A distinct Cu₄-thiolate cluster of human metallothionein-3 is located in the N-terminal domain

Abstract.

Metallothionein-3 (MT-3), also known as neuronal growth inhibitory factor, is a metalloprotein expressed almost exclusively in the brain. Isolated MT-3 contains four Cu(I) and three Zn(II) ions organized in homometallic metal-thiolate clusters located in two independent protein domains. In this work a Cu(I) binding to metal-free MT-3 has been studied, aiming at the better understanding of the domain specificity for this metal ion. The cluster formation was followed by electronic absorption, circular dichroism, and by luminescence spectroscopy at room temperature and 77 K. The stepwise incorporation of Cu(I) into recombinant human apo-MT-3 revealed the cooperative formation of two Cu₄S₉ clusters in succession, formed in both protein domains, i.e. Cu₄- and Cu₈-MT-3. Further binding of four Cu(I) caused an expansion of these Cu(I) cores, leading to fully metal-loaded Cu₁₂-MT-3 containing Cu₆S₉ and Cu₆S₁₁ clusters in the β- and α-domains of the protein, respectively. The location of the preferentially formed Cu₄ cluster in the protein was established by immunochemistry. Using domain-specific antibodies, in combination with limited tryptic digestion of a partially metal-occupied Cu₄-MT-3, we could demonstrate that the Cu₄S₉ cluster is located in the N-terminal β-domain of the protein that contains a total of nine cysteine ligands. Electronic supplementary material to this paper, comprising Table S1 (amino acid sequences of peptides used in immunization) and Fig. S1 (luminescence spectra of Cu(I) titrated apo-MT-3), can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-002-0339-1.