Metallothioneins (MTs) are a class of low molecular weight and cysteine-rich metal binding proteins present in all the branches of the tree of life. MTs efficiently bind with high affinity several essential and toxic divalent and monovalent transition metals by forming characteristic polynuclear metalthiolate clusters within their structure. MTs fulfil multiple biological functions related to their metal binding properties, with essential roles in both Zn(II) and Cu(I) homeostasis as well as metal detoxification. Depending on the organism considered, the primary sequence, and the specific physiological and The presence of nonsulfur coordination residues (e.g., histidine) in some members of the superfamily, such as bacterial and plant MTs, has broken the dogma of the exclusive metal coordination by cysteine thiolate residues in MTs. According to Binz and K€ agi (2), MTs are classified into 15 families based on taxonomic parameters and the patterns of distribution of Cys residues in their sequence. The recognized fundamental functions of MTs arise from their capabilities to bind transition metals with high affinity, and their primary biological roles include homeostasis of essential trace metals zinc and copper, and sequestration and protection from environmental toxic metals such as cadmium, mercury, and lead (1). In addition, in light of the reactivity of the metal-coordinating thiolate ligands, MTs play fundamental roles in protection against oxidative stress including reactive oxygen and nitrogen species and other free radicals (1,3-5). However, additional specific MT functions arise from specific biological needs and complexity of the organisms in which they are expressed. Since 60 years from their discovery, it emerged that metallothoineins indeed possess complex pleiotropic functions. This is exemplified by the better-studied mammalian MTs for which additional specialized roles in adaptation to stress, protection against brain injury, regulation of neuronal outgrowth, antiapoptotic effects, and reactivity and inactivation of metal-based chemotherapeutics leading to resistance have been demonstrated (4).As a result of the high number of thiolate coordinating residues in MTs and the lack of defined three-dimensional (3D) structures in the metal depleted apo forms, MTs can bind a number of different monovalent and divalent metals both in vitro and in vivo. The affinity of the metal ions for the binding
Copper is controlled by a sophisticated network of transport and storage proteins within mammalian cells, yet its uptake and efflux occur with rapid kinetics. Present as Cu(I) within the reducing intracellular environment, the nature of this labile copper pool remains elusive. While glutathione is involved in copper homeostasis and has been assumed to buffer intracellular copper, we demonstrate with a ratiometric fluorescent indicator, crisp-17, that cytosolic Cu(I) levels are buffered to the vicinity of 1 aM, where negligible complexation by glutathione is expected. Enabled by our phosphine sulfide-stabilized phosphine (PSP) ligand design strategy, crisp-17 offers a Cu(I) dissociation constant of 8 aM, thus exceeding the binding affinities of previous synthetic Cu(I) probes by four to six orders of magnitude. Two-photon excitation microscopy with crisp-17 revealed rapid, reversible increases in intracellular Cu(I) availability upon addition of the ionophoric complex CuGTSM or the thiol-selective oxidant 2,2′-dithiodipyridine (DTDP). While the latter effect was dramatically enhanced in 3T3 cells grown in the presence of supplemental copper and in cultured Menkes mutant fibroblasts exhibiting impaired copper efflux, basal Cu(I) availability in these cells showed little difference from controls, despite large increases in total copper content. Intracellular copper is thus tightly buffered by endogenous thiol ligands with significantly higher affinity than glutathione. The dual utility of crisp-17 to detect normal intracellular buffered Cu(I) levels as well as to probe the depth of the labile copper pool in conjunction with DTDP provides a promising strategy to characterize perturbations of cellular copper homeostasis.
Mammalian metallothioneins (MT-1 through MT-4) are a class of metal binding proteins containing two metal-thiolate clusters formed through the preferential coordination of d10 metals, Cu(I) and Zn(II), by 20 conserved cysteine residues located in two protein domains. MT metalation (homometallic or heterometallic Zn(II)/Cu(I) species) appears to be isoform specific and controlling zinc and copper concentrations to perform specific and distinct biological functions. Structural and functional relationships, and in vivo metalation studies, identified evolutionary features defining the metal-selectivity nature for MTs. Metallothionein-3 (MT-3) has been shown to possess the most pronounced Cu-thionein character forming Cu(I)-containing species more favorably than metallothionein-2 (MT-2), which possesses the strongest Zn-thionein character. In this work, we identify isoform-specific determinants which control metal binding selectivity bias in different MTs isoforms. By studying the reactivity of Zn7MT-2, Zn7MT-3 and Zn7MT-3 mutants towards Cu(II) to form Cu(I)4Zn4MTs, we have identified isoform-specific key non-coordinating residues governing folding/outer sphere control of metal selectivity bias in MTs metal clusters. By mutating selected residues and motifs in MT-3 to the corresponding MT-2 amino acids, we dissected key roles in modulating cluster dynamic and metal exchange rates, in increasing the Cu(I)-affinity in MT-3 N-terminal β-domain and/or modulating the higher stability of the Zn(II)-thiolate cluster in MT-2 β-domain. We thus engineered MT-3 variants in which the copper-thionein character is converted into a zinc-thionein. These results provide new insights into the molecular determinants governing metal selectivity in metal-thiolate clusters.
Copper complexes are of medicinal and biological interest, including as anticancer drugs designed to cleave intracellular biomolecules by O 2 activation. To exhibit such activity,the copper complex must be redox active and resistant to dissociation. Metallothioneins (MTs) and glutathione (GSH) are abundant in the cytosol and nucleus.Because they are thiol-richr educing molecules with high Cu I affinity,t hey are potential competitors for ac opper ion bound in ac opper drug. Herein, we report the investigation of apanel of Cu I /Cu II complexes often used as drugs,w ith diverse coordination chemistries and redox potentials.W ee valuated their catalytic activity in ascorbate oxidation based on redox cycling between Cu I and Cu II ,a sw ell as their resistance to dissociation or inactivation under cytosolically relevant concentrations of GSH and MT.O 2 -activating Cu I /Cu II complexes for cytosolic/nuclear targets are generally not stable against the GSH/MT system, whichcreates achallenge for their future design.
Edited by Ruma BanerjeeCopper (Cu) is essential for the survival of aerobic organisms through its interaction with molecular oxygen (O 2 ). However, Cu's chemical properties also make it toxic, requiring specific cellular mechanisms for Cu uptake and handling, mediated by Cu chaperones. CCS1, the budding yeast (S. cerevisiae) Cu chaperone for Cu-zinc (Zn) superoxide dismutase (SOD1) activates by directly promoting both Cu delivery and disulfide formation in SOD1. The complete mechanistic details of this transaction along with recently proposed molecular chaperone-like functions for CCS1 remain undefined. Here, we present combined structural, spectroscopic, kinetic, and thermodynamic data that suggest a multifunctional chaperoning role(s) for CCS1 during SOD1 activation. We observed that CCS1 preferentially binds a completely immature form of SOD1 and that the SOD1⅐CCS1 interaction promotes high-affinity Zn(II) binding in SOD1. Conserved aromatic residues within the CCS1 C-terminal domain are integral in these processes. Previously, we have shown that CCS1 delivers Cu(I) to an entry site at the SOD1⅐CCS1 interface upon binding. We show here that Cu(I) is transferred from CCS1 to the entry site and then to the SOD1 active site by a thermodynamically driven affinity gradient. We also noted that efficient transfer from the entry site to the active site is entirely dependent upon the oxidation of the conserved intrasubunit disulfide bond in SOD1. Our results herein provide a solid foundation for proposing a complete molecular mechanism for CCS1 activity and reclassification as a first-of-its-kind "dual chaperone."Copper (Cu) 2 is required for the activation of dioxygen, a function essential for the survival of aerobic organisms (1). Figure 4. Kinetic analysis of CCS1 mediated copper delivery to sites on SOD1. Stopped-flow kinetic traces at 260 nm and corresponding curve fittings (red line) obtained upon rapid mixing of Cu(I)-Ccs (20 M) with equal volumes of apo H46R/H48Q (X,Zn-SOD1 SH ) or WT E,Zn-SOD1 SH (20 M) under aerobic or anaerobic conditions are shown. The decreased absorbance at 260 nm (top left) corresponds to the copper ion moving from the cysteine coordination of CCS1 and/or SOD1 and entering the histidine coordination of the SOD1 active site. Increased absorbance at 260 nm shows the copper ion transfer from CCS1 cysteines to the SOD1⅐CCS1 2Cys/1His entry site.
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.
Zinc (II) ions (hereafter simplified as zinc) are important for the structural and functional activity of many proteins. For Cu, Zn superoxide dismutase (Sod1), zinc stabilizes the native structure of each Sod1 monomer, promotes homo-dimerization and plays an important role in activity by “softening” the active site so that copper cycling between Cu(I) and Cu(II) can rapidly occur. Previously, we have reported that binding of Sod1 by its copper chaperone (Ccs) stabilizes a conformation of Sod1 that promotes site-specific high-affinity zinc binding. While there are a multitude of Sod1 mutations linked to the familial form of amyotrophic lateral sclerosis (fALS), characterizations by multiple research groups have been unable to realize strong commonalities among mutants. Here, we examine a set of fALS-linked Sod1 mutations that have been well-characterized and are known to possess variation in their biophysical characteristics. The zinc affinities of these mutants are evaluated here for the first time and then compared with the previously established value for wild-type Sod1 zinc affinity. Ccs does not have the same ability to promote zinc binding to these mutants as it does for the wild-type version of Sod1. Our data provides a deeper look into how (non)productive Sod1 maturation by Ccs may link a diverse set of fALS-Sod1 mutations.
Proteinaceous nanomaterials and, in particular, virus-like particles (VLPs) have emerged as robust and uniform platforms that are seeing wider use in biomedical research. However, there are a limited number of bioconjugation reactions for functionalizing the capsids, and very few of those involve functionalization across the supramolecular quaternary structure of protein assemblies. In this work, we exploit the recently described dibromomaleimide moiety as part of a bioconjugation strategy on VLP Qβ to break and rebridge the exposed and structurally important disulfides in good yields. Not only was the stability of the quaternary structure retained after the reaction, but the newly functionalized particles also became brightly fluorescent and could be tracked in vitro using a commercially available filter set. Consequently, we show that this highly efficient bioconjugation reaction not only introduces a new functional handle "between" the disulfides of VLPs without compromising their thermal stability but also can be used to create a fluorescent probe.
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