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

[Image: see text] 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