We report here the crystal structure of yeast copper thionein (Cu-MT), determined at 1.44-Å resolution. The Cu-MT structure shows the largest known oligonuclear Cu(I) thiolate cluster in biology, consisting of six trigonally and two digonally coordinated Cu(I) ions. This is at variance with the results from previous spectroscopic determinations, which were performed on MT samples containing seven rather than eight metal ions. The protein backbone has a random coil structure with the loops enfolding the copper cluster, which is located in a cleft where it is bound to 10 cysteine residues. The protein structure is somewhat different from that of Ag7-MT and similar, but not identical, to that of Cu7-MT. Besides the different structure of the metal cluster, the main differences lie in the cysteine topology and in the conformation of some portions of the backbone. The present structure suggests that Cu-MT, in addition to its role as a safe depository for copper ions in the cell, may play an active role in the delivery of copper to metal-free chaperones.copper metabolism ͉ metallothionein ͉ Saccharomyces cerevisiae ͉ x-ray structure

The first member of the metallothionein (MT) family was isolated in 1957 [1]. Since then, a large number of proteins have been described featuring common characteristics. They include ubiquitous small cysteine-rich proteins (50-70 amino acids) that are able to bind many d 10 metal ions [2]. A wealth of different biological functions has been proposed and continues to be discovered. Obviously, MTs play important roles in minimizing the uncontrolled reactions of heavy metal ions like cadmium and the homeostasis of essential metal ions including copper(I) and zinc(II) ions [2,3]. They are known to successfully cope with oxidative stress and ionizing radiation [4,5]. Other functions may be associated with the occurrence of tissue-specific isoforms, such as the brain-specific isoform MT-3, which acts as neuronal growth inhibitory factor [6,7].Both mammalian MT-1 and MT-2 are composed of the N-terminal b-and the C-terminal a-domain. They are predominantly isolated containing zinc or cadmium exclusively bound to cysteine thiolates. The nine cysteine residues of the b-domain accommodate a metal (M)(II) 3 S 9 cluster, while 11 cysteine residues contribute to the formation of a M(II) 4 Vertebrate metallothioneins are found to contain Zn(II) and variable amounts of Cu(I), in vivo, and are believed to be important for d 10 -metal control. To date, structural information is available for the Zn(II) and Cd(II) forms, but not for the Cu(I) or mixed metal forms. Cu(I) binding to metallothionein-1 has been investigated by circular dichroism, luminescence and 1 H NMR using two synthetic fragments representing the a-and the b-domain. The 1 H NMR data and thus the structures of Zn 4 a metallothionein (MT)-1 and Zn 3 bMT-1 were essentially the same as those already published for the corresponding domains of native Cd 7 MT-1. Cu(I) titration of the Zn(II)-reconstituted domains provided clear evidence of stable polypeptide folds of the three Cu(I)-containing a-and the four Cu(I)-containing b-domains. The solution structures of these two species are grossly different from the structures of the starting Zn(II) complexes. Further addition of Cu(I) to the two single domains led to the loss of defined domain structures. Upon mixing of the separately prepared aqueous three and four Cu(I) loaded a-and b-domains, no interaction was seen between the two species. There was neither any indication for a net transfer of Cu(I) between the two domains nor for the formation of one large single Cu(I) cluster involving both domains.Abbreviations M, metal; MT, metallothionein.

Kidney contains glycogen. Glycogen is degraded by glycogen phosphorylase (GP). This enzyme comes in three isoforms, one of which, the brain isozyme (GP BB), is known to occur in kidney. Its pattern of distribution in rat kidney was studied in comparison to that of the muscle isoform (GP MM) with the aim to see if for GP BB and GP MM there were functional similarities in brain and kidney. In immunoblotting and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) experiments, both isozymes and their respective mRNAs were found in kidney homogenates. GP BB was immunocytochemically detected in collecting ducts which were identified by the marker protein aquaporin-2. GP MM was localized exclusively in interstitial cells of cortex and outer medulla. These cells were identified as fibroblasts by their expression of 5'-ectonucleotidase (cortex) or by their morphology (outer medulla). The physiological role of both isozymes is discussed in respect to local demands of energy and of proteoglycan building blocks.