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The p53 protein plays a major role in cancer prevention, and over 50% of cancer diagnoses can be attributed to p53 malfunction. p53 incorporates a structural Zn site that is required for proper protein folding and function, and in many cases point mutations can result in loss of the Zn2+ ion, destabilization of the tertiary structure, and eventual amyloid aggregation. Herein, we report a series of compounds designed to act as small molecule stabilizers of mutant p53, and feature Zn-binding fragments to chaperone Zn2+ to the metal depleted site and restore wild-type (WT) function. Many Zn metallochaperones (ZMCs) have been shown to generate intracellular reactive oxygen species (ROS), likely by chelating redox-active metals such as Fe2+/3+ and Cu+/2+ and undergoing associated Fenton chemistry. High levels of ROS can result in off-target effects and general toxicity, and thus, careful tuning of ligand Zn2+ affinity, in comparison to the affinity for other endogenous metals, is important for selective mutant p53 targeting. In this work we show that by using carboxylate donors in place of pyridine we can change the relative Zn2+/Cu2+ binding ability in a series of ligands, and we investigate the impact of donor group changes on metallochaperone activity and overall cytotoxicity in two mutant p53 cancer cell lines (NUGC3 and SKGT2).

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The p53 protein, known as the 'guardian of the genome', plays an important role in cancer prevention. Unfortunately, p53 mutations result in compromised activity with over 50% of cancers resulting from point mutations to p53. There is considerable interest in mutant p53 reactivation, with the development of small-molecule reactivators showing promise. We have focused our efforts on the common p53 mutation Y220C, which causes protein unfolding, aggregation, and can result in the loss of a structural Zn from the DNA-binding domain. In addition, the Y220C mutant creates a surface pocket that can be stabilized using small molecules. We previously reported the bifunctional ligand L5 as a Zn metallochaperone and reactivator of the p53-Y220C mutant. Herein we report two new ligands L5-P and L5-O that are designed to act as Zn metallochaperones and non-covalent binders in the Y220C mutant pocket. For L5-P the distance between the Zn-binding di-(2-picolyl)amine function and the pocket-binding diiodophenol was extended in comparison to L5, while for L5-O we extended the pocket-binding moiety via attachment of an alkyne function. While both new ligands displayed similar Zn-binding affinity to L5, neither acted as efficient Zn-metallochaperones. However, the new ligands exhibited significant cytotoxicity in the NCI-60 cell line screen as well as in the NUGC3 Y220C mutant cell line. We identified that the primary mode of cytotoxicity is likely reactive oxygen species (ROS) generation for L5-P and L5-O, in comparison to mutant p53 reactivation for L5, demonstrating that subtle changes to the ligand scaffold can change the toxicity pathway.

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The bioavailability of copper (Cu) in human cells may depend on a complex interplay with zinc (Zn) ions. We investigated the ability of the Zn ion to target the human Cu-chaperone Atox1, a small cytosolic protein capable of anchoring Cu(I), by a conserved surface-exposed Cys-X-X-Cys (CXXC) motif, and deliver it to Cu-transporting ATPases in the trans-Golgi network. The crystal structure of Atox1 loaded with Zn displays the metal ion bridging the CXXC motifs of two Atox1 molecules in a homodimer. The identity and location of the Zn ion were confirmed through the anomalous scattering of the metal by collecting X-ray diffraction data near the Zn K-edge. Furthermore, soaking experiments of the Zn-loaded Atox1 crystals with a strong chelating agent, such as EDTA, caused only limited removal of the metal ion from the tetrahedral coordination cage, suggesting a potential role of Atox1 in Zn metabolism and, more generally, that Cu and Zn transport mechanisms could be interlocked in human cells.

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A large percentage of transcription factors require zinc to bind DNA. In this review, we discuss what makes p53 unique among zinc-dependent transcription factors. The conformation of p53 is unusually malleable: p53 binds zinc extremely tightly when folded, but is intrinsically unstable in the absence of zinc at 37°C. Whether the wild-type protein folds in the cell is largely determined by the concentration of available zinc. Consequently, zinc dysregulation in the cell as well as a large percentage of tumorigenic p53 mutations can cause p53 to lose zinc, misfold, and forfeit its tumor suppressing activity. We highlight p53's noteworthy biophysical properties that give rise to its malleability and how proper zinc binding can be restored by synthetic metallochaperones to reactivate mutant p53. The activity and mechanism of metallochaperones are compared to those of other mutant p53-targeted drugs with an emphasis on those that have reached the clinical trial stage.

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Metalloenzymes catalyze a diverse set of challenging chemical reactions that are essential for life. These metalloenzymes rely on a wide range of metallocofactors, from single metal ions to complicated metallic clusters. Incorporation of metal ions and metallocofactors into apo-proteins often requires the assistance of proteins known as metallochaperones. Nucleoside triphosphate hydrolases (NTPases) are one important class of metallochaperones and are found widely distributed throughout the domains of life. These proteins use the binding and hydrolysis of nucleoside triphosphates, either adenosine triphosphate or guanosine triphosphate, to carry out highly specific and regulated roles in the process of metalloenzyme maturation. Here, we review recent literature on NTPase metallochaperones and describe the current mechanistic proposals and available structural data. By using representative examples from each type of NTPase, we also illustrate the challenges in studying these complicated systems. We highlight open questions in the field and suggest future directions. This minireview is part of a special collection of articles in memory of Professor Deborah Zamble, a leader in the field of nickel biochemistry.

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Metal ion dysregulation has been implicated in a number of diseases from neurodegeneration to cancer. While defective metal ion transport mechanisms are known to cause specific diseases of genetic origin, the role of metal dysregulation in many diseases has yet to be elucidated due to the complicated function (both good and bad!) of metal ions in the body. A breakdown in metal ion speciation can manifest in several ways from increased reactive oxygen species (ROS) generation to an increase in protein misfolding and aggregation. In this review, we will discuss the role of Zn in the proper function of the p53 protein in cancer. The p53 protein plays a critical role in the prevention of genome mutations via initiation of apoptosis, DNA repair, cell cycle arrest, anti-angiogenesis, and senescence pathways to avoid propagation of damaged cells. p53 is the most frequently mutated protein in cancer and almost all cancers exhibit malfunction along the p53 pathway. Thus, there has been considerable effort dedicated to restoring normal p53 expression and activity to mutant p53. This includes understanding the relative populations of the Zn-bound and Zn-free p53 in wild-type and mutant forms, and the development of metallochaperones to re-populate the Zn binding site to restore mutant p53 activity. Parallels will be made to the development of multifunctional metal binding agents for modulating the aggregation of the amyloid-beta peptide in Alzheimer's Disease (AD).

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Metallochaperones are a class of unique protein families that was originally found to interact with cellular metal ions by metal delivery to specific target proteins such as metal enzymes. Recently, some members of metallochaperones receive much attention owning to their multi-biological functions in mediating plant growth, development and biotic or abiotic stress responses, particularly in the aspects of metal transport and accumulation in plants. For example, some non-essential toxic heavy metals (e.g. cadmium and mercury) accumulating in farmland due to the industrial and agronomic activities, are a constant threat to crop production, food safety and human health. Digging genetic resources and functional genes like metallochaperones is critical for understanding the metal detoxification in plants, and may help develop cleaner crops with minimal toxic metals in leafy vegetables and grains, or plants for metal-polluted soil phytoremediation. In this review, we highlight the current advancement of the research on functions of metallochaperones in metal accumulation, detoxification and homeostasis. We also summarize the recent progress of the research on the critical roles of the metal-binding proteins in regulating plant responses to some other biological processes including plant growth, development, pathogen stresses, and abiotic stresses such salt, drought, cold and light. Finally, an additional capacity of some members of metallochaperones involved in the resistance to the pathogen attack and possibly regulatory roles was reviewed.

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The mitochondrial cytochrome c oxidase, the terminal enzyme of the respiratory chain, contains heme and copper centers for electron transfer. The conserved COX2 subunit contains the CuA site, a binuclear copper center. The copper chaperones SCO1, SCO2, and COA6, are required for CuA center formation. Loss of function of these chaperones and the concomitant cytochrome c oxidase deficiency cause severe human disorders. Here we analyzed the molecular function of COA6 and the consequences of COA6 deficiency for mitochondria. Our analyses show that loss of COA6 causes combined complex I and complex IV deficiency and impacts membrane potential-driven protein transport across the inner membrane. We demonstrate that COA6 acts as a thiol-reductase to reduce disulfide bridges of critical cysteine residues in SCO1 and SCO2. Cysteines within the CX3CXNH domain of SCO2 mediate its interaction with COA6 but are dispensable for SCO2-SCO1 interaction. Our analyses define COA6 as thiol-reductase, which is essential for CuA biogenesis.

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Cancer cells cope with high oxidative stress levels, characterized by a shift toward the oxidized form (GSSG) of glutathione (GSH) in the redox couple GSSG/2GSH. Under these conditions, the cytosolic copper chaperone Atox1, which delivers Cu(I) to the secretory pathway, gets oxidized, i.e., a disulfide bond is formed between the cysteine residues of the Cu(I)-binding CxxC motif. Switching to the covalently-linked form, sulfur atoms are not able to bind the Cu(I) ion and Atox1 cannot play an antioxidant role. Atox1 has also been implicated in the resistance to platinum chemotherapy. In the presence of excess GSH, the anticancer drug cisplatin binds to Cu(I)-Atox1 but not to the reduced apoprotein. With the aim to investigate the interaction of cisplatin with the disulfide form of the protein, we performed a structural characterization in solution and in the solid state of oxidized human Atox1 and explored its ability to bind cisplatin under conditions mimicking an oxidizing environment. Cisplatin targets a methionine residue of oxidized Atox1; however, in the presence of GSH as reducing agent, the drug binds irreversibly to the protein with ammine ligands trans to Cys12 and Cys15. The results are discussed with reference to the available literature data and a mechanism is proposed connecting platinum drug processing to redox and copper homeostasis.

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The di-copper center CuA is an essential metal cofactor in cytochrome oxidase (Cox) of mitochondria and many prokaryotes, mediating one-electron transfer from cytochrome c to the site for oxygen reduction. CuA is located in subunit II (CoxB) of Cox and protrudes into the periplasm of Gram-negative bacteria or the mitochondrial intermembrane space. How the two copper ions are brought together to build CoxB·CuA is the subject of this review. It had been known that the reductase TlpA and the metallochaperones ScoI and PcuC are required for CuA formation in bacteria, but the mechanism of copper transfer has emerged only recently for the Bradyrhizobium diazoefficiens system. It consists of the following steps: (a) TlpA keeps the active site cysteine pair of CoxB in its dithiol state as a prerequisite for metal insertion; (b) ScoI·Cu2+ rapidly forms a transient complex with apo-CoxB; (c) PcuC, loaded with Cu1+ and Cu2+ , dissociates this complex to CoxB·Cu2+ , and a second PcuC·Cu1+ ·Cu2+ transfers Cu1+ to CoxB·Cu2+ , yielding mature CoxB·CuA . Variants of this pathway might exist in other bacteria or mitochondria.

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Helicobacter pylori HypA (HpHypA) is a metallochaperone necessary for maturation of [Ni,Fe]-hydrogenase and urease, the enzymes required for colonization and survival of H. pylori in the gastric mucosa. HpHypA contains a structural Zn(II) site and a unique Ni(II) binding site at the N-terminus. X-ray absorption spectra suggested that the Zn(II) coordination depends on pH and on the presence of Ni(II). This study was performed to investigate the structural properties of HpHypA as a function of pH and Ni(II) binding, using NMR spectroscopy combined with DFT and molecular dynamics calculations. The solution structure of apo,Zn-HpHypA, containing Zn(II) but devoid of Ni(II), was determined using 2D, 3D and 4D NMR spectroscopy. The structure suggests that a Ni-binding and a Zn-binding domain, joined through a short linker, could undergo mutual reorientation. This flexibility has no physiological effect on acid viability or urease maturation in H. pylori. Atomistic molecular dynamics simulations suggest that Ni(II) binding is important for the conformational stability of the N-terminal helix. NMR chemical shift perturbation analysis indicates that no structural changes occur in the Zn-binding domain upon addition of Ni(II) in the pH 6.3-7.2 range. The structure of the Ni(II) binding site was probed using 1H NMR spectroscopy experiments tailored to reveal hyperfine-shifted signals around the paramagnetic metal ion. On this basis, two possible models were derived using quantum-mechanical DFT calculations. The results provide a comprehensive picture of the Ni(II) mode to HpHypA, important to rationalize, at the molecular level, the functional interactions of this chaperone with its protein partners.

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Immature copper-zinc superoxide dismutase (Sod1) is activated by its copper chaperone (Ccs1). Ccs1 delivers a single copper ion and catalyzes oxidation of an intra-subunit disulfide bond within each Sod1 monomer through a mechanistically ambiguous process. Here, we use residue specific fluorescent labeling of immature Sod1 to quantitate the thermodynamics of the Sod1•Ccs1 interaction while determining a more complete view of Ccs1 function. Ccs1 preferentially binds a completely immature form of Sod1 that is metal deficient and disulfide reduced (E, E-Sod1SH). However, binding induces structural changes that promote high-affinity zinc binding by the Ccs1-bound Sod1 molecule. This adds further support to the notion that Ccs1 likely plays dual chaperoning roles during the Sod1 maturation process. Further analysis reveals that in addition to the copper-dependent roles during Sod1 activation, the N- and C-terminal domains of Ccs1 also have synergistic roles in securing both Sod1 recognition and its own active conformation. These results provide new and measurable analyses of the molecular determinants guiding Ccs1-mediated Sod1 activation.

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Restoration of wild-type structure and function to mutant p53 with a small molecule (hereafter referred to as "reactivating" mutant p53) is one of the holy grails in cancer therapeutics. The majority of TP53 mutations are missense which generate a defective protein that is targetable. We are currently developing a new class of mutant p53 reactivators called zinc metallochaperones (ZMCs) and, here, we review our current understanding of them. The p53 protein requires the binding of a single zinc ion, coordinated by four amino acids in the DNA binding domain, for proper structure and function. Loss of the wild-type structure by impairing zinc binding is a common mechanism of inactivating p53. ZMCs reactivate mutant p53 using a novel two-part mechanism that involves restoring the wild-type structure by reestablishing zinc binding and activating p53 through post-translational modifications induced by cellular reactive oxygen species (ROS). The former causes a wild-type conformation change, the later induces a p53-mediated apoptotic program to kill the cancer cell. ZMCs are small molecule metal ion chelators that bind zinc and other divalent metal ions strong enough to remove zinc from serum albumin, but weak enough to donate it to mutant p53. Recently we have extended our understanding of the mechanism of ZMCs to the role of cells' response to this zinc surge. We found that cellular zinc homeostatic mechanisms, which normally function to maintain free intracellular zinc levels in the picomolar range, are induced by ZMCs. By normalizing zinc levels, they function as an OFF switch to ZMCs because zinc levels are no longer sufficiently high to maintain a wild-type structure. This on/off switch leads to a transient nature to the mechanism of ZMCs in which mutant p53 activity comes on in a few hours and then is turned off. This finding has important implications for the translation of ZMCs to the clinic because it indicates that ZMC concentrations need not be maintained at high levels for their activity. Indeed, we found that short exposures (as little as 15 min) were adequate to observe the mutant p53 reactivating activity. This switch mechanism imparts an advantage over other targeted therapeutics in that efficacy can be accomplished with minimal exposure which minimizes toxicity and maximizes the therapeutic window. This on/off switch mechanism is unique in targeted cancer therapeutics and will impact the design of human clinical trials.

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Copper-transporting P-type ATPases, which play important roles in trafficking Cu(I) across membranes for the biogenesis of copper proteins or for copper detoxification, contain a variable number of soluble metal-binding domains at their N-termini. It is increasingly apparent that these play an important role in regulating copper transport in a Cu(I)-responsive manner, but how they do this is unknown. CopA, a Cu(I)-transporter from Bacillus subtilis, contains two N-terminal soluble domains that are closely packed, with inter-domain interactions at two principal regions. Here, we sought to determine the extent to which the domains interact in the absence of their inter-domain covalent linker, and how their Cu(I)-binding properties are affected. Studies of a 1:1 mixture of separate CopAa and CopAb domains showed that the domains do not form a stable complex, with only indirect evidence of a weak interaction between them. Their Cu(I)-binding behaviour was distinct from that of the two domain protein and consistent with a lack of interaction between the domains. Cu(I)-mediated protein association was observed, but this occurred only between domains of the same type. Thus, the inter-domain covalent link between CopAa and CopAb is essential for inter-domain interactions and for Cu(I)-binding behaviour.

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The ability of metallochaperones to allosterically regulate the binding/release of metal ions and to switch protein-binding partners along the metal delivery pathway is essential to the metallation of the metalloenzymes. Urease, catalyzing the hydrolysis of urea into ammonia and carbon dioxide, contains two nickel ions bound by a carbamylated lysine in its active site. Delivery of nickel ions for urease maturation is dependent on GTP hydrolysis and is assisted by four urease accessory proteins UreE, UreF, UreG, and UreH(UreD). Here, we determined the crystal structure of the UreG dimer from Klebsiella pneumoniae in complex with nickel and GMPPNP, a nonhydrolyzable analog of GTP. Comparison with the structure of the GDP-bound Helicobacter pylori UreG (HpUreG) in the UreG2F2H2 complex reveals large conformational changes in the G2 region and residues near the 66CPH68 metal-binding motif. Upon GTP binding, the side chains of Cys66 and His68 from each of the UreG protomers rotate toward each other to coordinate a nickel ion in a square-planar geometry. Mutagenesis studies on HpUreG support the conformational changes induced by GTP binding as essential to dimerization of UreG, GTPase activity, in vitro urease activation, and the switching of UreG from the UreG2F2H2 complex to form the UreE2G2 complex with the UreE dimer. The nickel-charged UreE dimer, providing the sole source of nickel, and the UreG2F2H2 complex could activate urease in vitro in the presence of GTP. Based on our results, we propose a mechanism of how conformational changes of UreG during the GTP hydrolysis/binding cycle facilitate urease maturation.

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Cyclic and acyclic peptides with sequences derived from metallochaperone binding sites, but differing at position 2, were analyzed for their inhibitory reactivity towards cellular ROS (reactive oxygen species) formation and catalytic activity towards oxidation with H2 O2 , in comparison with three commercial drugs clinically employed in chelation therapy for Wilson's disease. Acyclic peptides were more effective inhibitors than the cyclic ones, with one leading peptide with threonine at position 2 systematically showing the highest efficiency in reducing cellular ROS levels and in inhibiting Cu oxidation. This peptide was more effective than all commercial drugs in all aspects analyzed, and showed no toxicity towards human colon HT-29 cancer cells at concentrations 10-100 times higher than the IC50 of the commercial drugs, corroborating its high medicinal potential.

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Maturation of cytochrome oxidases is a complex process requiring assembly of several subunits and adequate uptake of the metal cofactors. Two orthologous Sco proteins (Sco1 and Sco2) are essential for the correct assembly of the dicopper CuA site in the human oxidase, but their function is not fully understood. Here, we report an in vitro biochemical study that shows that Sco1 is a metallochaperone that selectively transfers Cu(I) ions based on loop recognition, whereas Sco2 is a copper-dependent thiol reductase of the cysteine ligands in the oxidase. Copper binding to Sco2 is essential to elicit its redox function and as a guardian of the reduced state of its own cysteine residues in the oxidizing environment of the mitochondrial intermembrane space (IMS). These results provide a detailed molecular mechanism for CuA assembly, suggesting that copper and redox homeostasis are intimately linked in the mitochondrion.

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Copper is an important micronutrient required as a redox co-factor in the catalytic centers of enzymes. However, free copper is a potential hazard because of its high chemical reactivity. Consequently, organisms exert a tight control on Cu(+) transport (entry-exit) and traffic through different compartments, ensuring the homeostasis required for cuproprotein synthesis and prevention of toxic effects. Recent studies based on biochemical, bioinformatics, and metalloproteomics approaches, reveal a highly regulated system of transcriptional regulators, soluble chaperones, membrane transporters, and target cuproproteins distributed in the various bacterial compartments. As a result, new questions have emerged regarding the diversity and apparent redundancies of these components, their irregular presence in different organisms, functional interactions, and resulting system architectures.

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