Cu(I) binds to Zn7-MT2 via two parallel pathways
Adyn Melenbacher1, Martin J Stillman1
1Department of Chemistry, The University of Western Ontario, London, Ontario, Canada.
Metallomics : Integrated Biometal Science
|September 12, 2023
Summary
Metallothionein-2 (MT2) protein binds copper and zinc ions through two distinct pathways, influencing metal homeostasis and detoxification. This study reveals specific Cu:Zn ratios and binding domains within MT2, crucial for understanding its role in disease.
Area of Science:
- Biochemistry
- Proteomics
- Spectroscopy
Background:
- Metallothionein proteins (MTs) are vital for maintaining copper (Cu) and zinc (Zn) homeostasis and detoxifying heavy metals.
- MT2's diverse expression and disease correlations (cancers, neurological, respiratory) highlight the importance of understanding its metallation properties.
- Isotopically pure 63Cu(I) and 68Zn(II) are crucial for resolving mass spectral complexities in Cu, Zn-MT2 studies.
Purpose of the Study:
- To precisely determine Cu(I) and Zn(II) stoichiometries bound to MT2 at physiological pH.
- To elucidate the parallel pathways of Cu(I) metallation in Zn7-MT2.
- To establish formation constants (KF) for various Cu, Zn-MT2 species and assign spectral bands.
Main Methods:
- Electrospray ionization (ESI)-mass spectrometry was used to analyze Cu, Zn-MT2 complexes.
- Mass spectral simulations were employed to determine exact Cu:Zn ratios during titration.
- Room temperature phosphorescence and circular dichroism (CD) spectroscopy provided parallel data for species assignment.
Main Results:
- Two parallel pathways of Cu(I) metallation for Zn7-MT2 were identified, yielding specific Cu:Zn ratios.
- Pathway ① produced Cu5Zn5-MT2 and Cu9Zn3-MT2; Pathway ② yielded major products Cu6Zn4-MT2 and Cu10Zn2-MT2.
- CD spectral analysis suggests Cu(I) initially binds to the β domain, forming Cu5Zn1 or Cu6 clusters, leaving the α domain with Zn4.
Conclusions:
- The study precisely quantifies Cu(I) and Zn(II) binding stoichiometries to MT2 using isotopically pure metals and mass spectrometry.
- Metallation occurs via two distinct pathways, leading to defined Cu, Zn-MT2 species.
- Cu(I) preferentially binds to the β domain of MT2, influencing the overall protein structure and function.
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