroism spectroscopy, we report the pH dependencies that control metal binding mechanisms of these domains. We report that the α-domain mechanism is driven by the cooperative formation of the Cd4MT cluster at slightly acidic pH (≤6.9) switching binding mechanisms over a physiologically relevant pH range, whereas the β-domain metalation mechanism is dominated by terminal coordination of cadmium in a non-cooperative manner above pH5.5. These results suggest that, in some acidic sub-cellular compartments, cadmium could be sequestered in the α-domain, leaving zinc or copper bound in the β-domain and available for donation to other metalloproteins. We propose that these results can be explained by the intrinsic nature of the two domains, the four-metal α-cluster being more resistant to proton attack due to its lower charge-to-metal ratio, compared with the three-metal β-domain.

Putting the pieces into place: Properties of intact zinc metallothionein 1A determined from interaction of its isolated domains with carbonic anhydrase.

Mammalian metallothioneins (MTs) bind up to seven Zn(2+) using a large number of cysteine residues relative to their small size and can act as zinc-chaperones. In metal-saturated Zn7-MTs, the seven zinc ions are co-ordinated tetrahedrally into two distinct clusters separated by a linker; the N-terminal β-domain [(Zn3Cys9)(3-)] and C-terminal α-domain [(Zn4Cys11)(3-)]. We report on the competitive zinc metalation of apo-carbonic anhydrase [CA; metal-free CA (apo-CA)] in the presence of apo-metallothionein 1A domain fragments to identify domain specific determinants of zinc binding and zinc donation in the intact two-domain Znn-βαMT1A (human metallothionein 1A isoform; n=0-7). The apo-CA is shown to compete effectively only with Zn2-3-βMT and Zn4-αMT. Detailed modelling of the ESI mass spectral data have revealed the zinc-binding affinities of each of the zinc-binding sites in the two isolated fragments. The three calculated equilibrium zinc affinities [log(KF)] of the isolated β-domain were: 12.2, 11.7 and 11.4 and the four isolated α-domain affinities were: 13.5, 13.2, 12.7 and 12.6. These data provide guidance in identification of the location of the strongest-bound and weakest-bound zinc in the intact two-domain Zn7βαMT. The β-domain has the weakest zinc-binding site and this is where zinc ions are donated from in the Zn7-βαMT. The α-domain with the highest affinity binds the first zinc, which we propose leads to an unscrambling of the cysteine ligands from the apo-peptide bundle. We propose that stabilization of the intact Zn6-MT and Zn7-MT, relative to that of the sum of the separated fragments, is due to the availability of additional cysteine ligand orientations (through interdomain interactions) to support the clustered structures.

Kinetics of Zinc and Cadmium Exchanges between Metallothionein and Carbonic Anhydrase.

The flexible coordination stoichiometry of a relatively high number of metal ions is a property unique to the metallothionein (MT) family of proteins. Mammalian MTs, for example, accommodate up to seven divalent metal ions in tetrahedral coordination geometries, using its complement of 20 cysteine ligands. The lability of the metals from these metalloclusters has been used to support the proposal of MTs acting as metal chaperones, by donating to other metal-binding proteins. The metal exchange kinetics between human MT1A and carbonic anhydrase (CA) were examined using time-dependent electrospray ionization mass spectrometry (ESI-MS). The time dependence of three different reaction conditions were studied: (i) zinc donation from partially metalated zinc-MT to apoCA; (ii) metal exchange between zinc saturated MTs and cadmium saturated CA (Cd-CA); and (iii) metal exchange between partially metalated zinc-MTs and Cd-CA. The results show that zinc donation from Zn-MTs to apo-zinc-dependent enzymes is dependent on the metal loading of the Znn-MT (where n = 1-7) and that this is a direct consequence of the increasing metal affinity for smaller values of n. Partially metalated MTs are also shown to extract cadmium from Cd-CA with significantly faster rates than metal saturated MTs and that even under zinc limiting conditions, mammalian Cd-CA would not coexist with MT. On the basis of these and previously published results, we suggest that protein-protein interactions between MT and CA facilitate metal transfers through favorable electrostatic interactions and hypothesize that the metal could be transferred between the MT and the enzyme active site using nearby metal-binding functionalities along the transfer pathway.

Domain Selection in Metallothionein 1A: Affinity-Controlled Mechanisms of Zinc Binding and Cadmium Exchange.

Mammalian metallothioneins (MTs) are small, metal binding proteins implicated in cellular metal ion homeostasis and heavy metal detoxification. Divalent, metal-saturated MTs form two distinct domains; the N-terminal β domain binds three metals using nine Cys residues, and the C-terminal α domain binds four metals with 11 Cys residues. Domain selection during zinc binding and cadmium exchange to human MT1A was examined using a series of competition reactions with mixtures of the isolated domain fragments. These experiments were conducted at two biologically significant pH conditions where MTs exist in vivo. Neither zinc binding nor cadmium exchange showed any significant degree of specificity or selectivity based on detailed analysis of electrospray ionization mass spectrometric and circular dichroic data. Under acidic conditions, zinc binding and cadmium exchange showed slight α domain selectivity because of the increased preference for cooperative clustering of the α domain. Modeling of the reactions showed that at both physiological (7.4) and acidic (5.8) pHs, zinc binding and cadmium exchanges occur essentially randomly between the two fragments. The metal binding affinity distributions between the domain fragments are comingled and not significantly separated as required for a domain specific mechanism. The models show rather that the order of the binding events follows the order of the binding affinities that are distributed across both domains and that this can be considered quantitatively by the KF(Cd)/KF(Zn) binding constant ratio for each metal bound.