Structural Characterization of Cu(I)/Zn(II)-metallothionein-3 by Ion Mobility Mass Spectrometry and Top-Down Mass Spectrometry

Manuel David Peris-Díaz, Sylwia Wu, Karolina Mosna, Ellen Liggett, Alexey Barkhanskiy, Alicja Orzeł, Perdita Barran, Artur Krężel

Research output: Contribution to journalArticlepeer-review


Mammalian zinc metallothionein-3 (Zn7MT3) plays an important role in protecting against copper toxicity by scavenging free Cu(II) ions or removing Cu(II) bound to β-amyloid and α-synuclein. While previous studies reported that Zn7MT3 reacts with Cu(II) ions to form Cu(I)4Zn(II)4MT3ox containing two disulfides (ox), the precise localization of the metal ions and disulfides remained unclear. Here, we undertook comprehensive structural characterization of the metal-protein complexes formed by the reaction between Zn7MT3 and Cu(II) ions using native ion mobility mass spectrometry (IM-MS). The complex formation mechanism was found to involve the disassembly of Zn3S9 and Zn4S11 clusters from Zn7MT3 and reassembly into Cu(I)xZn(II)yMT3ox complexes rather than simply Zn(II)-to-Cu(I) exchange. At neutral pH, the β-domain was shown to be capable of binding up to six Cu(I) ions to form Cu(I)6Zn(II)4MT3ox, although the most predominant species was the Cu(I)4Zn(II)4MT3ox complex. Under acidic conditions, four Zn(II) ions dissociate, but the Cu(I)4-thiolate cluster remains stable, highlighting the MT3 role as a Cu(II) scavenger even at lower than the cytosolic pH. IM-derived collision cross sections (CCS) reveal that Cu(I)-to-Zn(II) swap in Zn7MT3 with concomitant disulfide formation induces structural compaction and a decrease in conformational heterogeneity. Collision-induced unfolding (CIU) experiments estimated that the native-like folded Cu(I)4Zn(II)4MT3ox conformation is more stable than Zn7MT3. Native top-down MS demonstrated that the Cu(I) ions are exclusively bound to the β-domain in the Cu(I)4Zn(II)4MT3ox complex as well as the two disulfides, serving as a steric constraint for the Cu(I)4-thiolate cluster. In conclusion, this study enhances our comprehension of the structure, stability, and dynamics of Cu(I)xZn(II)yMT3ox complexes.

Original languageEnglish
Pages (from-to)10966-10974
Number of pages9
JournalAnalytical Chemistry
Issue number29
Early online date13 Jul 2023
Publication statusPublished - 25 Jul 2023


  • Animals
  • Coordination Complexes/chemistry
  • Copper/chemistry
  • Disulfides
  • Mammals/metabolism
  • Mass Spectrometry
  • Metallothionein 3
  • Metallothionein/chemistry
  • Zinc/chemistry


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