Metallothionein‐3: <scp>63Cu</scp>(I) binds to human <scp>68Zn7</scp>‐βα <scp>MT3</scp> with no preference for Cu4‐β cluster formation

Melenbacher, Adyn; Stillman, Martin J.

doi:10.1111/febs.16812

Cited by 5 publications

(4 citation statements)

References 96 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Their findings revealed the existence of several species including αCu 4 MT2, βCu 6 MT2, and βCu 6 αCu 4 MT2. These results are in line with those from the Stillman group, who studied Cu(I) binding to apoMT1a and showed that the α-domain binds four Cu(I) ions, while the β-domain binds six Cu(I), forming copper-thiolate clusters. , In their recent research, they showed that Cu(I) (not Cu(II)) binds to Zn 7 MT3 forming multiple species, with no preference for Cu 4 Zn 4 MT3 . In another study, Melenbacher et al studied Cu(I) binding to Zn 7 MT1a instead of apoMT1a .…”

supporting

confidence: 83%

“… 36 , 37 In their recent research, they showed that Cu(I) (not Cu(II)) binds to Zn 7 MT3 forming multiple species, with no preference for Cu 4 Zn 4 MT3. 38 In another study, Melenbacher et al studied Cu(I) binding to Zn 7 MT1a instead of apoMT1a. 39 Their results suggested the formation of two main complexes, βZn 1 Cu 5 αZn 4 MT1a and βCu 6 αZn 4 MT1a.…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Peris-Díaz,

Wu,

Mosna

et al. 2023

Anal. Chem.

View full text Add to dashboard Cite

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

show abstract

supporting

confidence: 83%

mentioning

confidence: 99%

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

Peris-Díaz,

Wu,

Mosna

et al. 2023

Anal. Chem.

View full text Add to dashboard Cite

show abstract

“…When using Cu(I) as a metal source rather than Cu(II), no domain preference was observed. 47,48 Therefore, these results indicate that the intramolecular disulfide formation formed upon Cu(II) reduction to Cu(I) dictates the domain preference and not the nature of the metal ion.…”

Section: Cid Experiments On Cu(i)/zn(ii)-nem X Mt3 Ox Speciesmentioning

confidence: 90%

“…43−46 Interestingly, no preference for a specific domain was found when using Cu(I) titrations on Zn 7 MT3. 47,48 Using this protein, we demonstrated that NEM could be employed in the initial labeling step to dissociate all Zn(II) ions while Cu(I) remains bound due to its higher affinity for Cys. In the second labeling step, an excess of IAM is utilized to label Cu(I)-bound Cys residues, leading to Cu(I) dissociation.…”

Section: ■ Introductionmentioning

confidence: 99%

Combining Native Mass Spectrometry and Proteomics to Differentiate and Map the Metalloform Landscape in Metallothioneins

Peris-Díaz,

Orzeł,

et al. 2024

J. Proteome Res.

View full text Add to dashboard Cite

Within the intricate landscape of the proteome, approximately 30% of all proteins bind metal ions. This repertoire is even larger when considering all the different forms of a protein, known as proteoforms. Here, we propose the term "metalloforms" to refer to different structural or functional variations of a protein resulting from the binding of various hetero-or homogeneous metal ions. Using human Cu(I)/Zn(II)-metallothionein-3 as a representative model, we developed a chemical proteomics strategy to simultaneously differentiate and map Zn(II) and Cu(I) metal binding sites. In the first labeling step, Nethylmaleimide reacts with Cysteine (Cys), resulting in the dissociation of all Zn(II) ions while Cu(I) remains bound to the protein. In the second labeling step, iodoacetamide is utilized to label Cu(I)-bound Cys residues. Native mass spectrometry (MS) was used to determine the metal/labeling protein stoichiometries, while bottom-up/top-down MS was used to map the Cys-labeled residues. Next, we used a developed methodology to interrogate an isolated rabbit liver metallothionein fraction containing three metallothionein-2 isoforms and multiple Cd(II)/Zn(II) metalloforms. The approach detailed in this study thus holds the potential to decode the metalloproteoform diversity within other proteins.

show abstract

Cu(I) binds to Zn7-MT2 via two parallel pathways

Melenbacher,

Stillman

2023

Metallomics

View full text Add to dashboard Cite

Metallothionein proteins are essential for Cu(I) and Zn(II) homeostasis as well as heavy metal detoxification. The metallation properties of MT2 are of great interest due to their wide patterns of expression and correlation with multiple diseases including cancers, neurological disorders, and respiratory diseases. Use of isotopically pure 63Cu(I) and 68Zn(II) eliminates the complexity of the Cu, Zn-MT2 mass spectral peaks due to significant overlap of naturally abundant isotopes. This allows for the resolution of the precise Cu(I) and Zn(II) stoichiometries when both Cu(I) and Zn(II) are bound to MT2 at physiological pH as expected in vivo. Exact Cu: Zn ratios were determined from mass spectral simulations carried out for every point in the titration. We report that Cu(I) metallation of Zn7-MT2 can only be understood in terms of two pathways occurring in parallel with pathway ① resulting in Cu5Zn5-MT2 and Cu9Zn3-MT2. Pathway ② results in Cu6Zn4-MT2 and Cu10Zn2-MT2, which are the major products of the reaction. From the electrospray ionization (ESI)–mass spectral data we report a series of formation constants (KF) for species starting from Zn7-MT2 up to Cu11Zn2-MT2. Room temperature phosphorescence and circular dichroism (CD) spectra were measured in parallel with the ESI–mass spectrometry data allowing for the assignment of specific species to specific spectral bands. Through analysis of the CD spectral bands, we propose that Cu(I) binds to the β domain first to form a Cu5Zn1 cluster or Cu6 cluster with emission at 670 and 750 nm, respectively, leaving the Zn4 cluster in the α domain.

show abstract

Metallothionein‐3: ⁶³Cu(I) binds to human ⁶⁸Zn₇‐βα MT3 with no preference for Cu₄‐β cluster formation

Cited by 5 publications

References 96 publications

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

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

Combining Native Mass Spectrometry and Proteomics to Differentiate and Map the Metalloform Landscape in Metallothioneins

Cu(I) binds to Zn7-MT2 via two parallel pathways

Contact Info

Product

Resources

About

Metallothionein‐3: 63Cu(I) binds to human 68Zn7‐βα MT3 with no preference for Cu4‐β cluster formation

Cited by 5 publications

References 96 publications

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

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

Combining Native Mass Spectrometry and Proteomics to Differentiate and Map the Metalloform Landscape in Metallothioneins

Cu(I) binds to Zn7-MT2 via two parallel pathways

Contact Info

Product

Resources

About

Metallothionein‐3: ⁶³Cu(I) binds to human ⁶⁸Zn₇‐βα MT3 with no preference for Cu₄‐β cluster formation