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Metal exchange in metallothioneins – a novel structurally significant Cd 5 species in the alpha domain of human metallothionein 1a
Author(s) -
Rigby Duncan Kelly E.,
Kirby Christopher W.,
Stillman Martin J.
Publication year - 2008
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2008.06375.x
Subject(s) - metallothionein , cadmium , chemistry , metal ions in aqueous solution , metal , cysteine , crystallography , stereochemistry , biochemistry , enzyme , organic chemistry
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 α domain of metallothionein isoform 1a, which encompasses the metal‐binding cysteines between Cys33 and Cys60 of the α 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 α‐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 α species following addition of 5.0 molar equivalents of Cd 2+ to a solution of Cd 4 α‐MT. Significantly, the structurally sensitive CD spectrum shows a sharp monophasic peak at 254 nm for the Cd 5 α species in contrast to the derivative‐shaped spectrum of the Cd 4 α‐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 α 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 α‐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 α‐MT, but has two thiolate ligands as part of its ligand environment, with additional coordination to either water or anions in solution.