title: Modeling the Zn(2+) and Cd(2+) metalation mechanism in mammalian metallothionein 1a.
abstract: 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 Zn(2+) or Cd(2+) ions into two distinct domains: an N-terminal β-domain that binds 3 Zn(2+) or Cd(2+), and a C-terminal α-domain that binds 4 Zn(2+) or Cd(2+). Although stepwise metalation to the saturated M(7)-MT (where M=Zn(2+) or Cd(2+)) 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 (M(n)-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: K(1)<K(2)<K(3)<···<K(7)), weakly cooperative (where K(1)=K(2)=K(3)=···=K(7)), and noncooperative, (where K(1)>K(2)>K(3)>···>K(7)). Detailed ESI-MS metalation data of human recombinant MT 1a by Zn(2+) and Cd(2+) 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 Zn(2+) and Cd(2+) binding to rhMT 1a.
authors.affiliation: Department of Biology, The University of Western Ontario, London, Ontario, Canada N6A 5B7.
authors.forename: Duncan E K
authors.initials: DE
authors.lastname: Sutherland
authors.forename: Kelly L
authors.initials: KL
authors.lastname: Summers
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Stillman
mesh_terms: D000595:Amino Acid Sequence; D002104:Cadmium / Q000737:chemistry*; D006801:Humans; D008668:Metallothionein / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D015032:Zinc / Q000737:chemistry*
chemical_list: C530099:MT1A protein, human; D011994:Recombinant Proteins; D002104:Cadmium; D008668:Metallothionein; D015032:Zinc