Peptide folding, metal-binding mechanisms, and binding site structures in metallothioneins

This minireview specifically focuses on recent studies carried out on structural aspects of metal-free metallothionein (MT), the mechanism of metal binding for copper and arsenic, structural studies using x-ray absorption spectroscopy and molecular mechanics modeling, and speciation studies of a novel cadmium and arsenic binding algal MT. Molecular mechanics-molecular dynamics calculations of apo-MT show that significant secondary structural features are retained by the polypeptide backbone upon sequential removal of the metal ions, which is stabilized by a possible H-bonding network. In addition, the cysteinyl sulfurs were shown to rotate from within the domain core, where they are found in the metallated state, to the exterior surface of the domain, suggesting an explanation for the rapid metallation reactions that were measured. Mixing Cu6beta-MT with Cd4alpha-MT and Cu6alpha-MT with Cd3beta-MT resulted in redistribution of the metal ions to mixed metal species in each domain; however, the Cu+ ions preferentially coordinated to the beta domain in each case. Reaction of As3+ with the individual metal-free beta and alpha domains of MT resulted in three As3+ ions coordinating to each of the domains, respectively, in a proposed distorted trigonal pyramid structure. Kinetic analysis provides parameters that allow simulation of the binding of each of the As3+ ions. X-ray absorption spectroscopy provides detailed information about the coordination environment of the absorbing element. We have combined measurement of x-ray absorption near edge structure (XANES) and extended x-ray absorption fine structure (EXAFS) data with extensive molecular dynamics calculations to determine accurate metal-thiolate structures. Simulation of the XANES data provides a powerful technique for probing the coordination structures of metals in metalloproteins. The metal binding properties of an algal MT, Fucus vesiculosus, has been investigated by UV absorption and circular dichroism spectroscopy and electrospray ionization-mass spectrometry. The 16 cysteine residues of this algal MT were found to coordinate six Cd2+ ions in two domains with stoichiometries of a novel Cd3S7 cluster and a beta-like Cd3S9 cluster.     

Metal-dependent protein folding: metallation of metallothionein

Metallothionein (MT), a low molecular mass, cysteine-rich protein, is a model system for metal ion-directed folding due to its diverse metal binding properties. In this minireview, the current status of theoretical and experimental studies that have focused primarily on the initial metallation steps involving the metal-free, or apo, MT and divalent metals, Zn²⁺ and Cd²⁺ is described. Apo-MT has recently been reported to be present in the cell in quantities equal to that of the metallated protein, which might indicate a potential role for the protein in the absence of metals. Molecular mechanics-molecular dynamics (MM3/MD) calculations carried out on the demetallation of cadmium-coordinated MT isoform 1a indicate structural stability of the metal-free protein with significant retention of the backbone conformation imposed by the metal-thiolate clusters present in the metallated holo-protein. Significantly, the cysteinyl sulfurs were found inverted to the outside of a quite compact sphere. In contrast, MM3/MD calculations of apo-MT starting from a linear strand did not possess any structural stability and can be described as a random coil conformation. Evidence for the sequence of metallation is discussed, together with current experimental data to support either a cooperative or sequential binding mechanisms.     

Histidine ligands in bacterial metallothionein enhance cluster stability

The cyanobacterial metallothionein (MT) SmtA is the prototype for bacterial MTs and protects against elevated levels of zinc. In contrast to mammalian MTs, bacterial MTs coordinate to metal ions not only via cysteine sulfurs, but unusually for MTs, also via histidine nitrogens. To investigate whether histidine coordination in these metal-sulfur clusters provides advantages over S-coordination only, we mutated the two metal-binding histidine residues in the cyanobacterial MT SmtA from Synechococcus PCC7942 to cysteines. We show that the mutant proteins are still capable of binding up to four zinc ions as is the wild-type protein. However, the mutations perturb protein folding and metal-binding dynamics. Interestingly, several homologues of SmtA also show variations in these two residues. We conclude that histidine residues in Synechococcus PCC7942 SmtA have a stabilising effect due to electrostatic interactions that impact on protein folding and metal cluster charge, and are involved in fine-tuning the reactivity of the bound metal ions.     

植物类金属硫蛋白半胱氨酸富含区结构的建模

详细了解蛋白质的三级结构信息有助于理解其生物学功能.随着植物基因组研究的进展,已发现了50多个植物类金属硫蛋白(Metallothionein-Like, MT-L)基因.但至今只有少数几个MT-L蛋白得到了纯化,而其结构尚无报道,因此有必要建立分析这类蛋白结构特征的方法.本研究根据已知的哺乳动物MT的结构数据,分析得出了CXC、CXXC模式和金属-硫络合簇结构原子间的距离限制条件,并用距离几何算法计算得出预测蛋白可能的构象;然后通过统计分析筛选出目标函数值显著较小、构象能低的结构作为这些蛋白半胱氨酸富含区的预测结构,由此建成了适合于植物类金属硫蛋白半胱氨酸富含区的结构预测方法.从应用该方法正确地预测出了已知结构的蓝蟹MT的结构来看,该方法是可行的.并用该方法预测了油菜MT-L蛋白的半胱氨酸富含区的结构.     

用距离几何算法和同源建模法对水稻类金属硫蛋白(rgMT)的三维结构建模

水稻类金属硫蛋白(rgMT)的两端是高度保守的半胱氨酸富含区的结构域(CR区),中间是不含半胱氨酸的间隔区,呈典型的三段式结构.本研究分别采用距离几何算法和同源建模相结合的方法对水稻类金属硫蛋白进行三级结构建模.在排列出CR区的所有可能的半胱氨酸-金属硫络合的组合方式,并对每一种组合方式给出一定的限制条件后各生成20个随机构象.根据生成的随机构象是否能形成金属硫络合结构,从900个随机构象中最终选出6个构象(N端4种,C端2种组合)作为可能的结构模型.另一方面,采用GOR方法对间隔区进行了二级结构预测,随后用同源建模法对其建模.将上述建成的三部分模型连接起来后形成rgMT的整体三维构象.结果表明rgMT能像哺乳动物MT蛋白一样,可形成两个独立的、在结构和能量上均没有障碍的金属-硫络合结构.介于所有植物类金属硫蛋白都具有典型的三段式结构,其中的一部分还具有与rgMT相同的半胱氨酸排列方式,所以rgMT三维结构模型的建立对于其他植物类金属硫蛋白的结构研究具有重要的参考价值.     

用距离几何算法和同源建模法对水稻类金属硫蛋白（rgMT）的三维结构建模

水稻类金属硫蛋白(rgMT)的两端是高度保守的半胱氨酸富含区的结构域(CR区),中间是不含半胱氨酸的间隔区,呈典型的三段式结构。本研究分别采用距离几何算法和同源建模相结合的方法对水稻类金属硫蛋白进行三级结构建模。在排列出CR区的所有可能的半胱氨酸-金属硫络合的组合方式,并对每一种组合方式给出一定的限制条件后各生成20个随机构象。根据生成的随机构象足否能形成金属硫络合结构,从900个随机构象中最终选出6个构象(N端4种,C端2种组合)作为可能的结构模型。另一方面,采用GOR方法对间隔区进行了二级结构预测,随后用同源建模法对其建模。将上述建成的三部分模型连接起来后形成rgMT的整体三维构象。结果表明rgMT能像哺乳动物MT蛋白一样,可形成两个独立的、在结构和能量上均没有障碍的金属-硫络合结构。介于所有植物类金属硫蛋白都具有典型的三段式结构,其中的一部分还具有与rgMT相同的半胱氨酸排列方式,所以rgMT三维结构模型的建立对于其他植物类金属硫蛋白的结构研究具有重要的参考价值。     

Reversible folding of cysteine-rich metallothionein by an overcritical reaction path

A first-order-like state transition is considered to be involved in the restoration of the activities of a few proteins by correctly folding the protein [Phys. Rev. E 66 (2002) 021903]. In order to understand the general applicability of this mechanism, we studied a metallothionein (MT) protein with an unconventional structure, i.e., without any alpha-helix or beta-sheet. MT is a 61 amino-acid peptide. There are 6-7 Zn(2+) ions, which bind avidly to 20 conserved cysteines (Cys) of MT. These properties indicate that the structure of MT is quite different from those of the other proteins. Similar to our previous findings, the denatured MT can be folded without any aggregation via a designated stepwise quasi-static process (an over-critical reaction path). The particle size of folded MT intermediates, determined by dynamic light scattering, shrank right after the first folding stage. It is consistent with a collapse-model. In addition, results from both atomic absorption and circular dichroism (CD) indicate that the stable intermediates may fold to the native conformation but with only partial Zn(2+) binding, which in turn implies that those folding intermediates are in a molten globular state. These reversible unfolding and folding processes indicate that Cys-rich protein, MT, may also be folded by way of a first-order-like state transition mechanism. We suspect that this process may likely be involved in the reaction of the metal substitution process in metal containing enzymes.     

Cadmium binding studies to the earthworm Lumbricus rubellus metallothionein by electrospray mass spectrometry and circular dichroism spectroscopy

The earthworm Lumbricus rubellus has been found to inhabit cadmium-rich soils and accumulate cadmium within its tissues. Two metallothionein (MT) isoforms (1 and 2) have been identified and cloned from L. rubellus. In this study, we address the metalation status, metal coordination, and structure of recombinant MT-2 from L. rubellus using electrospray ionization mass spectrometry (ESI-MS), UV absorption, and circular dichroism (CD) spectroscopy. This is the first study to show the detailed mass and CD spectral properties for the important cadmium-containing earthworm MT. We report that the 20-cysteine L. rubellus MT-2 binds seven Cd(2+) ions. UV absorption and CD spectroscopy and ESI-MS pH titrations show a distinct biphasic demetalation reaction, which we propose results from the presence of two metal-thiolate binding domains. We propose stoichiometries of Cd(3)Cys(9) and Cd(4)Cys(11) based on the presence of 20 cysteines split into two isolated regions of the sequence with 11 cysteines in the N-terminal and 9 cysteines in the C-terminal. The CD spectrum reported is distinctly different from any other metallothionein known suggesting quite different binding site structure for the peptide.     

Isolation and characterization of a self-sufficient one-domain protein. (Cd)-metallothionein from Eisenia foetida.

Earthworms have been shown to accumulate trace elements in general, and particularly high amounts of metal ions such as cadmium, copper and zinc. The earthworm's response to metal contamination has been linked to the induction and expression of metallothionein (MT) proteins, a detoxification strategy analogous to that found in other biological systems. The present study focuses on an inducible Cd-MT isolated from the compost-dwelling brandling worm Eisenia foetida (Savigny). A full characterization of the protein (including protein induction, MT cDNA, amino-acid sequence and metal stoichiometry) revealed a new dimension of knowledge to the molecular genetic information available to date. Whereas the elucidated cDNA codes for a putative protein which possesses 80 amino-acid residues, the characterized protein bears only 41 amino acids. The isolated product has evidently attained its size and shape by cleavage near the N-terminal site and at the linker region between the two putative metal-binding domains of the translated product, yielding a small MT moiety which contains 12 Cys residues (including one triple Cys-motif) binding four cadmium ions. It can be shown that the isolated MT molecule represents a self-sufficient one-domain MT which is stable in vitro. The isolation of the single-domain MT peptide raises the question about the method of formation and significance in vivo of such small MT moieties from tissues of E. foetida and possibly other terrestrial invertebrates. In this respect, two hypotheses are discussed: firstly, the possibility of formation of small MT peptides due to enzymatic cleavage of the intact protein during the process of preparation and isolation; and secondly, the possibility of deliberate post-translational processing of the translated gene product to yield functional one-domain MT moieties.     

Investigation of metal complexes with metallothionein in rat tissues by hyphenated techniques.

The potential of hyphenated techniques based on a combination of microbore reversed-phase (RP) HPLC or capillary zone electrophoresis (CZE) with inductively coupled plasma (ICP) or electrospray (ES) mass spectrometry (MS) was demonstrated for the characterization of metal complexes with metallothionein in rat liver and kidney. The mixture of MT complexes was isolated from the tissues by size-exclusion LC and further characterized in neutral pH conditions (pH 6.8-7.2) by RP-HPLC or CZE. The metal stoichiometry and the molar mass of the eluted complexes was measured by ICP-MS and ES-MS, respectively. An additional dimension to the analysis was achieved by post-column acidification of the chromatographic eluent that allowed the determination of the molecular weight of the demetallated complexes with 10-fold higher sensitivity. The approach allowed the detection of two major metallothionein (MT) isoforms (MT-1 and MT-2) in liver and one MT isoform in kidney. The actual number of peaks in chromatograms and electropherograms was bigger because of the formation of mixed Cd-Cu complexes of the same MT isoform that showed different hydrophobicities.     

Identification of mercury and other metals complexes with metallothioneins in dolphin liver by hydrophilic interaction liquid chromatography with the parallel detection by ICP MS and electrospray hybrid linear/orbital trap MS/MS

A novel analytical procedure for the identification of metal (Hg, Cd, Cu, Zn) complexes with individual metallothionein (MT) isoforms in biological tissues by electrospray MS/MS was developed. The sample preparation was reduced to three rapid steps: the two-fold dilution of the sample cytosol with acetonitrile, the recovery of the supernatant containing MT-complexes by centrifugation and its concentration under nitrogen flow. The replacement of reversed phase HPLC by hydrophilic interaction LC (HILIC) allowed the preservation of the unstable and low abundant metallothionein zinc-mercury mixed complexes (MT-Zn(6)Hg). The MT complexes eluted were detected by ICP MS and identified in terms of molecular mass by electrospray high resolution (100,000) MS. The identification was completed by on line demetallation and the determination of the molecular mass of the apoform, followed by amino acid sequencing in the top-down mode using high energy collision fragmentation (HCD). The method was applied to the identification of MT complexes in a white-sided dolphin (Lagenorhynchus acutus) liver homogenate. The Zn complex of the N-acetylated MT2 isoform was found to be predominant, the presence of mixed complexes with Cd, Cu and, for the first time ever, Hg, was demonstrated. The latter finding has the potential to shed new light on the mercury detoxification mechanism in marine organisms.     

Triticum durum metallothionein. Isolation of the gene and structural characterization of the protein using solution scattering and molecular modeling

A novel gene sequence, with two exons and one intron, encoding a metallothionein (MT) has been identified in durum wheat Triticum durum cv. Balcali85 genomic DNA. Multiple alignment analyses on the cDNA and the translated protein sequences showed that T. durum MT (dMT) can be classified as a type 1 MT. dMT has three Cys-X-Cys motifs in each of the N- and C-terminal domains and a 42-residue-long hinge region devoid of cysteines. dMT was overexpressed in Escherichia coli as a fusion protein (GSTdMT), and bacteria expressing the fusion protein showed increased tolerance to cadmium in the growth medium compared with controls. Purified GSTdMT was characterized by SDS- and native-PAGE, size exclusion chromatography, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. It was shown that the recombinant protein binds 4 +/- 1 mol of cadmium/mol of protein and has a high tendency to form stable oligomeric structures. The structure of GSTdMT and dMT was investigated by synchrotron x-ray solution scattering and computational methods. X-ray scattering measurements indicated a strong tendency for GSTdMT to form dimers and trimers in solution and yielded structural models that were compatible with a stable dimeric form in which dMT had an extended conformation. Results of homology modeling and ab initio solution scattering approaches produced an elongated dMT structure with a long central hinge region. The predicted model and those obtained from x-ray scattering are in agreement and suggest that dMT may be involved in functions other than metal detoxification.     

Full quantum-mechanical structure of the human protein Metallothionein-2

Metallothioneins (MT) are small, metal-binding proteins with diverse functions related to metal ion homeostasis. This paper presents the full 384-388-atom structures of the two native Zn(II)- and the Cd(II)-containing domains of human MT2, optimized with density functional theory. The presented structures are accurate to ~ 0.03 Å for bond lengths and thus provide new physical insight into the detailed electronic structures of MTs, in particular with accurate accounts of bridging vs. terminal bonds not available from NMR or EXAFS. The MT protein enhances the asymmetry, as compared to the protein-free clusters, causing a hierarchy in binding that most likely allows MTs to transfer ions to multiple targets in vivo. The protein polarization is substantial and occurs primarily via the terminal sulfurs, a key mechanism in providing domain-specific electronic structures. The β-domain polarizes its smaller cluster less on average, due to its less polarizable, higher negative charge density, as reflected in longer MS bond lengths and smaller bond orders. This may explain why MT2β is more reactive and dynamic and why MTs have evolved two different-size, asymmetric domains with different metal binding affinities fit for different molecular targets of metal ion transfer.     

High resolution solution structure of the protein part of Cu7 metallothionein.

The three-dimensional solution structure of the protein part of Cu7 metallothionein (Cu7MT) of Saccharomyces cerevisiae has been attempted by 1H two-dimensional NMR spectroscopy at 800 MHz. The protein part constitutes 53 amino acids. A total of 1192 NOEs, of which 1048 are meaningful, were used to determine the solution structure of the first 40 residues, the last 13 residues being disordered. A family of 30 structures was generated. Root-mean-square deviation (rmsd) values from the average structure of 0.32 +/- 0.13 A and 0.61 +/- 0.15 A for backbone and all heavy atoms, respectively, were obtained for the residues 2-40. The ten copper-coordinating cysteine sulfurs and the empty spaces around them are well defined. The structure of the protein part is similar but not identical to the available ones of the same holoprotein and of the Ag7 metallothionein, and is qualitatively superior. If the same metal-sulfur connectivities reported in the literature from 1H-109Ag heteronuclear multiple quantum coherence spectroscopy are assumed to hold for the present copper derivative, a peptide structure is obtained which is again similar, but still not identical, within indetermination, to that available. The structure of the copper polymetallic center may well be different from that proposed for the silver derivative, and indeed a number of different arrangements of the seven copper ions are consistent with the present highly refined structure of the protein part.     

Structural studies of metal-free metallothionein

We report the first molecular dynamics calculations on the structure of metal-free betaalpha recombinant human metallothionein, with comparison to the two isolated fragments, alpha-rhMT and beta-rhMT, starting from a linear synthesized strand as well as a demetallated conformation. Following a 5000 ps MM3/MD calculation, the cysteine side chains were found to populate the outside surface of the metal-free protein, regardless of the initial conformation. The polypeptide backbone adopted a random coil conformation when starting from the linear strand, however, it retained a significant amount of secondary structure when starting from the demetallated conformation. We propose that the inverted cysteinyl sulfur orientation facilitates the binding of the metal ions to form the proteolytically stable, metallated protein.     

Properties of the reaction of cis-dichlorodiammineplatinum(II) with metallothionein.

Properties of the reactions of cis-dichlorodiammine Pt(II) and related complexes with zinc metallothionein or apometallothionein have been investigated. During these reactions, platinum binds stoichiometrically to protein sulfhydryl groups and zinc, if present, is displaced. The ammine ligands are also lost in the process, suggesting that Pt(II) has tetrathiolate coordination in metallothionein. This conclusion is supported by extended x-ray absorption-fine structure studies which indicate that there are 4 sulfurs in the first coordination sphere of the platinum centers. The product contains 10 +/- 2 Pt(II) per mol of protein and migrates over Sephadex G-75 as a structure of similar size to zinc metallothionein. The kinetics of reaction are biphasic as monitored by the formation of Pt-thiolate bonds or by the release of zinc from the protein. Both methods yield identical rate laws for the reaction. The first step is independent of Pt(II) concentration but involves the binding of as many as four platinum atoms to the protein with little Pt-sulfhydryl bond formation and without much loss of zinc. The second rate process is first order in both zinc or sulfhydryl binding sites and Pt(II). Neither kinetic step is sensitive to the chloride ion concentration over the range 0-0.5 M. However, the reaction is sensitive to pH between 5.5 and 8.0. trans-Dichlorodiammineplatinum(II) reacts with zinc metallothionein with similar kinetics.     

Cadmium-induced conformational changes in type 2 metallothionein of medicinal plant Coptis japonica: insights from molecular dynamics studies of apo,partially and fully metalated forms

Plants play an important role in the removal of excess heavy metals from soil and water. Medicinal plants can also have non-traditional use in phytoremediation technologies. Among the heavy metals, Cadmium (Cd) is the most abundant and readily taken up by the crop plants. Plant metallothioneins (MTs) are small proteins having cysteine-rich residues and appear to play key roles in metal homoeostasis. Plant metallothionein 2 (MT 2) from Coptis japonica (Gold-thread; CjMT 2) is a typical member of this subfamily and features two cysteine-rich regions containing eight and six cysteine residues, respectively, separated by 42 amino acids long linker region. In-silico analysis of MT 2 protein sequences of C. japonica was performed. In this study, ab initio methods were utilised for the prediction of three-dimensional structure of CjMT 2. After structure validation, heavy metal-binding sites were predicted for the selected modelled structures of CjMT 2. To obtain Cd_i-CjMT 2 (i = 1–7), metalated complex individual docking experiments were performed. The stability of the metalated docked structures was assessed by molecular dynamics (MD) simulation studies. Our study showed that CjMT 2 binds up to 4 Cd²⁺ ions in two distinct domains: a N-terminal β-domain that binds to 2 Cd²⁺ ions and a C-terminal α-domain that binds with 2 Cd²⁺ ions. Our analysis revealed that Cys residues of alpha and beta domain and some residues of spacer region of CjMT 2 protein might be important for the cadmium interaction. MD simulation studies provided insight into metal-induced conformational changes and mechanism of metalation of CjMT 2, an intrinsically disordered protein. This study provides useful insights into mechanism of cadmium-type 2 metallothionein interaction.     

Structural basis for copper transfer by the metallochaperone for the Menkes/Wilson disease proteins

The Hah1 metallochaperone protein is implicated in copper delivery to the Menkes and Wilson disease proteins. Hah1 and the N-termini of its target proteins belong to a family of metal binding domains characterized by a conserved MT/HCXXC sequence motif. The crystal structure of Hah1 has been determined in the presence of Cu(I), Hg(II), and Cd(II). The 1.8 A resolution structure of CuHah1 reveals a copper ion coordinated by Cys residues from two adjacent Hah1 molecules. The CuHah1 crystal structure is the first of a copper chaperone bound to copper and provides structural support for direct metal ion exchange between conserved MT/HCXXC motifs in two domains. The structures of HgHah1 and CdHah1, determined to 1.75 A resolution, also reveal metal ion coordination by two MT/HCXXC motifs. An extended hydrogen bonding network, unique to the complex of two Hah1 molecules, stabilizes the metal binding sites and suggests specific roles for several conserved residues. Taken together, the structures provide models for intermediates in metal ion transfer and suggest a detailed molecular mechanism for protein recognition and metal ion exchange between MT/HCXXC containing domains.     

Copper speciation in the alpha and beta domains of recombinant human metallothionein by electrospray ionization mass spectrometry.

ESI-MS data are reported for Cu(I) binding to the metal-free and cadmium-alpha and beta domains of recombinant human metallothionein. These data provide information on the stoichiometric ratios of copper and cadmium that bind to the 11 thiolate sulfurs in the alpha fragment and the nine thiolate sulfurs in the beta fragment. The data show the effects of the existing three-dimensional structure on the formation of different Cu(I)-thiolate clusters. Charge-state spectra are reported for a range of Cu(I) binding at low and neutral pH to the isolated alpha and beta domains. There is an uneven distribution of charge states that show that changes in the three-dimensional structure take place as a function of Cu(I) loading. Metallation of the alpha domain at low pH takes place in a series of steps with the Cu7 species dominating until at higher levels of Cu(I) the clusters become unstable resulting in increased concentrations of the metal-free being detected. We interpret this behavior as being the result of the expansion of the Cu-S domain structure to accommodate digonal co-ordination for the increased Cu(I) loading. This larger structure is unstable in the mass spectrometer and demetallation takes place. Metallation of the beta domain at low pH proceeds in steps that involve initial formation of a Cu5S9 cluster, followed by the Cu6S9 at higher concentrations of Cu(I). The charge state spectra indicate a significant change in exposure of protonatable amino acids between Cu5S9 and Cu6S9 clusters, which indicates a change in peptide conformation when the Cu6S9 cluster forms. Metallation at neutral pH follows this same trend, namely, a much greater range of copper species is found during titrations of the Cd4S11-alpha fragment compared with the number of species that form when Cu(I) is added to Cd3S9-beta. The mass spectral data indicate that at neutral pH, the presence of the tetrahedral geometry of the Cd(II) facilitates formation of mixed trigonal and digonal geometries for the incoming Cu(I) so that the most prominent species in the beta fragment is Cd1Cu5S9 which transforms into Cu7S9 at higher concentrations of Cu(I), and finally to Cu9S9 at saturation, all species involving a number of Cu(I) in digonal geometries. The observation that the metallation patterns of the alpha and beta clusters follow different pathways at both low and neutral pH's, suggests that the structures in the two domains are quite different, in agreement with previous proposals