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

Mammalian zinc metallothionein-3 (ZnMT3) 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 ZnMT3 reacts with Cu(II) ions to form Cu(I)Zn(II)MT3ox 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 ZnMT3 and Cu(II) ions using native ion mobility mass spectrometry (IM-MS). The complex formation mechanism was found to involve the disassembly of ZnS and ZnS clusters from ZnMT3 and reassembly into Cu(I)Zn(II)MT3 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)Zn(II)MT3, although the most predominant species was the Cu(I)Zn(II)MT3 complex. Under acidic conditions, four Zn(II) ions dissociate, but the Cu(I)-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 ZnMT3 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)Zn(II)MT3 conformation is more stable than ZnMT3. Native top-down MS demonstrated that the Cu(I) ions are exclusively bound to the β-domain in the Cu(I)Zn(II)MT3 complex as well as the two disulfides, serving as a steric constraint for the Cu(I)-thiolate cluster. In conclusion, this study enhances our comprehension of the structure, stability, and dynamics of Cu(I)Zn(II)MT3 complexes.