Effects of Zn2+, Ca2+, and Mg2+ on the Structure of Zn7Metallothionein-3: Evidence for an Additional Zinc Binding Site

Human metallothionein-3 (Zn7MT-3), an intra- and extracellularly occurring metalloprotein, is highly expressed in the brain, where it plays an important role in the homeostasis of the essential metal ions Cu+ and Zn2+. Like other mammalian metallothioneins (MT-1 and -2), the protein contains a MII3(CysS)9 and a MII4(CysS)11 cluster localized in two independent protein domains linked by a flexible hinge region. However, there is a substantially increased number of acidic residues in MT-3 (11 residues) compared with MT-2 (four residues) which may act as binding ligands for additional metal ions. In this study, the binding of Zn2+, Ca2+, and Mg2+ to human Zn7MT-3 and its mutant lacking an acidic hexapeptide insert, Zn7MT-3Δ55−60, was investigated and compared with the binding of Zn7MT-2. By using spectroscopic and spectrometric techniques, we demonstrate that one additional Zn2+ binds with an apparent binding constant (Kapp) of ∼100 μM to Zn7MT-3 and Zn7MT-3Δ55−60, but not to Zn7MT-2. The changes in spectros...