Organization and assembly of metal-thiolate clusters in epithelium-specific metallothionein-4

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 Cd2+ and Co2+ ions. Both the Cd-113 NMR studies of (Cd7MT)-Cd-113-4 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 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 Cd7MT-4 is cooperative and sequential, with the Cd4Cys11 cluster being formed first, and that a distinct single-metal nucleation intermediate Cd1MT-4 is required in the cluster formation process. Conversely, the absorption and circular dichroism features of metal-thiolate clusters in Cd7MT-4 indicate that marked differences in the cluster geometry exist when compared with those in Cd7MT-1/2. The biological implication of our studies as to the role of MT-4 in zinc metabolism of stratified epithelia is discussed.