Metal binding and interdomain thermodynamics of mammalian metallothionein-3: enthalpically favoured Cu+ supplants entropically favoured Zn2+ to form Cu-4(+) clusters under physiological conditions

Metallothioneins (MTs) are a ubiquitous class of small metal-binding proteins involved in metal homeostasis and detoxification. While known for their high affinity for d(10) metal ions, there is a surprising dearth of thermodynamic data on metals binding to MTs. In this study, Zn2+ and Cu+ binding to mammalian metallothionein-3 (MT-3) were quantified at pH 7.4 by isothermal titration calorimetry (ITC). Zn2+ binding was measured by chelation titrations of Zn7MT-3, while Cu+ binding was measured by Zn2+ displacement from Zn7MT-3 with competition from glutathione (GSH). Titrations in multiple buffers enabled a detailed analysis that yielded condition-independent values for the association constant (K) and the change in enthalpy (Delta H) and entropy (Delta S) for these metal ions binding to MT-3. Zn2+ was also chelated from the individual alpha and beta domains of MT-3 to quantify the thermodynamics of inter-domain interactions in metal binding. Comparative titrations of Zn7MT-2 with Cu+ revealed that both MT isoforms have similar Cu+ affinities and binding thermodynamics, indicating that Delta H and Delta S are determined primarily by the conserved Cys residues. Inductively coupled plasma mass spectrometry (ICP-MS) analysis and low temperature luminescence measurements of Cu-replete samples showed that both proteins form two Cu-4(+)-thiolate clusters when Cu+ displaces Zn2+ under physiological conditions. Comparison of the Zn2+ and Cu+ binding thermodynamics reveal that enthalpically-favoured Cu+, which forms Cu-4(+)-thiolate clusters, displaces the entropically-favoured Zn2+. These results provide a detailed thermodynamic analysis of d(10) metal binding to these thiolate-rich proteins and quantitative support for, as well as molecular insight into, the role that MT-3 plays in the neuronal chemistry of copper.