Jeremias H. R. Kägi

Three-dimensional structure of rabbit liver [Cd7]metallothionein-2a in aqueous solution determined by nuclear magnetic resonance

In previous work the metal-polypeptide co-ordinative bonds in the major protein species of a reconstituted [113Cd7]metallothionein-2 preparation from rabbit liver in aqueous solution were determined, the secondary polypeptide structure was found to contain several half-turns and 3(10)-helical segments, and a preliminary characterization of the overall polypeptide backbone fold in the beta-domain containing the three-metal cluster, and the alpha-domain containing the four-metal cluster, was obtained. Using a new, more extensive set of nuclear magnetic resonance data these earlier structures were improved by new structure calculations. The new experimental data consist of distance constraints from measurements of nuclear Overhauser effects, and dihedral angle constraints derived from both coupling constants and nuclear Overhauser effects. The structure calculations were performed with the program DISMAN. Since no information on the orientation of the two domains relative to each other could be obtained, the structure calculations were performed separately for the alpha-domain and the beta-domain. The average of the pairwise root-mean-square distances among the 20 structures with the least residual violations of input constraints was 2.9 A for the beta-domain and 1.4 A for the alpha-domain (1 A = 0.1 nm). The overall chirality of the polypeptide fold is right-handed for the beta-domain and left-handed for the alpha-domain. For each of the seven metal ions the local chirality of the co-ordination of the four cysteinyl Sy atoms is clearly defined. The improved structures of both domains show the previously noted differences relative to the recently published crystal structure of metallothionein-2a from rat liver.

Thionein (apometallothionein) can modulate DNA-binding and transcription activation by zinc finger containing factor-Spl

A number of transcription factors contain so‐called zinc finger domains for the interaction with their cognate DNA sequence. It has been shown that removal of the zinc ions complexed in these zinc fingers abrogates DNA binding and transcription activation. Therefore we wanted to test the hypothesis that the activity of transcription factors could be regulated by physiological chelators of zinc. A prominent candidate for such a chelator is the Cys‐rich protein thionein (apometallothionein) that is inducible by heavy metal loads, and by other environmental stimuli. Here we show with DNA binding and in vitro transcriptions assays that thionein indeed can inactive the zinc finger‐containing Spl in a reversible manner. By contrast, transcription factor Oct‐l, which binds DNA via a homeo‐domain, i.e. a helix‐turn‐helix motif not involving zinc ions, is refractory to thionein action. We propose that modulation of intracellular thionein concentration is used for the coordinated regulation of a large subset of genes whose transcription depends on zinc finger proteins.

Human hepatic metallothioneins.

Metallothioneins are sulfur-rich proteins first recognized in equine and human renal cortex by their unusual content of cadmium [ 1,2] . All preparations contained in addition, however, equally appreciable amounts of zinc and the variations in the relative amounts of the two metals suggested even in the earliest studies that they may replace each other. A naturally occurring only zinc-containing form of metallothionein was recently identified in equine liver and recognized as one of the major zinc proteins of this organ [3,4] . In the present study we report the isolation from human liver and characterization of two similar, only zinc-containing forms of metallothionein.

Conformation of [Cd7]-metallothionein-2 from rat liver in aqueous solution determined by nuclear magnetic resonance spectroscopy.

The three-dimensional structure of [Cd7]-metallothionein-2 from rat liver was determined in aqueous solution, using nuclear magnetic resonance spectrometry and distance geometry calculations. The experimental data provided proton-proton distance constraints from measurements of nuclear Overhauser effects, constraints on the geometry of the metal-cysteine clusters determined by heteronuclear correlation spectroscopy, and dihedral angle constraints derived from both coupling constants and nuclear Overhauser effects. The structure calculations were performed with the program DISMAN. As in previous studies with rabbit liver metallothionein-2a, the structure calculations were performed separately for the alpha and beta-domains containing the 4 and 3-metal clusters, respectively, since no interdomain constraints were found. For both domains, the global polypeptide fold, the location of polypeptide secondary structure elements, the architecture of the metal-sulfur cluster and the local chirality of the metal co-ordination are very similar to the solution structure of rabbit metallothionein-2a, but show considerable difference relative to the crystal structure of rat metallothionein-2.

Metallothionein: Molecular evolution and classification

The nomenclature system for metallothionein was first adopted in 1978 [1] and extended in 1985 by introducing a subdivision of MTs into three classes [2]. According to this convention class I includes all proteinaceous MTs with locations of Cys closely related to those in the mammalian forms. Class II comprises proteinaceous MTs which lack this property. Class III subsumes metallopolyisopeptides containing gammaglutamyl-cysteinyl units resembling in their features proteinaceous MTs.

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.

Three-dimensional structure of human [113Cd7]metallothionein-2 in solution determined by nuclear magnetic resonance spectroscopy.

The three-dimensional structure of human [113Cd7]metallothionein-2 was determined by nuclear magnetic resonance spectroscopy in solution. Sequence-specific 1H resonance assignments were obtained using the sequential assignment method. The input for the structure calculations consisted of the metal-cysteine co-ordinative bonds identified with heteronuclear correlation spectroscopy, 1H-1H distance constraints from nuclear Overhauser enhancement spectroscopy, and spin-spin coupling constants 3JHN alpha and 3J alpha beta. The molecule consists of two domains, the beta-domain including amino acid residues 1 to 30 and three metal ions, and the alpha-domain including residues 31 to 61 and four metal ions. The nuclear magnetic resonance data present no evidence for a preferred relative orientation of the two domains. The polypeptide-to-metal co-ordinative bonds in human metallothionein-2 are identical to those in the previously determined solution structures of rat metallothionein-2 and rabbit metallothionein-2a, and the polypeptide conformations in the three proteins are also closely similar.

Primary structure of human hepatic metallothionein.

Metallothioneins constitute a family of low molecular weight proteins (6000-7000) of unusually high metal and cysteine content which occur in parenchymatous tissues of vertebrates and which are most abundant in liver and kidney [l] . Depending on the source, they contain variable amounts of Zn, Cd and Cu which add up to a stoichiometric ratio of 6 or 7 g-atom metal/m01 [2] . In experimental animals the formation of metallothionein can be induced by the administration of a variety of metals (Zn, Cd, Cu, Hg, Ag) and, hence, they have been suggested to play a role in metal detoxication [3]. However, recent studies have shown that the naturally occurring metallothioneins of human liver contain zinc as the only significant metallic component thus suggesting that their primary biological function is related to the metabolism of this essential element [4] . All metallothioneins examined thus far are single chain proteins of about 60 amino acid residues [5] . Besides their high cysteine content they are unusual by a total lack of aromatic amino acids and histidine (see table 1). In general, each metal ion is bound by three cysteinyl residues forming a negatively-charged trimercaptide complex [l] . In this paper we report the first amino acid sequence of a human zinc metallothionein and compare it to the known primary structure of equine metallothionein (MT-1 B) [ 11.

Comparison of the solution conformations of human [Zn7]-metallothionein-2 and [Cd7]-metallothionein-2 using nuclear magnetic resonance spectroscopy

The solution structure of native human [Zn7]-metallothionein-2 has been compared with the previously determined structure of human [Cd7]-metallothionein-2. The comparison was based on complete sequence-specific 1H nuclear magnetic resonance assignments for human [Zn7]-metallothionein-2 obtained using the sequential assignment method. The secondary structure was found to be very similar in the [Zn7]- and [Cd7]- forms of the protein. Only seven amide protons in [Zn7]- metallothionein-2 were found to have exchange rates lower than approximately 0.2 min-1 at pH 7.0 and 10 degrees C, which corresponds closely to the results of amide proton exchange studies with the [Cd7]- form of the protein. Finally, the 1H-1H distance constraints determined from nuclear Overhauser enhancement spectroscopy for human [Zn7]-metallothionein-2 were checked for compatibility with the [Cd7]-metallothionein-2 structure. Overall, although no direct method is available for identifying the metal-polypeptide co-ordinative bonds in the Zn(2+)-containing protein, these measurements provided several independent lines of evidence showing that the [Zn7]- and [Cd7]- forms of human metallothionein-2 have the same molecular architecture.

Modulation of DNA Binding of a Tramtrack Zinc Finger Peptide by the Metallothionein-Thionein Conjugate Pair

The ability of metallothionein (MT) to modulate DNA binding by a two-finger peptide of Tramtrack (TTK), a CCHH zinc transcription factor, was investigated using metal-bound and metal-deficient forms of rabbit MT-2 and the TTK peptide. Thionein inhibited DNA binding by zinc-bound TTK, and Zn-MT restored DNA-binding by zinc-deficient apo-TTK. “Free” zinc at low concentrations was as effective as Zn-MT in restoring DNA binding by apopeptide but was inhibitory at concentrations equal to zinc bound to 2 mol eq and higher of Zn-MT. Substitution of cadmium for zinc reduced the affinity of the peptide for its DNA binding site. This effect was reversed by incubation with Zn-MT. The circular dichroic spectra of the TTK peptide indicated that zinc removal resulted in loss of α-helical structures, which are sites of DNA contact points. Reconstitution with cadmium resulted in stoichiometric substitution of 2 mol of Cd/mol of peptide but not recovery of α-helical structures. Incubation of Cd-TTK with Zn-MT restored the secondary structure expected for zinc-bound TTK. The ability of Zn-MT and thionein to restore or inhibit DNA-binding by TTK was associated with effects on the metallation status of the peptide and related alterations in its secondary structure.