Elucidating the metalation pathway of the Ec-1 metallothionein βE-domain: Insights into ZnII binding and protein folding

Abstract

Metallothioneins (MTs) are small cysteine-rich proteins that preferentially bind d¹⁰ metal ions such as Zn^II, Cu^I, and Cd^II, playing essential roles in metal ion homeostasis and detoxification. The E_c-1 metallothionein from Triticum aestivum (common bread wheat) was the first plant metallothionein for which a 3D structure was successfully determined, although this structure represents only the fully metalated state of the protein. In this study, we aim to elucidate the metalation pathway of the β_E-domain of wheat E_c-1. This domain features a mononuclear Zn^II binding site composed of two cysteine and two highly conserved histidine residues, reminiscent of the Zn-finger motifs found in certain proteins. Moreover, the domain forms a trinuclear Zn₃Cys₉ cluster, similar to the β-cluster motif observed, for example, in vertebrate MTs. To investigate the metalation pathway of the β_E-domain, we combined nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and targeted cysteine modification techniques. Our results confidently identify the sequential binding site regions for each of the four Zn^II ions and reveal intriguing, unexpected insights into the folding pathway of the peptide backbone.