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

Abstract

Human metallothionein-3 (Zn(7)MT-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 Zn(2+). Like other mammalian metallothioneins (MT-1 and -2), the protein contains a M(II)(3)(CysS)(9) and a M(II)(4)(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 Zn(2+), Ca(2+), and Mg(2+) to human Zn(7)MT-3 and its mutant lacking an acidic hexapeptide insert, Zn(7)MT-3(Delta55-60), was investigated and compared with the binding of Zn(7)MT-2. By using spectroscopic and spectrometric techniques, we demonstrate that one additional Zn(2+) binds with an apparent binding constant (K(app)) of approximately 100 microM to Zn(7)MT-3 and Zn(7)MT-3(Delta55-60), but not to Zn(7)MT-2. The changes in spectroscopic features of metal-thiolate clusters and gel filtration behavior reveal that the formation of Zn(8)MT-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 Zn(2+) is followed by a slow time-dependent protein dimerization. The binding of Zn(2+) to Zn(7)MT-3 is specific as in the presence of Ca(2+) and Mg(2+) only an alteration of the R(s) of Zn(7)MT-3 at substantially higher concentrations was observed. The significance of these findings for the biological role of MT-3 is discussed.

In vivo‐folded metal–metallothionein 3 complexes reveal the Cu–thionein rather than Zn–thionein character of this brain‐specific mammalian metallothionein

“…However, this domain independency would only apply to the major Zn 7 – and minor Zn 6 – and Zn 5 –complexes, if considering the speciation reached in the syntheses of the separate domains (Zn 3 –βMT3, Zn 2 –βMT3 and Zn 1 –βMT3 plus Zn 4 –αMT3). This suggests that the overmetalated complexes (Zn 8 –MT3 and Zn 9 –MT3) arise from an MT3 behavior determined by the two domains linked in the full‐length structure, which is in agreement with the incorporation of the eighth (and perhaps further) Zn 2+ ions proposed in the literature .…”

“…Hence, on the one hand, a single-residue insertion (Thr) at position 5 confers bioactivity to bMT3 through its unique Thr-Cys-Pro-Cys-Pro pattern [21], as confirmed by site-directed mutagenesis of Thr5, Pro7, and Pro9 [22], so that MT3 bioactivity appears to be totally dependent on this sequence motif [21,23]. On the other hand, aMT3 encompasses an insertion of six residues (Glu-Gly_or_Ala-Ala-Glu_or_Lys-Ala-Glu) that, after being discarded as an additional binding site, was shown to be responsible for the peculiar low stability of the aMT3 metal clusters, probably by increasing the solvent-exposed polypeptide surface [24][25][26]. The higher affinity of MT3 than of MT1 and MT2 for Cu + [27], together with the relevance of the presence of Cu in the CNS, prompted a detailed study of the MT3 Cu-binding behavior.…”

Function of Metallothionein-3 in Neuronal Cells: Do Metal Ions Alter Expression Levels of MT3?

“…He et al speculate that lead forms a trigonal pyramidal coordination when bound to MT2 with three sulfurs and electron donating oxygens (Pb–S 3 O) [57]. The known differences in metal binding preferences between MT3 and other metallothioneins, including the presence of an 8th metal binding site in MT2, is likely to effect the overall structure of metal-replete MT3 [54,61,62]. Work is ongoing to determine if these structural differences lead to functional differences in the way that MT3 interacts with partner proteins [9].…”

“…Mt3 was isolated via HiLoad 26/600 75 pg size exclusion column. Mt3 concentration was determined by measuring the absorbance of the samples in 1 mM HCl at 220 nm and calculating using an extinction coefficient of 53,000 M −1 ·cm −1 [62]. The presence of Mt3 was confirmed by SDS-PAGE on a 15% polyacrylamide gel, either with silver or Coomassie staining and florescence imaging on bromobimane-modified MT3 samples [72].…”

The Functions of Metamorphic Metallothioneins in Zinc and Copper Metabolism

“…It is important to realize that these structures are based on either a species induced in vivo with Cd 2+ [9], which remarkably has two Cd 2+ ions in the N-terminal β-domain at defined positions, or a species where all the metal ions had been removed, all the cysteines reduced, and the protein was reconstituted with seven Zn 2+ or Cd 2+ ions. In vitro, MTs can bind more than seven equivalents of metal ions [10,11]. Thus, for structural studies the proteins were brought into chemically defined “homogeneous” forms—a requirement for most biophysical methods to obtain molecular structure—whereas the inherent heterogeneity of the protein in vivo provides a clue to its functions.…”