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

ABSTRACT: 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., Ag₄-MT. The results for Ag₄-MT are then compared to similar results obtained for Cd₄-MT. The results show that Ag₄-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 Ag₄-MT are bound to the β-domain. The binding sites are identified as Cys13, Cys15, Cys19, Cys21, Cys24, and Cys26. While both Ag⁺ and Cd²⁺ react with MT to yield cooperative products, i.e., Ag₄-MT and Cd₄-MT, these products are very different; Ag⁺ ions of Ag₄-MT are located in the β-domain, whereas Cd²⁺ ions of Cd₄-MT are located in the α-domain. Ag₆-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 Ag₄NEM₁₄-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.

SUBMITTER: Dong S 

PROVIDER: S-EPMC8114364 | biostudies-literature |

REPOSITORIES: biostudies-literature