Zinc transfer potentials of the α- and β-clusters of metallothionein are affected by domain interactions in the whole molecule

Abstract

The ␣-and ␤-polypeptides of human metallothionein (isoform 2), obtained by chemical synthesis, were converted into their respective zinc͞thiolate clusters, and each domain was investigated separately. Proton titration data for the N-terminal ␤-domain fit a simple model with three ionizations of the same apparent pK a value of 4.9 and a collective binding constant for zinc of 5 ؋ 10 ؊12 M at pH 7.0. The zinc cluster in the C-terminal ␣-domain is more stable than that in the ␤-domain. Its pH titration is also more complex, indicating at least two classes of zinc sites with different affinities. The whole molecule is stabilized with regard to the individual domains. Chemical modification implicates lysine side chains in both the stabilization of the ␤-domain cluster and the mutual stabilization of the domains in the whole molecule. The two zinc clusters also differ in the reactivity of their cysteine sulfurs and their potential to donate zinc to an acceptor molecule dependent on its type and characteristics. The isolated ␤-domain cluster reacts faster with Ellman's reagent and is a better zinc donor toward zinc-depleted sorbitol dehydrogenase than is the isolated ␣-domain cluster, whereas the reverse is observed when a chelating agent is the zinc acceptor. Thus, although each cluster assembles independently of the other, the cumulative properties of the individual domains do not suffice to describe metallothionein either structurally or functionally. The two-domain structure of the whole molecule is important for its interaction with ligands and for control of its reactivity and overall conformation.

Zinc efflux through lysosomal exocytosis prevents zinc-induced toxicity

“…The degree of pH change necessary to cause an effect on Zn 2+ handling, by contrast, significantly exceeds that reported to be caused by Baf. A pH drop below 6.7 is required to trigger an increase in intracellular Zn 2+ according to one set of studies (Kiedrowski, 2012), whereas another set has shown that metallothioneins release Zn 2+ only after cytoplasmic pH drops below 5.0 (Jiang et al, 2000). Thus, the increase in cytoplasmic Zn 2+ caused by Baf likely correlates with the loss of lysosomal function, rather than cytosolic pH changes.…”

Structural features specific to plant metallothioneins

“…Differences in properties however are apparent when comparing the full-length human MT2 protein with its two separate domains in vitro. The fully metal ion loaded full-length protein was found to be more stable towards oxidation and Zn II transfer [52], and remetallation studies with the apo-forms and As III revealed the faster metallation of the full-length form [53]. In addition, the apparent pK a values of the Cys residues in the Zn II -and the Cd II -forms, which are indicative for the metal ion affinities of the proteins, are lower for the full-length human MT2 compared to the average values obtained from separate measurements with the individual domains [52].…”

“…The fully metal ion loaded full-length protein was found to be more stable towards oxidation and Zn II transfer [52], and remetallation studies with the apo-forms and As III revealed the faster metallation of the full-length form [53]. In addition, the apparent pK a values of the Cys residues in the Zn II -and the Cd II -forms, which are indicative for the metal ion affinities of the proteins, are lower for the full-length human MT2 compared to the average values obtained from separate measurements with the individual domains [52]. In summary, while the influence of the length of the linker region is less clear as variations of the amino acid composition might have a difficult to predict influence, it seems obvious that the physical connection of both domains plays a crucial role for the stability of the protein.…”

Insights into Zn2+ homeostasis in neurons from experimental and modeling studies

“…Unfortunately, the binding and release kinetics of Zn 2ϩ with MT/T are still poorly understood, which, in turn, limits our understanding of its role as a cytosolic Zn 2ϩ buffer and/or chaperone. Studies of binding and release kinetics of the MT-2 isoform have suggested that the two domains differ significantly in Zn 2ϩ affinity (33,39). Two different Zn 2ϩ transporter gene families [solutelinked carrier (SLC) 39 and SLC30] have been identified; each family probably has a unique transport mechanism, function, and cellular location.…”