Challenging conventional wisdom: single domain metallothioneins

Metallothioneins (MT) are a family of small cysteine rich proteins that have been implicated in a range of roles including toxic metal detoxification, protection against oxidative stress, and as metallochaperones are undoubtedly involved in the homeostasis of both essential zinc and copper. While complete details of all possible cellular functions are still unknown, it is clear that they must be directly related to both the accessibility and the metal-binding properties of the many cysteine residues in the protein. The most well studied MTs are of mammalian origin and consist of two domains: a β-domain with 9 cysteine residues that sequesters 3 Cd(2+), 3 Zn(2+) or 6 Cu(+) ions, and an α-domain with 11 cysteine residues that sequesters 4 Cd(2+), 4 Zn(2+) or 6 Cu(+) ions. The key to understanding the cellular importance of MT in these different roles is in a precise description of the metallation status before and during reactions. An assessment of all possible and all biologically accessible metallation states is necessary before the functional mechanistic details can be fully determined. Conventionally, it has been considered that metal ions bind in a domain-specific and, therefore, cooperative manner, where the apparently isolated domains fill with their full complement of metal ions immediately with no discernible or measurable intermediates. A number of detailed mechanistic studies of the metallation reactions of mammalian MTs have provided significant insight into the metallation reactions. Recent results from electrospray ionization mass spectrometric studies of the stepwise metallation of the two fragments and the whole protein with Zn(2+), Cd(2+), As(3+) and Bi(3+) indicate a noncooperative mechanism of a declining series of K(F)'s. Of particular note are new details about the early stages of the stepwise metallation reactions, specifically the stability of partially metallated species for As(3+), Cd(2+), and Zn(2+) that do not correspond to the two-domain model. In addition, at the other end of the coordination spectrum are the supermetallated species of MT, where supermetallation defines metallation in excess of traditional levels. It has been reported that with metal ion excess the formation of a single 'super domain' is possible and again this deviates from the two-domain model of MT. In both cases, these results suggest that the structural view of mammalian MT that is of two essentially isolated domains may be the exceptional case and that under the normal conditions of cellular metal-ion concentrations the two domain structure might coexist in equilibrium with various single domain, multi-metal site structures. This review specifically focuses on providing context for these recent studies and the new ideas concerning metallation prior to the establishment of domain-based clusters that these studies suggest.