Joint forces of mass spectrometric techniques (ICP-MS and MALDI-TOF-MS) and fluorescence spectrometry in the study of platinum-based cytostatic drugs interactions with metallothionein MT2 and MT3.

Herby, the interaction of metallothioneins with commonly used Pt-based anticancer drugs - cisplatin, carboplatin, and oxaliplatin - was investigated using the combined power of elemental (i.e. LA-ICP-MS, CE-ICP-MS) and molecular (i.e. MALDI-TOF-MS) analytical techniques providing not only required information about the interaction, but also the benefit of low sample consumption. The amount of Cd and Pt incorporated within the protein was determined for protein monomers and dimer/oligomers formed by non-oxidative dimerization. Moreover, fluorescence spectrometry using Zn2+-selective fluorescent indicator - FluoZin3 - was employed to monitor the ability of Pt drugs to release natively occurring Zn from the protein molecule. The investigation was carried out using two protein isoforms (i.e. MT2, MT3), and significant differences in behaviour of these two isoforms were observed. The main attention was paid to elucidating whether the protein dimerization/oligomerization may be the reason for the potential failure of the anticancer therapy based on these drugs. Based on the results, it was demonstrated that the interaction of MT2 (both monomers and dimers) interacted with Pt drugs significantly less compared to MT3 (both monomers and dimers). Also, a significant difference between monomeric and dimeric forms (both MT2 and MT3) was not observed. This may suggest that dimer formation is not the key factor leading to the inactivation of Pt drugs.

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

Mammalian zinc metallothionein-3 (Zn7MT3) plays an important role in protecting against copper toxicity by scavenging free Cu(II) ions or removing Cu(II) bound to ß-amyloid and α-synuclein. While previous studies reported that Zn7MT3 reacts with Cu(II) ions to form Cu(I)4Zn(II)4MT3ox 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 Zn7MT3 and Cu(II) ions using native ion mobility mass spectrometry (IM-MS). The complex formation mechanism was found to involve the disassembly of Zn3S9 and Zn4S11 clusters from Zn7MT3 and reassembly into Cu(I)xZn(II)yMT3ox complexes rather than simply Zn(II)-to-Cu(I) exchange. At neutral pH, the ß-domain was shown to be capable of binding up to six Cu(I) ions to form Cu(I)6Zn(II)4MT3ox, although the most predominant species was the Cu(I)4Zn(II)4MT3ox 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 Zn7MT3 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)4Zn(II)4MT3ox conformation is more stable than Zn7MT3. Native top-down MS demonstrated that the Cu(I) ions are exclusively bound to the ß-domain in the Cu(I)4Zn(II)4MT3ox 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)xZn(II)yMT3ox complexes.

The connection of α- and ß-domains in mammalian metallothionein-2 differentiates Zn(II) binding affinities, affects folding, and determines zinc buffering properties.

Mammalian metallothioneins (MTs) are small Cys-rich proteins involved in Zn(II) and Cu(I) homeostasis. They bind seven Zn(II) ions in two distinct ß- and α-domains, forming Zn3Cys9 and Zn4Cys11 clusters, respectively. After six decades of research, their role in cellular buffering of Zn(II) ions has begun to be understood recently. This is because of different affinities of bound ions and the proteins' coexistence in variously Zn(II)-loaded Zn4-7MT species in the cell. To date, it has remained unclear how these mechanisms of action occur and how the affinities are differentiated despite the Zn(S-Cys)4 coordination environment being the same. Here, we dissect the molecular basis of these phenomena by using several MT2 mutants, hybrid protein, and isolated domains. Through a combination of spectroscopic and stability studies, thiol(ate) reactivity, and steered molecular dynamics, we demonstrate that both protein folding and thermodynamics of Zn(II) ion (un)binding significantly differ between isolated domains and the whole protein. Close proximity reduces the degrees of freedom of separated domains, making them less dynamic. It is caused by the formation of intra- and interdomain electrostatic interactions. The energetic consequence of domains connection has a critical impact on the role of MTs in the cellular environment, where they function not only as a zinc sponge but also as a zinc buffering system keeping free Zn(II) in the right concentrations. Any change of that subtle system affects the folding mechanism, zinc site stabilities, and cellular zinc buffer components.