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

Abstract

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

Phytochelatins Bind Zn(II) with Micro- to Picomolar Affinities without the Formation of Binuclear Complexes, Exhibiting Zinc Buffering and Muffling Rather than Storing Functions

“…Mass spectrometry was employed to verify the identity and purity of the investigated thiol peptides, as outlined in Table S1, and to monitor the metal binding stoichiometry of the PC series. , It is important to note that the qualitative analysis was conducted in the gas phase, providing limited information about equilibria in solution. Nevertheless, this information should not be extrapolated into a quantitative model, as the ratios of detected species, influenced by their disparate ionization coupled with dissociation of species, vary during the phase transition . For this reason, irrespective of the molar Zn­(II)-load of the ligand, only equimolar complex species were identified, as biscomplexes are typically unstable under experimental conditions .…”

“…Nevertheless, this information should not be extrapolated into a quantitative model, as the ratios of detected species, influenced by their disparate ionization coupled with dissociation of species, vary during the phase transition. 55 For this reason, irrespective of the molar Zn(II)-load of the ligand, only equimolar complex species were identified, as biscomplexes are typically unstable under experimental conditions. 56 Binuclear species were detected for only the longest studied homologue.…”

Copper trafficking systems in cells: insights into coordination chemistry and toxicity

“…3b, native mass spectrometric investigation in conjunction with the isotopic distribution analysis of Zn(II) 7 MT3 further supported the Cu(II) reduction to Cu(I). 78 This process can be coupled with the oxidation of four Cys residues of Zn(II) 7 MT3, forming two intramolecular disulfide bonds, followed by metal replacement to generate Cu(I) 4 Zn(II) 4 MT3. 78 In addition, the conformational compactness of Cu(I) 4 Zn(II) 4 MT3 in comparison with Zn(II) 7 MT3 was confirmed through ion-mobility mass spectrometric studies, that is attributed to the stronger Cu(I)-thiolate bonds and the structural rigidity of disulfide bonds within the resul- 90 The GSH/GSSG pair can regulate the redox state of thiol-disulfide oxidoreductase enzymes as well as redox signaling.…”

“…Perspective tant species. 78 Moreover, MT can be indirectly involved in copper metabolism by regulating the trafficking of ATP7A from the trans-Golgi to the cellular membrane upon impairment of copper homeostasis, which ultimately controls the cell viability. 79…”

“…78 This process can be coupled with the oxidation of four Cys residues of Zn( ii ) 7 MT3, forming two intramolecular disulfide bonds, followed by metal replacement to generate Cu( i ) 4 Zn( ii ) 4 MT3. 78 In addition, the conformational compactness of Cu( i ) 4 Zn( ii ) 4 MT3 in comparison with Zn( ii ) 7 MT3 was confirmed through ion-mobility mass spectrometric studies, that is attributed to the stronger Cu( i )–thiolate bonds and the structural rigidity of disulfide bonds within the resultant species. 78 Moreover, MT can be indirectly involved in copper metabolism by regulating the trafficking of ATP7A from the trans -Golgi to the cellular membrane upon impairment of copper homeostasis, which ultimately controls the cell viability.…”