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 N -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 4 -MT. The results for Ag 4 -MT are then compared to similar results obtained for Cd 4 -MT. The results show that Ag 4 -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 4 -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 2+ react with MT to yield cooperative products, i.e., Ag 4 -MT and Cd 4 -MT, these products are very different; Ag + ions of Ag 4 -MT are located in the β-domain, whereas Cd 2+ ions of Cd 4 -MT are located in the α-domain. Ag 6 -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 i -MT complexes ( i = 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 Ag 4 NEM 14 -MT. Results from CIU suggest that Ag i -MT complexes are structurally more ordered and that the energy required to unfold these complexes increases as the number of coordinated Ag + increases.