Ag Ion Binding to Human Metallothionein-2A Is Cooperative and Domain Specific.

Metallothioneins (MTs) constitute a family of cysteine-rich proteins that play key biological roles for a wide range of metal ions, but unlike many other metalloproteins, the structures of apo- and partially metalated MTs are not well understood. Here, we combine nano-electrospray ionization-mass spectrometry (ESI-MS) and nano-ESI-ion mobility (IM)-MS with collision-induced unfolding (CIU), chemical labeling using -ethylmaleimide (NEM), and both bottom-up and top-down proteomics in an effort to better understand the metal binding sites of the partially metalated forms of human MT-2A, viz., Ag-MT. The results for Ag-MT are then compared to similar results obtained for Cd-MT. The results show that Ag-MT is a cooperative product, and data from top-down and bottom-up proteomics mass spectrometry analysis combined with NEM labeling revealed that all four Ag ions of Ag-MT are bound to the β-domain. The binding sites are identified as Cys13, Cys15, Cys19, Cys21, Cys24, and Cys26. While both Ag and Cd react with MT to yield cooperative products, i.e., Ag-MT and Cd-MT, these products are very different; Ag ions of Ag-MT are located in the β-domain, whereas Cd ions of Cd-MT are located in the α-domain. Ag-MT has been reported to be fully metalated in the β-domain, but our data suggest the two additional Ag ions are more weakly bound than are the other four. Higher order Ag-MT complexes ( = 7-17) are formed in solutions that contain excess Ag ions, and these are assumed to be bound to the α-domain or shared between the two domains. Interestingly, the excess Ag ions are displaced upon addition of NEM to this solution to yield predominantly AgNEM-MT. Results from CIU suggest that Ag-MT complexes are structurally more ordered and that the energy required to unfold these complexes increases as the number of coordinated Ag increases.