Organization and Assembly of Metal-Thiolate Clusters in Epithelium-specific Metallothionein-4

Abstract

Mammalian metallothionein-4 (MT-4) was found to be specifically expressed in stratified squamous epithelia where it plays an essential but poorly defined role in regulating zinc or copper metabolism. Here we report on the organization, stability, and the pathway of metal-thiolate cluster assembly in MT-4 reconstituted with Cd 2؉ and Co 2؉ ions. Both the 113 Cd NMR studies of 113 Cd 7 MT-4 and the spectroscopic characterization of Co 7 MT-4 showed that, similar to the classical MT-1 and MT-2 proteins, metal ions are organized in two independent Cd 4 Cys 11 and Cd 3 Cys 9 clusters with each metal ion tetrahedrally coordinated by terminal and bridging cysteine ligands. Moreover, we have demonstrated that the cluster formation in Cd 7 MT-4 is cooperative and sequential, with the Cd 4 Cys 11 cluster being formed first, and that a distinct single-metal nucleation intermediate Cd 1 MT-4 is required in the cluster formation process. Conversely, the absorption and circular dichroism features of metal-thiolate clusters in Cd 7 MT-4 indicate that marked differences in the cluster geometry exist when compared with those in Cd 7 MT-1/2. The biological implication of our studies as to the role of MT-4 in zinc metabolism of stratified epithelia is discussed. Metallothioneins (MTs)2 is a superfamily of low molecular mass cysteine-and metal-rich proteins or polypeptides conserved through evolution and present in all eukaryotes and certain prokaryotes (reviewed in Refs. 1 and 2). In mammals, the MT gene family consists of four subfamilies designated MT-1 through MT-4. Whereas in mouse only one member of each subfamily is present ( Fig. 1), in primates a significant genetic polymorphism exists with 17 genes and pseudogenes in humans. From these 17 genes, 10 are functional, including single MT-3 and MT-4 genes (3). Mammalian MTs are composed of a single polypeptide chain of 61-68 amino acids with a conserved array of 20 cysteines and no aromatic residues or histidine. In the structurally characterized MTs (MT-1-MT-3), all cysteines are present in reduced form and are involved in the binding of seven divalent (Zn 2ϩ , Cd 2ϩ ) and up to 12 monovalent metal ions (Cu ϩ ) forming two metal-thiolate clusters located in two independent protein domains (4 -6). Although still under debate, suggested functions for mammalian MTs include homeostasis and transport of physiologically essential metals (zinc, copper), detoxification of toxic metals (cadmium, mercury), protection against oxidative stress, regulation of cell proliferation and apoptosis, and the maintenance of intracellular redox balance (7-10). Differential expression of mammalian MT isoforms is tightly regulated during development and in pathological situations (9,11). The extensively studied mammalian MT-1 and MT-2 show ubiquitous expression regulated at the transcriptional level (12). Their biosynthesis is inducible by a variety of compounds and stress conditions, such as metals, glucocorticoids, cytokines, and reactive oxygen species (9). MT-3 and MT-4 are relatively unresponsiv...

Zinc binding ligands and cellular zinc trafficking: Apo-metallothionein, glutathione, TPEN, proteomic zinc, and Zn-Sp1

“…First recognized for its singular capacity to bind intracellular Cd 2+ , attention broadened to include its interaction with other toxic metal ions, its participation in Zn 2+ and Cu metabolism, and finally the role of its manifold sulfhydryl groups in electrophile and oxidant metabolism [5,[12][13][14]. From a chemical standpoint, the properties of its unique metal ion-thiolate clusters continue to be the subject of investigation [35,36]. Its apparent multifunctionality, symbolized by the pleotropic nature of the inducers of the protein, has left the protein without a consensus of well established roles within the cell [37].…”

Crosstalk of the structural and zinc buffering properties of mammalian metallothionein-2

“…It also reflects more pronounced differentiation of Cd(II) affinities towards the b-domain than the a-domain. Such differentiation was observed for MT4 84 and is less pronounced for MT3 81 and least visible for MT1 and MT2 isoforms. 9,82,83 This shows that the relative affinities of metal ions in the b-domain vary depending on the metallothionein isoform.…”

Metallothioneins and Platinum(II) Anti-Tumor Compounds

“…Little structural information is currently available about MT-4. Spectroscopic investigations suggest that, like in MT-1/-2, two individual clusters are formed with some differences in the cluster geometry [28].…”