Metal binding and interdomain thermodynamics of mammalian metallothionein-3: enthalpically favoured Cu+ supplants entropically favoured Zn2+ to form Cu4+ clusters under physiological conditions. Issue 18 (20th April 2022)

• Record Type: Journal Article
• Authors: Mehlenbacher, Matthew R.; Elsiesy, Rahma; Lakha, Rabina; Villones, Rhiza Lyne E.; Orman, Marina; Vizcarra, Christina L.; Meloni, Gabriele; Wilcox, Dean E.; Austin, Rachel N.
• Is Part Of: Chemical science. Volume 13:Issue 18(2022)
• Page Start: 5289
• Page End: 5304
• Publication Date: 2022-04-20
• Subjects: Chemistry -- Periodicals; 540.5
• DOI: 10.1039/d2sc00676f
• Languages: English
• ISSNs: 2041-6520

Abstract

Abstract : Metallothioneins (MTs) are a ubiquitous class of small metal-binding proteins involved in metal homeostasis and detoxification. Abstract : 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 Zn7 MT-3, while Cu + binding was measured by Zn 2+ displacement from Zn7 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 (Δ H ) and entropy (Δ S ) 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 Zn7 MT-2 with Cu + revealed that both MT isoforms have similar Cu + affinities and binding thermodynamics, indicating that Δ H and Δ 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 Cu4 + –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 Cu4 + –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.