Metal binding and interdomain thermodynamics of mammalian metallothionein-3: enthalpically favoured Cu(+) supplants entropically favoured Zn(2+) 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, Zn(2+) and Cu(+) binding to mammalian metallothionein-3 (MT-3) were quantified at pH 7.4 by isothermal titration calorimetry (ITC). Zn(2+) binding was measured by chelation titrations of Zn(7)MT-3, while Cu(+) binding was measured by Zn(2+) displacement from Zn(7)MT-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 (ΔH) and entropy (ΔS) for these metal ions binding to MT-3. Zn(2+) was also chelated from the individual α and β domains of MT-3 to quantify the thermodynamics of inter-domain interactions in metal binding. Comparative titrations of Zn(7)MT-2 with Cu(+) revealed that both MT isoforms have similar Cu(+) affinities and binding thermodynamics, indicating that ΔH and ΔS 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 Zn(2+) under physiological conditions. Comparison of the Zn(2+) and Cu(+) binding thermodynamics reveal that enthalpically-favoured Cu(+), which forms Cu(4)(+)–thiolate clusters, displaces the entropically-favoured Zn(2+). 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.