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 M-3(II)(CysS)(9) and a M-4(II)(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-3(Delta 55-60), 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 (K-app) of similar to 100 mu M to Zn7MT-3 and Zn7MT-3(Delta 55-60), but not to Zn7MT-2. The changes in spectroscopic features of metal-thiolate clusters and gel filtration behavior reveal that the formation of Zn8MT-3 is immediate and is accompanied by a decrease in the Stokes radius (R-s). The changes in the R-s suggest a mutual approach of both protein domains. The fast binding of Zn2+ is followed by a slow time-dependent protein dimerization. The binding of Zn2+ to Zn7MT-3 is specific as in the presence of Ca2+ and Mg2+ only an alteration of the R-s of Zn7MT-3 at substantially higher concentrations was observed. The significance of these findings for the biological role of MT-3 is discussed.