Elucidating the metalation pathway of the E-1 metallothionein β-domain: Insights into Zn binding and protein folding.

Metallothioneins (MTs) are small cysteine-rich proteins that preferentially bind d metal ions such as Zn, Cu, and Cd, playing essential roles in metal ion homeostasis and detoxification. The E-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-domain of wheat E-1. This domain features a mononuclear Zn 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 ZnCys cluster, similar to the β-cluster motif observed, for example, in vertebrate MTs. To investigate the metalation pathway of the β-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 ions and reveal intriguing, unexpected insights into the folding pathway of the peptide backbone.