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


  • 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 -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 -domain, whereas Cd2+ ions of Cd4-MT are located in the -domain. Ag6-MT has been reported to be fully metalated in the -domain, but our data suggest the two additional Ag+ ions are more weakly bound than are the other four. Higher order Agi-MT complexes (i = 7-17) are formed in solutions that contain excess Ag+ ions, and these are assumed to be bound to the -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 Agi-MT complexes are structurally more ordered and that the energy required to unfold these complexes increases as the number of coordinated Ag+ increases.

published proceedings

  • Anal Chem

author list (cited authors)

  • Dong, S., Shirzadeh, M., Fan, L., Laganowsky, A., & Russell, D. H.

citation count

  • 12

complete list of authors

  • Dong, Shiyu||Shirzadeh, Mehdi||Fan, Liqi||Laganowsky, Arthur||Russell, David H

publication date

  • July 2020