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

Kairit Zovo,Jekaterina Kazantseva,Peep Palumaa,Milan Vašák,Gabriele Meloni

doi:10.1074/jbc.m601724200

Kairit Zovo, Jekaterina Kazantseva

Open Access

https://doi.org/10.1074/jbc.m601724200

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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.

Highlights

Maintenance of intracellular redox balance [7,8,9,10]
The presented studies were conducted with the aim of gaining insight into the metal binding properties and the structural features of MT-4, which are likely responsible for its function in zinc metabolism
Considering the results obtained in this investigation on Cd2ϩ and Co2ϩ binding to MT-4, it can be concluded that this protein possesses divalent metal binding properties similar to the ubiquitously expressed MT-1/2 isoforms

Summary

Introduction

Maintenance of intracellular redox balance [7,8,9,10]. Differential expression of mammalian MT isoforms is tightly regulated during development and in pathological situations [9,11]. Both the 113Cd NMR studies of 113Cd7MT-4 and the spectroscopic characterization of Co7MT-4 showed that, similar to the classical MT-1 and MT-2 proteins, metal ions are organized in two independent Cd4Cys11 and Cd3Cys9 clusters with each metal ion tetrahedrally coordinated by terminal and bridging cysteine ligands.

Results

Conclusion