Supermetalation of Cd-MT3 beyond the two-domain model

The flexibility of mammalian metallothioneins (MTs) has contributed to the difficulty in obtaining structural information for this family of metalloproteins that bind divalent metals with its twenty cysteines. While the two-domain structure for Cd7MT is well-established as a Cd4S11 and Cd3S9, a third structure has been reported when 8 Cd(II) ions bind to MT1. Isoform 3 of the MT family, MT3, has been of interest to the research community since its isolation as a growth inhibitory factor isolated in brain tissue, and has since been noted as a prominent participant in the mediation of neurodegenerative diseases and regular brain development. The differences between MT3 and the other isoforms of MT include an additional hexapeptide insertion of acidic residues in the α domain as well as the introduction of two prolines in the β domain. It is unclear whether these changes impact the metalation properties of MT3. We report the formation of a Cd8MT3 species is characterized by electrospray ionization mass spectrometry and UV–visible absorption spectroscopy. We report that the spectroscopic properties of this supermetalated Cd8MT3 are similar to those of the supermetalated Cd8MT1, with a clear indication of changes in structure from “fully-metalated” Cd7MT3 to supermetalated Cd8MT3 from circular dichroism spectra and both 1D 113Cd and 2D 1H-113Cd HSQC NMR spectra. We conclude that the metalation properties are not impacted significantly due to the amino acid changes in MT3, and that the cysteinyl thiols are the key players in determining the capacity of metal-binding and the structure of metal-thiolate clusters. Metallothionein-3 was found to be supermetalated to Cd8-metallothionein-3, forming a single domain incorporating both the α and β domains, supported by circular dichroism spectroscopy, UV–visible absorption spectroscopy, and NMR spectroscopy. [Display omitted] •Metallothionein-3 (MT3) can be supermetalated to Cd8MT3.•Structural changes of Cd7 to Cd8MT3 are characterized by circular dichroism spectra.•Cd7MT3 and Cd8MT3 are characterized by 1D 113Cd and 2D 1H113Cd HSQC NMR spectra.•Cd8MT3 NMR spectra similar to single-domain supermetalated Cd8MT1.