Metal exchange in metallothioneins - a novel structurally significant Cd₅ species in the alpha domain of human metallothionein 1a

• Published in: The FEBS journal 2008-05, Vol.275 (9), p.2227-2239
• Main Authors: Rigby Duncan, Kelly E, Kirby, Christopher W, Stillman, Martin J
• Format: Article
• Language: English
• Subjects: 113Cd NMR spectroscopy; Binding sites; Biochemistry; circular dichroism spectroscopy; ESI mass spectrometry; Genetic recombination; Ions; metal exchange; metallothionein; Metals; Proteins
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2008.06375.x

Metallothioneins (MTs) are cysteine-rich, metal-binding proteins known to provide protection against cadmium toxicity in mammals. Metal exchange of Zn²⁺ ions for Cd²⁺ 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 Cd²⁺ ions to the 11 cysteinyl sulfurs, and is shown to bind an additional Cd²⁺ ion to form a novel Cd₅α-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 Cd₅α species following addition of 5.0 molar equivalents of Cd²⁺ to a solution of Cd₄α-MT. Significantly, the structurally sensitive CD spectrum shows a sharp monophasic peak at 254 nm for the Cd₅α species in contrast to the derivative-shaped spectrum of the Cd₄α-MT species, with peak maxima at 260 nm (+) and 240 nm (-), indicating Cd-induced disruption of the exciton coupling between the original four Cd²⁺ ions in the Cd₄α species. The ¹¹³Cd chemical shift of the fifth Cd²⁺ is significantly shielded (approximately 400 p.p.m.) when compared with the data for the Cd²⁺ ions in Cd₄α-MT by both direct and indirect ¹¹³Cd NMR spectroscopy. Three of the four original NMR peaks move significantly upon binding the fifth cadmium. Evidence from indirect ¹H-¹¹³Cd HSQC NMR spectra suggests that the coordination environment of the additional Cd²⁺ is not tetrahedral to four thiolates, as is the case with the four Cd²⁺ ions in the Cd₄α-MT, but has two thiolate ligands as part of its ligand environment, with additional coordination to either water or anions in solution.