Challenging conventional wisdom: single domain metallothioneins

Abstract

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.

Metalation Kinetics of the Human α‐Metallothionein 1a Fragment Is Dependent on the Fluxional Structure of the apo‐Protein

“…The conformation of apoMT, because of its fluxional nature, [38b] has not been described extensively. FRET experiments provided evidence that at neutral pH both a-and b-MT fragments retained their compact structure upon demetalation [22,23] and molecular models [41] and ESI-MS data [42] support this conclusion. However, it is important to understand the dependence of metalation on the conformation of the apo-MT in order to determine metalation mechanism and the directors for metal specificity.…”

The Coordination Chemistry of Copper Uptake and Storage for Methane Oxidation

“…A homologue (MymT) has been identified in pathogenic mycobacteria that provided, prior to the discovery of the Csps, a very rare example of Cu storage in bacteria. Eukaryotic MTs are also able to store Zn II , and can bind other metals with a d 10 electronic configuration, including the highly toxic Cd II and Hg II . Due to difficulties in obtaining suitable crystals, NMR has been extensively used to study solution structures of Cu I ‐MTs, but provides limited information about the Cu I sites .…”

“…The similar arrangement of Cu I sites in Cup1 and Mt Csp3, and also presumably other members of these families of proteins, indicates that cluster formation is driven by Cu I ‐thiolate chemistry. This is perhaps to be expected for MTs whose apo‐forms are flexible, and is probably the key reason why they can bind a range of other metal ions . The ability to sequester both Cu I and Zn II in vivo results in the physiological role of MTs in Cu and Zn homeostasis being intertwined .…”

“…Eukaryotic MTsa re also able to store Zn II ,a nd can bind other metalsw ithad 10 electronic configuration, including the highly toxic Cd II and Hg II . [56,57,59,[61][62][63][64] Duet od ifficulties in obtaining suitable crystals, [52] NMR has been extensively used to study solution structures of Cu I -MTs, but provides limited information about the Cu I sites. [65][66][67][68][69][70] The only crystal structureso f metallated MTsa re for Cu I -Cup1 from Saccharomyces cerevisiae (Figure 6a) [52] andr at liver MT-2 binding five Cd II and two Zn II ions.…”

“…[44,52,53] The similara rrangement of Cu I sites in Cup1 and MtCsp3, and also presumablyo ther members of these families of proteins, indicates that cluster formation is driven by Cu I -thiolate chemistry.T his is perhaps to be expected for MTsw hose apo-forms are flexible, [56,63,71] andi sp robably the key reason why they can bind ar ange of other metal ions. [56,57,59,[61][62][63][64] The ability to sequester both Cu I and Zn II in vivo results in the physio- 6 ]c luster [52] involving Cu39, Cu40,Cu41 and Cu44 is shown in (a) and (b) in two orientations. For MtCsp3, the intermediate [Cu 4 (S-Cys) 4 (O-Asn)] (c and d, 1-2 equiv structure, PDB file 5NQM) [53] and the final[ Cu 4 (S-Cys) 5 (O-Asn)] (e and f, 19 equiv structure,P DB file 5ARN) [44] Cu11-Cu14 clusters are shown.The representation and labelling of the structures is as describedi nthe legendstoF igures3,5and6.…”