Metal exchange in metallothioneins: a novel structurally significant Cd(5) species in the alpha domain of human metallothionein 1a

FEBS J. 2008 May;275(9):2227-39. doi: 10.1111/j.1742-4658.2008.06375.x.

Abstract

Metallothioneins (MTs) are cysteine-rich, metal-binding proteins known to provide protection against cadmium toxicity in mammals. Metal exchange of Zn(2+) ions for Cd(2+) ions in metallothioneins is a critical process for which no mechanistic or structural information is currently available. The recombinant human alpha domain of metallothionein isoform 1a, which encompasses the metal-binding cysteines between Cys33 and Cys60 of the alpha domain of native human metallothionein 1a, was studied. Characteristically this fragment coordinates four Cd(2+) ions to the 11 cysteinyl sulfurs, and is shown to bind an additional Cd(2+) ion to form a novel Cd(5)alpha-MT species. This species is proposed here to represent an intermediate in the metal-exchange mechanism. The ESI mass spectrum shows the appearance of charge state peaks corresponding to a Cd(5)alpha species following addition of 5.0 molar equivalents of Cd(2+) to a solution of Cd(4)alpha-MT. Significantly, the structurally sensitive CD spectrum shows a sharp monophasic peak at 254 nm for the Cd(5)alpha species in contrast to the derivative-shaped spectrum of the Cd(4)alpha-MT species, with peak maxima at 260 nm (+) and 240 nm (-), indicating Cd-induced disruption of the exciton coupling between the original four Cd(2+) ions in the Cd(4)alpha species. The (113)Cd chemical shift of the fifth Cd(2+) is significantly shielded (approximately 400 p.p.m.) when compared with the data for the Cd(2+) ions in Cd(4)alpha-MT by both direct and indirect (113)Cd NMR spectroscopy. Three of the four original NMR peaks move significantly upon binding the fifth cadmium. Evidence from indirect (1)H-(113)Cd HSQC NMR spectra suggests that the coordination environment of the additional Cd(2+) is not tetrahedral to four thiolates, as is the case with the four Cd(2+) ions in the Cd(4)alpha-MT, but has two thiolate ligands as part of its ligand environment, with additional coordination to either water or anions in solution.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cadmium / chemistry*
  • Cadmium / metabolism
  • Circular Dichroism
  • Humans
  • Metallothionein / chemistry*
  • Metallothionein / metabolism
  • Metals / metabolism*
  • Models, Molecular
  • Molecular Sequence Data
  • Nuclear Magnetic Resonance, Biomolecular
  • Protein Conformation
  • Protein Structure, Tertiary

Substances

  • Metals
  • Cadmium
  • Metallothionein