A distinct Cu(4)-thiolate cluster of human metallothionein-3 is located in the N-terminal domain.
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 Cu(4)S(9) clusters in succession, formed in both protein domains, i.e. Cu(4)- and Cu(8)-MT-3. Further binding of four Cu(I) caused an expansion of these Cu(I) cores, leading to fully metal-loaded Cu(12)-MT-3 containing Cu(6)S(9) and Cu(6)S(11) clusters in the beta- and alpha-domains of the protein, respectively. The location of the preferentially formed Cu(4) cluster in the protein was established by immunochemistry. Using domain-specific antibodies, in combination with limited tryptic digestion of a partially metal-occupied Cu(4)-MT-3, we could demonstrate that the Cu(4)S(9) cluster is located in the N-terminal beta-domain of the protein that contains a total of nine cysteine ligands.
Roschitzki
Bernd
B
Institute of Biochemistry, University of Zürich, Winterthurerstrasse 190, Switzerland.
Vasák
Milan
M
D000918:Antibody Specificity; D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008163:Luminescent Measurements; D008668:Metallothionein / Q000737:chemistry / Q000378:metabolism*; D000090023:Metallothionein 3; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D013054:Spectrophotometry, Atomic; D013379:Substrate Specificity; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
D000090023:Metallothionein 3; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C007121:copper thionein; D003300:Copper; D008668:Metallothionein; D015032:Zinc