Human Copper Chaperone Atox1 Research Articles

Crystal Structure of the Human Copper Chaperone ATOX1 Bound to Zinc Ion.

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.

The Caenorhabditis elegans homolog of human copper chaperone Atox1, CUC-1, aids in distal tip cell migration

Cell migration is a fundamental biological process involved in for example embryonic development, immune system and wound healing. Cell migration is also a key step in cancer metastasis and the human copper chaperone Atox1 was recently found to facilitate this process in breast cancer cells. To explore the role of the copper chaperone in other cell migration processes, we here investigated the putative involvement of an Atox1 homolog in Caenorhabditis elegans, CUC-1, in distal tip cell migration, which is a key process during the development of the C. elegans gonad. Using knock-out worms, in which the cuc-1 gene was removed by CRISPR-Cas9 technology, we probed life span, brood size, as well as distal tip cell migration in the absence or presence of supplemented copper. Upon scoring of gonads, we found that cuc-1 knock-out, but not wild-type, worms exhibited distal tip cell migration defects in approximately 10–15% of animals and, had a significantly reduced brood size. Importantly, the distal tip cell migration defect was rescued by a wild-type cuc-1 transgene provided to cuc-1 knock-out worms. The results obtained here for C. elegans CUC-1 imply that Atox1 homologs, in addition to their well-known cytoplasmic copper transport, may contribute to developmental cell migration processes.

Oxidation of Human Copper Chaperone Atox1 and Disulfide Bond Cleavage by Cisplatin and Glutathione.

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.

Tetrathiomolybdate inhibits the reaction of cisplatin with human copper chaperone Atox1.

Cisplatin is a widely used anticancer drug in clinic, and ammonium tetrathiomolybdate ([(NH4)2MoS4], TM) is a copper chelator used in clinic for the treatment of Wilson's disease. Recently, TM has been found to enhance the therapeutic effect of cisplatin; however, the origin of this effect is not clear. Here we found that TM can inhibit the reaction of cisplatin with Cu-Atox1 and prevent the protein unfolding and aggregation induced by cisplatin. Although Ag(i) binds to Atox1 in a way similar to Cu(i)-Atox1, TM does not prevent the reaction of Ag-Atox1 with cisplatin. This result indicates that the formation of a Mo-centered trimeric protein cluster in the TM-Cu-Atox1 system plays a role in the inhibitory effect. This work provides new insights into the mechanism by which TM enhances the cytotoxic efficacy of cisplatin and helps to circumvent cisplatin resistance of tumor cells.

Interactions between human copper chaperone Atox1 and cisplatin, carboplatin, nedaplatin and oxaliplatin studied by ESI mass spectrometry

Increasing evidence indicates that the copper chaperone Atox1 involves the mechanism of Pt-drugs. Here, the interactions of Atox1 with cisplatin and cisplatin analogues were studied. Results show that, like cisplatin, the other three Pt complexes can interact with Atox1 and CuI coordination obviously enhances the platination of Atox1. Cisplatin, carboplatin and nedaplatin formed some of adducts with the same composition due to having the same cis-diammine carrier ligands and differ only in the leaving groups. Nedaplatin, similar to carboplatin, was relatively inert with respect to cisplatin. Remarkably, an unexpected adduct with loss of the 1, 2-diaminocyclohexane (DACH) carrier ligand were observed in the reaction of oxaliplatin with holo-Atox1. These results indicate that both platination adducts and rates of platination are significantly influenced by the ligand and cuprous ion, suggesting the probably different influence of Atox1 on Pt-drugs in vivo.

The structural flexibility of the human copper chaperone Atox1: Insights from combined pulsed EPR studies and computations.

Metallochaperones are responsible for shuttling metal ions to target proteins. Thus, a metallochaperone's structure must be sufficiently flexible both to hold onto its ion while traversing the cytoplasm and to transfer the ion to or from a partner protein. Here, we sought to shed light on the structure of Atox1, a metallochaperone involved in the human copper regulation system. Atox1 shuttles copper ions from the main copper transporter, Ctr1, to the ATP7b transporter in the Golgi apparatus. Conventional biophysical tools such as X-ray or NMR cannot always target the various conformational states of metallochaperones, owing to a requirement for crystallography or low sensitivity and resolution. Electron paramagnetic resonance (EPR) spectroscopy has recently emerged as a powerful tool for resolving biological reactions and mechanisms in solution. When coupled with computational methods, EPR with site-directed spin labeling and nanoscale distance measurements can provide structural information on a protein or protein complex in solution. We use these methods to show that Atox1 can accommodate at least four different conformations in the apo state (unbound to copper), and two different conformations in the holo state (bound to copper). We also demonstrate that the structure of Atox1 in the holo form is more compact than in the apo form. Our data provide insight regarding the structural mechanisms through which Atox1 can fulfill its dual role of copper binding and transfer.

ChemInform Abstract: A Copper Story: From Protein Folding and Metal Transport to Cancer

AbstractReview: [73 refs.

A Copper Story: From Protein Folding and Metal Transport to Cancer

AbstractEnzymes need to fold into unique three‐dimensional structures in order to function. Copper ions are cofactors in many essential enzymes. Such enzymes need to couple polypeptide folding with metal incorporation, as the metal sites are often integrated within the folded structure. Since free copper ions are toxic, most organisms have highly specialized copper transport systems. The human cytoplasmic copper chaperone Atox1 delivers copper to P1B‐type ATPases in the Golgi, for incorporation into copper‐dependent enzymes following the secretory path. Copper plays key roles in cancer development, as copper‐dependent enzymes are needed for tumor growth and metastasis. In addition, platinum‐based drugs are exported out of cells by the copper transport machinery. Recent findings also imply that some copper transport proteins regulate cell growth and development. In this brief journey of my later career, I will discuss the roles of copper in protein folding, mechanisms of copper ion transport, and cisplatin hitchhiking. The identification of new partners for Atox1 underscore the importance of further research in this area for combating cancer.

Oxaliplatin Binding to Human Copper Chaperone Atox1 and Protein Dimerization.

Copper trafficking proteins have been implicated in the cellular response to platinum anticancer drugs. We investigated the reaction of the chaperone Atox1 with an activated form of oxaliplatin, the third platinum drug to reach worldwide approval. Unlike cisplatin, which contains monodentate ammines, oxaliplatin contains chelated 1,2-diaminocyclohexane (DACH), which is more resistant to displacement by nucleophiles. In solution, one or two {Pt(DACH)(2+)} moieties bind to the conserved CXXC metal-binding motif of Atox1; in the latter case the two sulfur atoms likely bridging the two platinum units. At longer reaction times, a dimeric species is formed whose composition, Atox12·Pt(2+)2, indicates complete loss of the diamine ligands. Such a dimerization process is accompanied by partial unfolding of the protein. Crystallization experiments aiming at the characterization of the monomeric species have afforded, instead, a dimeric species resembling that already obtained by Boal and Rosenzweig in a similar reaction performed with cisplatin. However, while in the latter case there was only one Pt-binding site (0.4 occupancy) made of four sulfur atoms of the CXXC motifs of the two Atox1 chains in a tetrahedral arrangement, we found, in addition, a secondary Pt-binding site involving Cys41 of the B chain (0.25 occupancy). Moreover, both platinum atoms have lost their diamines. Thus, there appears to be little relationship between what is observed in solution and what is formed in the solid state. Since full occupancy of the tetrahedral cavity is a common feature of all Atox1 dimeric structures obtained with other metal ions (Cu(+), Cd(2+), and Hg(2+)), we propose that in the case of platinum, where the occupancy is only 0.4, the remaining cavities are occupied by Cu(+) ions. Experimental evidence is reported in support of the latter hypothesis. Our proposal represents a meeting point between the initial proposal of Boal and Rosenzweig (0.4 Pt occupancy) and the reinterpretation of the original crystallographic data put forward by Shabalin et al. (1 Cu occupancy), and could apply to other cases.

Identification of New Potential Interaction Partners for Human Cytoplasmic Copper Chaperone Atox1: Roles in Gene Regulation?

The human copper (Cu) chaperone Atox1 delivers Cu to P1B type ATPases in the Golgi network, for incorporation into essential Cu-dependent enzymes. Atox1 homologs are found in most organisms; it is a 68-residue ferredoxin-fold protein that binds Cu in a conserved surface-exposed Cys-X-X-Cys (CXXC) motif. In addition to its well-documented cytoplasmic chaperone function, in 2008 Atox1 was suggested to have functionality in the nucleus. To identify new interactions partners of Atox1, we performed a yeast two-hybrid screen with a large human placenta library of cDNA fragments using Atox1 as bait. Among 98 million fragments investigated, 25 proteins were found to be confident interaction partners. Nine of these were uncharacterized proteins, and the remaining 16 proteins were analyzed by bioinformatics with respect to cell localization, tissue distribution, function, sequence motifs, three-dimensional structures and interaction networks. Several of the hits were eukaryotic-specific proteins interacting with DNA or RNA implying that Atox1 may act as a modulator of gene regulation. Notably, because many of the identified proteins contain CXXC motifs, similarly to the Cu transport reactions, interactions between these and Atox1 may be mediated by Cu.

Human Copper Chaperone Atox1 Translocates to the Nucleus but does not Bind DNA In Vitro.

After Ctr1-mediated cell uptake, copper (Cu) is transported by the cytoplasmic Cu chaperone Atox1 to P1B type ATPases ATP7A and ATP7B in the Golgi network, for incorporation into Cudependent enzymes. Atox1 is a small 68-residue protein that binds Cu in a conserved CXXC motif; it delivers Cu to target domains in ATP7A/B via direct protein-protein interactions. Specific transcription factors regulating expression of the human Cu transport proteins have not been reported although Atox1 was recently suggested to have dual functionality such that it, in addition to its cytoplasmic chaperone function, acts as a transcription factor in the nucleus. To examine this hypothesis, here we investigated the localization of Atox1 in HeLa cells using fluorescence imaging in combination with in vitro binding experiments to fluorescently labeled DNA duplexes harboring the proposed promotor sequence. We found that whereas Atox1 is present in the nucleus in HeLa cells, it does not bind to DNA in vitro. It appears that Atox1 mediates transcriptional regulation via additional (unknown) proteins.

Back Cover: Structural Biology of Cisplatin Complexes with Cellular Targets: The Adduct with Human Copper Chaperone Atox1 in Aqueous Solution (Chem. Eur. J. 37/2014)

Hybrid QM/MM simulations and molecular spectroscopy provide structural insights on cisplatin binding to the copper transporter Atox1 in water solution. A molecular model of the platinated protein is provided. In this model, the Pt atom binds to Cys12 and Cys15 while retaining its two cis ammine ligands. The calculated 1H, 13C, and 15N NMR chemical shifts and CD spectra of this model are in accord with experiment. For the complete story see the Full Paper by P. Carloni et al. on page 11719 ff.

Structural biology of cisplatin complexes with cellular targets: the adduct with human copper chaperone atox1 in aqueous solution.

Cisplatin is one of the most used anticancer drugs. Its cellular influx and delivery to target DNA may involve the copper chaperone Atox1 protein. Although the mode of binding is established by NMR spectroscopy measurements in solution-the Pt atom binds to Cys12 and Cys15 while retaining the two ammine groups-the structural determinants of the adduct are not known. Here a structural model by hybrid Car-Parrinello density functional theory-based QM/MM simulations is provided. The platinated site minimally modifies the fold of the protein. The calculated NMR and CD spectral properties are fully consistent with the experimental data. Our in silico/in vitro approach provides, together with previous studies, an unprecedented view into the structural biology of cisplatin-protein adducts.

Probing the structural flexibility of the human copper metallochaperone Atox1 dimer and its interaction with the CTR1 c-terminal domain.

Both the essentiality and the toxicity of copper in human, yeast, and bacteria cells require precise mechanisms for acquisition, intimately linked to controlled distribution, which have yet to be fully understood. This work explores one aspect in the copper cycle, by probing the interaction between the human copper chaperone Atox1 and the c-terminal domain of the copper transporter, CTR1, using electron paramagnetic resonance (EPR) spectroscopy and circular dichroism (CD). The data collected here shows that the Atox1 keeps its dimer nature also in the presence of the CTR1 c-terminal domain; however, two geometrical states are assumed by the Atox1. One is similar to the geometrical state reported by the crystal structure, while the latter has not yet been constructed. In the presence of the CTR1 c-terminal domain, both states are assumed; however, the structure of Atox1 is more restricted in the presence of the CTR1 c-terminal domain. This study also shows that the last three amino acids of the CTR1 c-terminal domain, HCH, are important for maintaining the crystal structure of the Atox1, allowing less structural flexibility and improved thermal stability of Atox1.

Copper binding modulates the platination of human copper chaperone Atox1 by antitumor trans-platinum complexes

The transport system of platinum-based anticancer agents is crucial for drug sensitivity. Increasing evidence indicates that the copper transport system is also involved in the cellular influx and efflux of platinum drugs. The copper chaperone Atox1 has been shown to bind to cisplatin in vitro and in cells. Previous results reveal that copper binding promotes the reaction between Atox1 and cisplatin. Here, we have performed detailed solution NMR and ESI-MS experiments to investigate the effect of Cu(i) binding on the reactions of Atox1 with two antitumor active trans-platinum agents, trans-EE and trans-PtTz. Results indicate that, similar to the reaction of cisplatin, copper coordination also enhances the platination of Atox1 by two trans-platinum complexes, and platinum binds to the copper coordinating residues. However, copper binding promotes the trans-platinum transfer from Atox1 to dithiothreitol (DTT). This result is in contrast to the reaction of Atox1 with cisplatin, in which the presence of copper largely suppresses the platination of DTT. Additionally, both apo- and Cu(I)-Atox1 react faster with trans-platinum complexes than with cisplatin, however, less protein aggregation is observed in the reaction of trans-platinum complexes. These results indicate that the roles of Atox1 in the regulation of cellular trafficking of platinum drugs are dependent on the coordination configurations.

Copper binding promotes the interaction of cisplatin with human copper chaperone Atox1

Cu(I) binding promotes the platination of Atox1, although cisplatin binds to the copper coordination sites. In addition, Cu(I) binding enhances the competition of Atox1 with DTT in the reaction of cisplatin. These results indicate that cuprous ions could regulate the cellular trafficking of cisplatin.

Conserved residue modulates copper-binding properties through structural dynamics in human copper chaperone Atox1

The human copper chaperone Atox1 plays a central role in the transport of copper in cells. It has been reported that the conserved residue Lys60 contributes to the heterocomplex stability of Atox1 with its target protein ATPase, and that the K60A mutation could diminish the copper transfer. In this work, we carried out the structure determination and dynamic analysis of Atox1 with the K60A mutation in order to elucidate the role of the conserved residue Lys60 in the copper transport. Results show that the K60A mutation results in crucial secondary structure rearrangements and side-chain orientation alteration of the metal-binding residues in Atox1. Protein dynamic studies reveal that the K60A mutation leads to increased overall flexibility, and a significant difference in dynamic properties of the metal-binding sites. The structure and dynamic changes cause a decrease in the copper-binding stability of the K60A mutant. In addition, Cu(i)-mediated hetero-protein interactions with ATP7A are present in the metal transfer of both Atox1 variants, although copper transfer is accompanied with smaller structural alteration in the K60A mutant. These results indicate that Lys60 is crucial in maintaining the structure and dynamic properties of Atox1.

In Vitro Thermodynamic Dissection of Human Copper Transfer from Chaperone to Target Protein

Transient protein-protein and protein-ligand interactions are fundamental components of biological activity. To understand biological activity, not only the structures of the involved proteins are important but also the energetics of the individual steps of a reaction. Here we use in vitro biophysical methods to deduce thermodynamic parameters of copper (Cu) transfer from the human copper chaperone Atox1 to the fourth metal-binding domain of the Wilson disease protein (WD4). Atox1 and WD4 have the same fold (ferredoxin-like fold) and Cu-binding site (two surface exposed cysteine residues) and thus it is not clear what drives metal transfer from one protein to the other. Cu transfer is a two-step reaction involving a metal-dependent ternary complex in which the metal is coordinated by cysteines from both proteins (i.e., Atox1-Cu-WD4). We employ size exclusion chromatography to estimate individual equilibrium constants for the two steps. This information together with calorimetric titration data are used to reveal enthalpic and entropic contributions of each step in the transfer process. Upon combining the equilibrium constants for both steps, a metal exchange factor (from Atox1 to WD4) of 10 is calculated, governed by a negative net enthalpy change of ∼10 kJ/mol. Thus, small variations in interaction energies, not always obvious upon comparing protein structures alone, may fuel vectorial metal transfer.

Predicting the Coordination Number within Copper Chaperones: Atox1 as Case Study

The concentration of copper ions in biological systems is tightly regulated by metallochaperone proteins which are responsible for Cu(I) delivery to designated locations in the cell. These proteins contain a unique motif (MXCXXC) that binds Cu(I) very tightly and specifically but at the same time allows efficient metal transfer to target proteins that often contain a similar copper binding motif. It was found that binding to Cu(I) is achieved through the two cysteine residues in a low coordination number of 2-3 due to possible binding of a third external ligand. Understanding copper transport requires better understanding of copper coordination. Here we therefore focused on establishing a computational method that can predict the coordination number of copper in copper chaperones. The method is shown to be successful in predicting the coordination of Cu(I) within the human copper chaperone (Atox1). Based on the results, a possible rationale for this unique Cu(I) dicoordination in Atox1 is suggested.

Probing the Interaction of Cisplatin with the Human Copper Chaperone Atox1 by Solution and In-Cell NMR Spectroscopy

Among anticancer therapeutics, platinum-based drugs have a prominent role. They carry out their antitumor activity by forming stable adducts with DNA, thus interfering with replication and transcription processes. Cellular uptake of these drugs is tightly connected to copper transport. The major Cu(I) influx transporter Ctr1 has been found to mediate transport of cisplatin and its analogues. Evidence also suggests that ATP7A and ATP7B mediate cisplatin sequestration and efflux from cells, thus influencing drug resistance. The copper-chaperone Atox1, which normally binds Cu(I) via two cysteines and delivers the metal to ATP7A/B, has also been reported to interact with cisplatin in in vitro experiments. In the present investigation we apply a combined approach, using solution and in-cell NMR spectroscopy methods, to probe intracellular drug delivery and interaction of cisplatin with Atox1. The intracellular environment provides itself the suitable conditions for the preservation of the protein in its active form. Initially a {Pt(NH(3))(2)}-Atox1 adduct is formed. At longer reaction time we observed protein dimerization and loss of the ammines. Such a process is reminiscent of the copper-promoted formation of Atox1 dimers which have been proposed to be able to cross the nuclear membrane and act as a transcription factor. We also show that overexpression of Atox1 in E. coli reduces the amount of DNA platination and, consequently, the degree of cell filamentation.