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

Mammalian zinc metallothionein-3 (Zn 7 MT3) 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 Zn 7 MT3 reacts with Cu(II) ions to form Cu(I) 4 Zn(II) 4 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 Zn 7 MT3 and Cu(II) ions using native ion mobility mass spectrometry (IM-MS). The complex formation mechanism was found to involve the disassembly of Zn 3 S 9 and Zn 4 S 11 clusters from Zn 7 MT3 and reassembly into Cu(I) x Zn(II) y MT3 ox 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) 6 Zn(II) 4 MT3 ox , although the most predominant species was the Cu(I) 4 Zn(II) 4 MT3 ox 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 Zn 7 MT3 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) 4 Zn(II) 4 MT3 ox conformation is more stable than Zn 7 MT3. Native top-down MS demonstrated that the Cu(I) ions are exclusively bound to the β-domain in the Cu(I) 4 Zn(II) 4 MT3 ox 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) x Zn(II) y MT3 ox complexes.