Issue 18, 2022

Metal binding and interdomain thermodynamics of mammalian metallothionein-3: enthalpically favoured Cu+ supplants entropically favoured Zn2+ to form Cu4+ clusters under physiological conditions

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 d10 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 (ΔH) and entropy (ΔS) for these metal ions binding to MT-3. Zn2+ 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 Zn7MT-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 Cu4+–thiolate clusters when Cu+ displaces Zn2+ under physiological conditions. Comparison of the Zn2+ and Cu+ binding thermodynamics reveal that enthalpically-favoured Cu+, which forms Cu4+–thiolate clusters, displaces the entropically-favoured Zn2+. These results provide a detailed thermodynamic analysis of d10 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.

Graphical abstract: Metal binding and interdomain thermodynamics of mammalian metallothionein-3: enthalpically favoured Cu+ supplants entropically favoured Zn2+ to form Cu4+ clusters under physiological conditions

Supplementary files

Article information

Article type
Edge Article
Submitted
02 Febr. 2022
Accepted
01 Apr. 2022
First published
04 Apr. 2022
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY license

Chem. Sci., 2022,13, 5289-5304

Metal binding and interdomain thermodynamics of mammalian metallothionein-3: enthalpically favoured Cu+ supplants entropically favoured Zn2+ to form Cu4+ clusters under physiological conditions

M. R. Mehlenbacher, R. Elsiesy, R. Lakha, R. L. E. Villones, M. Orman, C. L. Vizcarra, G. Meloni, D. E. Wilcox and R. N. Austin, Chem. Sci., 2022, 13, 5289 DOI: 10.1039/D2SC00676F

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

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