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...

Metallothionein-3 attenuates the effect of Cu2+ ions on actin filaments

“…We created a phospho-mimetic version of MT-3 with the mutation Ser33Asp (S33D). First, we measured the affinity of S33D MT-3 for Zn 2+ using a well-established isothermal titration calorimetry (ITC) chelation assay [34]. From these experiments, we determined the Zn 2+ binding free energy, ΔG° ITC,S33D = −10.1 ± 0.3 kcal·mol −1 (Fig.…”

“…Mouse MT-3 (UniProt P28184) was expressed in BL21-DE3-pLysS E. coli cells and purified as previously described [34]. To produce different metalation states (Zn 7 MT-3, Pb 7 MT-3, Cu 4 Zn 4 MT-3), stock solutions of either ZnCl 2 , Pb(NO 3 ) 2 , and/or CuCl 2 were added to apo-MT-3 in stoichiometric amounts.…”

“…To produce different metalation states (Zn 7 MT-3, Pb 7 MT-3, Cu 4 Zn 4 MT-3), stock solutions of either ZnCl 2 , Pb(NO 3 ) 2 , and/or CuCl 2 were added to apo-MT-3 in stoichiometric amounts. A non-GIF mutant (Pro7Ser/Pro9Ala) and a phospho-mimetic mutant (Ser33Asp) were both constructed by PCR-based mutagenesis [41] (DNA sequences in SI) and purified using the same protocol as WT MT-3 [34]. Rabbit skeletal muscle actin from acetone powder ( Pel-Freez Biologicals ) and recombinant S. pombe profilin were purified, and actin was labeled as previously described [42].…”

“…Protein was then eluted with glutathione elution buffer (50 mM Tris-HCl, 100 mM NaCl, 100 mM glutathione, 1 mM DTT, pH 8.0) and dialyzed into low salt buffer at 4 °C overnight. His 6 -tagged TEV protease (purified as described [34]) was added to the protein in dialysis tubing to cleave the fusion protein. The sample was nutated with glutathione Sepharose for 1 hr to separate the cleaved fascin from GST.…”

“…Furthermore, MT-3 is the most copper-philic of the mammalian MTs [30,31]. In general, copper is an essential and highly regulated trace element with a variety of roles in normal growth, development, and cellular metabolism [32] and is known to bind to both actin [33] and MT-3 [31,34]. In hippocampal neurons, copper is enriched in dendrites compared to somata, where nano X-ray fluorescence suggests it co-localizes with actin filaments in dendritic spine necks [35].…”