Distinct Metal−Thiolate Clusters in the N-Terminal Domain of Neuronal Growth Inhibitory Factor

Neuronal growth inhibitory factor (GIF), a brain-specific metallothionein-like protein (metallothionein-3), impairs the survival and neurite formation of cultured neurons. The metal distribution in isolated Cu4,Zn3-GIF is not known. In the present studies, the metal-thiolate clusters formed with monovalent and divalent metal ions in the N-terminal domain of human GIF [GIF(1-32)] were investigated. The cluster formation was followed by using electronic absorption, circular dichroism (CD), and magnetic circular dichroism (MCD), and in the case of Cu(I) complexes also by luminescence spectroscopy at 77 K. With Cu(I) ions, two well-defined clusters are formed involving the nine cysteine ligands of GIF(1-32), i.e., Cu4S9- and Cu6S9-clusters. In contrast to the Cu6S9-cluster, the Cu4S9-cluster shows a remarkable stability to air oxidation. As similar properties and spectral features have also been observed with isolated Cu4-5,Zn2-3-GIF, the presence of a Cu4-cluster in this GIF form is suggested. The studies with Zn(II), Cd(II), and Co(II) ions indicated the presence of a Me3S9-cluster in GIF(1-32). However, spectral features of these metal derivatives substantially differ from those reported for the corresponding Me3S9-cluster in the beta-domain of metallothioneins, suggesting structural differences. A large conformational flexibility of the Zn3- and Cd3-GIF(1-32) structures, characterized by short T2 proton relaxations, precluded their investigation by NMR methods. The significance of Cu- and Zn-clusters for the structure of biologically active GIF(1-32) is discussed.