Metallothioneins (MTs) are a ubiquitous class of small metal-binding proteins involved in metal homeostasis and detoxification. While known for their high affinity for d10 metal ions, there is a surprising...

The human copper-binding protein metallothionein-3 (MT-3) can reduce Cu­(II) to Cu­(I) and form a polynuclear Cu­(I)4-Cys5–6 cluster concomitant with intramolecular disulfide bonds formation, but the cluster is unusually inert toward O2 and redox-cycling. We utilized a combined array of rapid-mixing spectroscopic techniques to identify and characterize the transient radical intermediates formed in the reaction between Zn7MT-3 and Cu­(II) to form Cu­(I)4Zn­(II)4MT-3. Stopped-flow electronic absorption spectroscopy reveals the rapid formation of transient species with absorption centered at 430–450 nm and consistent with the generation of disulfide radical anions (DRAs) upon reduction of Cu­(II) by MT-3 cysteine thiolates. These DRAs are oxygen-stable and unusually long-lived, with lifetimes in the seconds regime. Subsequent DRAs reduction by Cu­(II) leads to the formation of a redox-inert Cu­(I)4-Cys5 cluster with short Cu–Cu distances (<2.8 Å), as revealed by low-temperature (77 K) luminescence spectroscopy. Rapid freeze-quench Raman and electron paramagnetic resonance (EPR) spectroscopy characterization of the intermediates confirmed the DRA nature of the sulfur-centered radicals and their subsequent oxidation to disulfide bonds upon Cu­(II) reduction, generating the final Cu­(I)4-thiolate cluster. EPR simulation analysis of the radical g- and A-values indicate that the DRAs are directly coupled to Cu­(I), potentially explaining the observed DRA stability in the presence of O2. We thus provide evidence that the MT-3 Cu­(I)4-Cys5 cluster assembly process involves the controlled formation of novel long-lived, copper-coupled, and oxygen-stable disulfide radical anion transient intermediates.

In eukaryotes, iron-sulfur clusters are essential cofactors for numerous physiological processes, but these clusters are primarily biosynthesized in mitochondria. Previous studies suggest mitochondrial ABCB7-type exporters are involved in maturation of cytosolic iron-sulfur proteins. However, the molecular mechanism for how the ABCB7-type exporters participate in this process remains elusive. Here, we report a series of cryo-electron microscopy structures of a eukaryotic homolog of human ABCB7, CtAtm1, determined at average resolutions ranging from 2.8 to 3.2 Å, complemented by functional characterization and molecular docking in silico. We propose that CtAtm1 accepts delivery from glutathione-complexed iron-sulfur clusters. A partially occluded state links cargo-binding to residues at the mitochondrial matrix interface that line a positively charged cavity, while the binding region becomes internalized and is partially divided in an early occluded state. Collectively, our findings substantially increase the understanding of the transport mechanism of eukaryotic ABCB7-type proteins.

Detection of anions in complex aqueous media is a fundamental challenge with practical utility that can be addressed by supramolecular chemistry. Biomolecular hosts such as proteins can be used and adapted as an alternative to synthetic hosts. Here, we report how the mutagenesis of the β-bulge residues (D137 and W138) in mNeonGreen, a bright, monomeric fluorescent protein, unlocks and tunes the anion preference at physiological pH for sulfate, resulting in the turn-off sensor SulfOFF-1. This unprecedented sensing arises from an enhancement in the kinetics of binding, largely driven by position 138. In line with these data, molecular dynamics (MD) simulations capture how the coordinated entry and gating of sulfate into the β-barrel is eliminated upon mutagenesis to facilitate binding and fluorescence quenching.

Cis-diamminedichloroplatinum(II) (cisplatin) is a widely used metal-based chemotherapeutic drug for the treatment of cancers. However, intrinsic and acquired drug resistance limit the efficacy of cisplatin-based treatments. Increased production of intracellular thiol-rich molecules, in particular metallothioneins (MTs), which form stable coordination complexes with the electrophilic cisplatin, results in cisplatin sequestration leading to pre-target resistance. MT-1/-2 are overexpressed in cancer cells, and their expression is controlled by the Metal Response Element (MRE)-binding Transcription factor-1 (MTF-1), featuring six Cys2His2-type zinc fingers which, upon zinc metalation, recognize specific MRE sequences in the promoter region of MT genes triggering their expression. Cisplatin can efficiently react with protein metal binding sites featuring nucleophilic cysteine and/or histidine residues, including MTs and zinc fingers proteins, but the preferential reactivity towards specific targets with competing binding sites cannot be easily predicted. In this work, by in vitro competition reactions, we investigated the thermodynamic and kinetic preferential reactivity of cisplatin towards human Zn7MT-2, each of the six MTF-1 zinc fingers, and the entire human MTF-1 zinc finger domain. By spectroscopic, spectrometric and electrophoretic mobility shift assays (EMSA), we demonstrated that cisplatin preferentially reacts with Zn7MT-2 to form Cys4-Pt(II) complexes, resulting in zinc release from MT-2. Zinc transfer from MT-2 to the MTF-1 triggers MTF-1 metalation, activation and binding to target MRE sequences, as demonstrated by EMSA with DNA oligonucleotides. The cisplatin-dependent MT-mediated MTF-1 activation leading to apo-MT overexpression potentially establishes one of the molecular mechanisms underlying the development and potentiation of MT-mediated pre-target resistance.