Studies on the assembly and stability of the metal-thiolate clusters of metallothionein in dimethyl sulfoxide.

Solvent perturbation studies in 100% dimethyl sulfoxide (Me2SO) have been carried out on rabbit liver metallothionein (MT) in an effort to learn more about the factors stabilizing the three-dimensional structure and the mechanism of cluster formation. As indicated by the electronic absorption spectra of Co7-metallothionein, the reconstituted protein preserves its structural integrity in this solvent. Minor spectral differences between water and Me2SO were fully reversible. The titration of apoMT with cobalt(II) in Me2SO, followed by UV-visible-near-infrared electronic absorption, circular dichroism, magnetic circular dichroism, and EPR spectroscopy, indicate that the protein can refold in this solvent. A comparison with the previous titration data in water reveals that the first four titration steps in both solvents are identical, indicating a thermodynamically controlled folding process. However, the reversed order of the cluster completion between Me2SO and water may suggest the involvement of a kinetically controlled folding process in the last three titration steps. A new cluster form developed with approximately nine Co(II) equivalents.     

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

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

Polypeptide fold in the two metal clusters of metallothionein-2 by nuclear magnetic resonance in solution

The solution conformation of rabbit liver Cd27+-metallothionein-2 was determined by nuclear magnetic resonance (n.m.r.) and distance geometry. The n.m.r. data are based on complete sequence-specific resonance assignments for the polypeptide chain. This letter describes the global arrangement of the polypeptide chain, which forms two distinct domains containing metal clusters of three and four Cd ions, respectively.     

Evidence for a dynamic structure of human neuronal growth inhibitory factor and for major rearrangements of its metal-thiolate clusters.

Human neuronal growth inhibitory factor (GIF), a metallothionein-like protein classified as metallothionein-3, impairs the survival and the neurite formation of cultured neurons. Despite its approximately 70% amino acid sequence identity with those of mammalian metallothioneins (MT-1 and MT-2 isoforms), only GIF exhibits growth inhibitory activity. In this study, structural features of the metal-thiolate clusters in recombinant Zn(7)- and Cd(7)-GIF, and in part also in synthetic GIF (68 amino acids), were investigated by using circular dichroism (CD) and (113)Cd NMR. The CD and (113)Cd NMR studies of recombinant Me(7)-GIF confirmed the existence of distinct Me(4)S(11)- and Me(3)S(9)-clusters located in the alpha- and beta-domains of the protein, respectively. Moreover, a mutual structural stabilization of both domains was demonstrated. The (113)Cd NMR studies of recombinant (113)Cd(7)-GIF were conducted at different magnetic fields (66.66 and 133.33 MHz) and temperatures (298 and 323 K). At 298 K the spectra revealed seven (113)Cd signals at 676, 664, 651, 644, 624, 622, and 595 ppm. A striking feature of all resonances is the absence of resolved homonuclear [(113)Cd-(113)Cd] couplings and large apparent line widths (between 140 and 350 Hz), which account for the absence of cross-peaks in [(113)Cd, (113)Cd] COSY. On the basis of a close correspondence in chemical shift positions of the (113)Cd signals at 676, 624, 622, and 595 ppm with those obtained in our previous studies of (113)Cd(4)-GIF(32-68) [Hasler, D. W., Faller, P., and Vasák, M. (1998) Biochemistry 37, 14966], these resonances can be assigned to a Cd(4)S(11)-cluster in the alpha-domain of (113)Cd(7)-GIF. Consequently, the remaining three (113)Cd signals at 664, 651, and 644 ppm originate from a Me(3)S(9) cluster in the beta-domain. However, the latter resonances show a markedly reduced and temperature-independent intensity (approximately 20%) when compared with those of the alpha-domain, indicating that the majority of the signal intensity remained undetected. To account for the observed NMR features of (113)Cd(7)-GIF, we suggest that dynamic processes acting on two different NMR time scales are present: (i) fast exchange processes among conformational cluster substates giving rise to broad, weight-averaged resonances and (ii) additional very slow exchange processes within the beta-domain associated with the formation of configurational cluster substates. The implications of the structure fluctuation for the biological activity of GIF are discussed.     

Stochastic assembly of chemoreceptor clusters in Escherichia coli

Chemoreceptors and cytoplasmic chemotaxis proteins in Escherichia coli form clusters that play a key role in signal processing. These clusters localize at cell poles and at specific positions along the cell body which correspond to future division sites, but the details of cluster formation and the mechanism of cluster distribution remain unclear. Here, we used fluorescence microscopy to investigate how the numbers and sizes of receptor clusters depend on the expression level of chemotaxis proteins and on the cell length. We show that the average cluster number saturates at high levels of protein expression at approximately 3.7 clusters per cell, well below the number of available positioning sites. Correspondingly, distances between clusters in filamentous cells saturate at an average of 1 mum but, even at saturating expression levels, individual cluster numbers and distances show a broad distribution around the mean. Our data imply a stochastic mode of cluster assembly, where a defined average interval between clusters along the cell body arises from competition between nucleation of new clusters and growth of existing clusters. Upon subsequent anchorage to defined lateral sites, clusters grow with rates that inversely depend on their size, and become polar upon several rounds of cell division.     

Organization and evolution of mitochondrial gene clusters in human

Currently, the spatial patterns of mitochondrial genes and how the genomic localization of (pseudo)genes originated from mitochondrial DNA remain largely unexplained. The aim of this study was to elucidate the organization of mitochondrial (pseudo)genes given their evolutionary origin. We used a keyword finding method and a bootstrapping method to estimate parameter values that represent the distribution pattern of mitochondrial genes in the nuclear genome. Almost half of mitochondrial genes showing physical clusters were located in the pericentromeric and subtelomeric regions of the chromosome. Most interestingly, the size of these clusters ranged from 0.085 to 3.2 Mb (average ± SD 1.3 ± 0.73 Mb), which coincides with the size of the evolutionary pocket, or the average size of evolutionary breakpoint regions. Our findings imply that the localization of mitochondrial genes in the human genome is determined independent of adaptation.     

Organization and assembly of the TRAPPII complex

Current models suggest that TRAPP tethering complexes exist in two forms. Whereas the seven-subunit TRAPPI complex mediates ER-to-Golgi transport, TRAPPII contains three additional subunits (Trs65, Trs120 and Trs130) and is required for distinct tethering events at Golgi membranes. It is not clear how TRAPPII assembly is regulated. Here, we show that Tca17 is a fourth TRAPPII-specific component, and that Trs65 and Tca17 interact with distinct domains of Trs130 and make different contributions to complex assembly. Whereas Tca17 promotes the stable association of TRAPPII-specific subunits with the core complex, Trs65 stabilizes TRAPPII in an oligomeric form. We show that Trs85, which was previously reported to be a subunit of both TRAPPI and TRAPPII, is not associated with the TRAPPII complex in yeast. However, we find that proteins related to Trs85, Trs65 and Tca17 are part of the same TRAPP complex in mammalian cells. These findings have implications for models of TRAPP complex formation and suggest that TRAPP complexes may be organized differently in yeast and mammals.     

NifS-mediated assembly of [4Fe-4S] clusters in the N- and C-terminal domains of the NifU scaffold protein

NifU is a homodimeric modular protein comprising N- and C-terminal domains and a central domain with a redox-active [2Fe-2S](2+,+) cluster. It plays a crucial role as a scaffold protein for the assembly of the Fe-S clusters required for the maturation of nif-specific Fe-S proteins. In this work, the time course and products of in vitro NifS-mediated iron-sulfur cluster assembly on full-length NifU and truncated forms involving only the N-terminal domain or the central and C-terminal domains have been investigated using UV-vis absorption and M?ssbauer spectroscopies, coupled with analytical studies. The results demonstrate sequential assembly of labile [2Fe-2S](2+) and [4Fe-4S](2+) clusters in the U-type N-terminal scaffolding domain and the assembly of [4Fe-4S](2+) clusters in the Nfu-type C-terminal scaffolding domain. Both scaffolding domains of NifU are shown to be competent for in vitro maturation of nitrogenase component proteins, as evidenced by rapid transfer of [4Fe-4S](2+) clusters preassembled on either the N- or C-terminal domains to the apo nitrogenase Fe protein. Mutagenesis studies indicate that a conserved aspartate (Asp37) plays a critical role in mediating cluster transfer. The assembly and transfer of clusters on NifU are compared with results reported for U- and Nfu-type scaffold proteins, and the need for two functional Fe-S cluster scaffolding domains on NifU is discussed.     

Ancient and essential: the assembly of iron-sulfur clusters in plants

In plants iron-sulfur (Fe-S) proteins are found in the plastids, mitochondria, cytosol and nucleus, where they are essential for numerous physiological and developmental processes. Recent mutant studies, mostly in Arabidopsis thaliana, have identified three pathways for the assembly of Fe-S clusters. The plastids harbor the SUF (sulfur mobilization) pathway and operate independently, whereas cluster assembly in the cytosol depends on the emerging CIA (cytosolic iron-sulfur cluster assembly) pathway and mitochondria. The latter organelles use the ISC (iron-sulfur cluster) assembly pathway. In all three pathways the assembly process can be divided into a first stage where S and Fe are combined on a scaffold protein, and a second stage in which the Fe-S cluster is transferred to a target protein. The second stage might involve different carrier proteins with specialized functions.     

Metal binding of metallothionein-3 versus metallothionein-2: lower affinity and higher plasticity

Mammalian metallothioneins (MTs) are involved in cellular metabolism of zinc and copper and in cytoprotection against toxic metals and reactive oxygen species. MT-3 plays a specific role in the brain and is down-regulated in Alzheimer's disease. To evaluate differences in metal binding, we conducted direct metal competition experiments with MT-3 and MT-2 using electrospray ionization mass spectroscopy (ESI-MS). Results demonstrate that MT-3 binds Zn2+ and Cd2+ ions more weakly than MT-2 but exposes higher metal-binding capacity and plasticity. Titration with Cd2+ ions demonstrates that metal-binding affinities of individual clusters of MT-2 and MT-3 are decreasing in the following order: four-metal cluster of MT-2>three-metal cluster of MT-2 approximately four-metal cluster of MT-3>three-metal cluster of MT-3>extra metal-binding sites of MT-3. To evaluate the reasons for weaker metal-binding affinity of MT-3 and the enhanced resistance of MT-3 towards proteolysis under zinc-depleted cellular conditions, we studied the secondary structures of apo-MT-3 and apo-MT-2 by CD spectroscopy. Results showed that apo-MT-3 and apo-MT-2 have almost equal helical content (approximately 10%) in aqueous buffer, but that MT-3 had slightly higher tendency to form alpha-helical secondary structure in TFE-water mixtures. Secondary structure predictions also indicated some differences between MT-3 and MT-2, by predicting random coil for common MTs, but 22% alpha-helical structure for MT-3. Combined, all results highlight further differences between MT-3 and common MTs, which may be related with their functional specificities.     

金属硫蛋白-3研究进展

金属硫蛋白－3（MT－3）是近年来发现的一种脑特异性金属硫蛋白。本文综述了其在分布、蛋白结构及基因调控方面的特点;着重介绍了其在中枢神经系统中的功能及结构与功能关系的研究进展;总结了国外对其与老年痴呆症的可能关系的研究。     

Mitochondrial tRNA gene clusters in Aspergillus nidulans: Organization and nucleotide sequence

The organization of tRNA genes on the circular 32 kb mitochondrial genome of the ascomycete Aspergillus nidulans has been studied by gel transfer hybridization and by DNA sequencing. Most of the tRNA genes are tightly clustered within two regions (1 kb each) flanking the split gene for the large ribosomal subunit RNA. The upstream cluster contains nine genes, the downstream cluster eleven genes. The twenty tRNA genes are on the same strand as the two rRNA genes and are separated from each other by AT-rich spacer sequences, usually consisting of only a few nucleotides. Two tRNA genes (leul and ala) are joined end to end. The occurrence of two tRNA^Gty genes is the first exception to the observation that in mitochondria all four-codon families are read by a single tRNA. Both genes are adjacent and show extensive sequence homology, suggesting relatively recent origin by gene duplication. The product of glyl has a U in the wobble position as do all other tRNA gene products specific for four-codon families, whereas the gly2 product, which has a rare A in the same position, should read only the codon GGU. The products of metl and thr have an A and G in positions 18 and 55, respectively, like the mitochondrial tRNA^fMet and tRNA^Thr of Neurospora crassa. Other unusual features are the replacement of the invariant G-C pair at positions 53 and 61 by A-T in met2, glyl and gly2, the replacement of the invariant T at position 8 by A in phe and G in pro and the deletion of a nucleotide at position 9 in ser2.     

A distinct Cu4-thiolate cluster of human metallothionein-3 is located in the N-terminal domain

Metallothionein-3 (MT-3), also known as neuronal growth inhibitory factor, is a metalloprotein expressed almost exclusively in the brain. Isolated MT-3 contains four Cu(I) and three Zn(II) ions organized in homometallic metal-thiolate clusters located in two independent protein domains. In this work a Cu(I) binding to metal-free MT-3 has been studied, aiming at the better understanding of the domain specificity for this metal ion. The cluster formation was followed by electronic absorption, circular dichroism, and by luminescence spectroscopy at room temperature and 77 K. The stepwise incorporation of Cu(I) into recombinant human apo-MT-3 revealed the cooperative formation of two Cu(4)S(9) clusters in succession, formed in both protein domains, i.e. Cu(4)- and Cu(8)-MT-3. Further binding of four Cu(I) caused an expansion of these Cu(I) cores, leading to fully metal-loaded Cu(12)-MT-3 containing Cu(6)S(9) and Cu(6)S(11) clusters in the beta- and alpha-domains of the protein, respectively. The location of the preferentially formed Cu(4) cluster in the protein was established by immunochemistry. Using domain-specific antibodies, in combination with limited tryptic digestion of a partially metal-occupied Cu(4)-MT-3, we could demonstrate that the Cu(4)S(9) cluster is located in the N-terminal beta-domain of the protein that contains a total of nine cysteine ligands.     

Organization of acetylcholine receptor clusters in cultured rat myotubes is calcium dependent

The effect of extracellular Ca2+ concentration and myasthenic globulin on the distribution and appearance of acetylcholine receptor (AChR) clusters on rat myotubes was studied with tetramethyl-rhodamine-labeled alpha BTX. Low Ca2+ medium (2.5 X 10(-5) M) caused a time-dependent loss of AChR clusters, and a concomitant increase in small punctate areas of fluorescence. High Ca2+ concentrations (1.5 X 10(-2) M) increased the size of AChR clusters without altering AChR synthesis. These changes were not observed with other divalent ions. In the presence of myasthenic globulin, the rate of AChR turnover increases, and AChR clusters are rapidly dispersed. High Ca2+ concentration partially protects the AChR clusters from dispersal and decreases the rate of receptor turnover.     

Organization of Xenopus histone gene variants within clusters and their transcriptional expression

Using previously cloned Xenopus nucleosomal core histone genes as hybridization probes, a genomic DNA library of Xenopus laevis was screened for histone gene clusters. From over 200 histone-gene containing clones identified, 36 were selected as possibly containing H1 histone genes by hybridization to a probe derived from a sea urchin H1 histone gene. These 36 clones were further analyzed by hybrid-selected translation for the definitive presence of H1 histone genes. The genes for three different H1 histone variants were found: H1A , H1B and H1C . Mapping of the histone genes within each clone showed that at least three different gene arrangements can occur within a cluster and that the type of H1 histone variant present in a cluster may be related to the cluster type. S1-mapping experiments indicated that histone genes found in different cluster-types can be expressed in oocytes. Also, the H1 gene found in one cluster-type was expressed in at least three different cell-types: oocytes, gastrula-stage embryos, and erythroblasts.     

IscU as a scaffold for iron-sulfur cluster biosynthesis: sequential assembly of [2Fe-2S] and [4Fe-4S] clusters in IscU

Iron-sulfur cluster biosynthesis in both prokaryotic and eukaryotic cells is known to be mediated by two highly conserved proteins, termed IscS and IscU in prokaryotes. The homodimeric IscS protein has been shown to be a cysteine desulfurase that catalyzes the reductive conversion of cysteine to alanine and sulfide. In this work, the time course of IscS-mediated Fe-S cluster assembly in IscU was monitored via anaerobic anion exchange chromatography. The nature and properties of the clusters assembled in discrete fractions were assessed via analytical studies together with absorption, resonance Raman, and M?ssbauer investigations. The results show sequential cluster assembly with the initial IscU product containing one [2Fe-2S](2+) cluster per dimer converting first to a form containing two [2Fe-2S](2+) clusters per dimer and finally to a form that contains one [4Fe-4S](2+) cluster per dimer. Both the [2Fe-2S](2+) and [4Fe-4S](2+) clusters in IscU are reductively labile and are degraded within minutes upon being exposed to air. On the basis of sequence considerations and spectroscopic studies, the [2Fe-2S](2+) clusters in IscU are shown to have incomplete cysteinyl ligation. In addition, the resonance Raman spectrum of the [4Fe-4S](2+) cluster in IscU is best interpreted in terms of noncysteinyl ligation at a unique Fe site. The ability to assemble both [2Fe-2S](2+) and [4Fe-4S](2+) clusters in IscU supports the proposal that this ubiquitous protein provides a scaffold for IscS-mediated assembly of clusters that are subsequently used for maturation of apo Fe-S proteins.     

Reaction of human metallothionein-3 with cisplatin and transplatin

Human metallothioneins, small cysteine- and metal-rich proteins, play an important role in the acquired resistance to platinum-based anticancer drugs. These proteins contain a M(II)₄(CysS)₁₁ cluster and a M(II)₃(CysS)₉ cluster localized in the α-domain and the β-domain, respectively. The noninducible isoform metallothionein-3 (Zn₇MT-3) is mainly expressed in the brain, but was found overexpressed in a number of cancer tissues. Since the structural properties of this isoform substantially differ from those of the ubiquitously occurring Zn₇MT-1/Zn₇MT-2 isoforms, the reactions of cis-diamminedichloridoplatinum(II) (cisplatin) and trans-diamminedichloridoplatinum(II) (transplatin) with human Zn₇MT-3 were investigated and the products characterized. A comparison of the reaction kinetics revealed that transplatin reacts with cysteine ligands of Zn₇MT-3 faster than cisplatin. In both binding processes, stoichiometric amounts of Zn(II) were released from the protein. Marked differences between the reaction rates of cisplatin and transplatin binding to Zn₇MT-3 and the formation of the Pt–S bonds suggest that the binding of both Pt(II) compounds is a complex process, involving at least two subsequent binding steps. The electrospray ionization mass spectrometry characterization of the products showed that whereas all ligands in cisplatin were replaced by cysteine thiolates, transplatin retained its carrier ammine ligands. The ¹¹³Cd NMR studies of Pt₁ ¹¹³Cd₆MT-3 revealed that cisplatin binds preferentially to the β-domain of the protein. The rates of reaction of cisplatin and transplatin with Zn₇MT-3 were much faster than those of cisplatin and transplatin with Zn₇MT-2. The biological consequences of a substantially higher reactivity of cisplatin toward Zn₇MT-3 than Zn₇MT-2 in the acquired resistance to platinum-based drugs are discussed.     

Organization and developmental timing of the Bombyx mori chorion gene clusters in strain C108

We have standardized the map of chorion structural gene clusters in Bombyx mori strain C108 by analyzing quantitative and qualitative chorion electrophoretic markers in recombinant progeny from four independent crosses. In all we assigned 22 markers to three gene clusters, representing about one-third of the total number of chorion genes: 2 to Ch 1, 9 to Ch 2, and 8 to Ch 3. Three additional markers belong either to Ch 7 or Ch 2. By referring to published chorion protein synthesis patterns, we show that the clusters are restricted in their developmental specificities: Ch 3 appears to be an early locus, carrying all of the mapped early markers (4) and half of the early middles (3/6), while Ch 1 and Ch 2 carry predominantly middle (4/5) and all late, Hc (6) markers, along with some early middle markers (3). We cite evidence to show that Ch 1 and Ch 2 compose the left and right halves of a single gene cluster, which we formally designate as Ch 1–2.