Modeling the Zn2+ and Cd2+ metalation mechanism in mammalian metallothionein 1a

Mammalian metallothioneins (MTs) are a family of small cysteine rich proteins believed to have a number of physiological functions, including both metal ion homeostasis and toxic metal detoxification. Mammalian MTs bind 7 Zn2+ or Cd2+ ions into two distinct domains: an N-terminal β-domain that binds 3 Zn2+ or Cd2+, and a C-terminal α-domain that binds 4 Zn2+ or Cd2+. Although stepwise metalation to the saturated M7-MT (where M = Zn2+ or Cd2+) species would be expected to take place via a noncooperative mechanism involving the 20 cysteine thiolate ligands, literature reports suggest a cooperative mechanism involving cluster formation prior to saturation of the protein. Electrospray ionization mass spectrometry (ESI-MS) provides this sensitivity through delineation of all species (Mn-MT, n = 0–7) coexisting at each step in the metalation process. We report modeled ESI-mass spectral data for the stepwise metalation of human recombinant MT 1a (rhMT) and its two isolated fractions for three mechanistic conditions: cooperative (where the binding affinities are: K1 < K2 < K3 < ··· < K7), weakly cooperative (where K1 = K2 = K3 = ··· = K7), and noncooperative, (where K1 > K2 > K3 > ··· > K7). Detailed ESI-MS metalation data of human recombinant MT 1a by Zn2+ and Cd2+ are also reported. Comparison of the experimental data with the predicted mass spectral data provides conclusive evidence that metalation occurs in a noncooperative fashion for Zn2+ and Cd2+ binding to rhMT 1a.

Graphical abstractFigure optionsDownload full-size imageDownload as PowerPoint slideHighlights► ESI-mass spectral data provide key information about the metalation mechanism of MT. ► Metal ion speciation as a function of metals added is clearly determined. ► Models for noncooperative, weakly cooperative, and strongly cooperative mechanisms. ► Metalation of MT 1a by Zn2+, Cd2+, As3+, and Bi3+ follow a noncooperative mechanism. ► Results provide the framework for analyzing the metalation reactions of MT.