Domains I and III of the human copper chaperone for superoxide dismutase interact via a cysteine-bridged Dicopper(I) cluster

Copper binding to the human copper chaperone for superoxide dismutase (hCCS) has been investigated by X-ray absorption spectroscopy. Stoichiometry measurements on the dialyzed, as-isolated protein indicated that up to 3.5 Cu ions bound per hCCS molecule. Reduction with either sodium dithionite or dithiothreitol decreased the copper binding ratio to 2 coppers per hCCS monomer. Analysis of the as-isolated EXAFS data indicated coordination of Cu by a mixture of S and N backscatterers, suggestive of heterogeneous binding of copper between Cu-cysteine binding sites of domain I or III and copper-histidine SOD1-like metal binding sites of domain II. The best fit was obtained with 1.6 Cu-S (cysteine) at 2.24 A (2sigma(2) = 0.011 A(2)) and 1.1 N (histidine) at 1.98 A (2sigma(2) = 0.005 A(2)). A peak of variable intensity in the Fourier transform (FT) of the as-isolated protein at 2.7 A was suggestive of the presence of a heavy atom scatterer such as Cu. Analysis of the dithionite- and DTT-reduced derivatives indicated that copper was lost from the histidine coordinating sites, resulting in a S-only environment with copper coordinated to three S backscatterers at 2. 26 A. The heavy atom scatterer peak was now prominent in the FT and could be well fit by a Cu-Cu interaction at 2.72 A. The data were best interpreted by a dinuclear mu(2)()-bridged cluster with doubly bridging cysteine ligands similar to the cluster proposed to exist in the cytochrome c oxidase chaperone COX17. Analysis of primary sequence and X-ray structural information on yeast CCS strongly suggests that this cluster bridges between domains I and III in hCCS. A mechanism for copper translocation is briefly discussed.     

Cysteine-to-serine mutants of the human copper chaperone for superoxide dismutase reveal a copper cluster at a domain III dimer interface

Cysteine-to-serine mutants of a maltose binding protein fusion with the human copper chaperone for superoxide dismutase (hCCS) were studied with respect to (i) their ability to transfer Cu to E,Zn superoxide dismutase (SOD) and (ii) their Zn and Cu binding and X-ray absorption spectroscopic (XAS) properties. Previous work has established that Cu(I) binds to four cysteine residues, two of which, C22 and C25, reside within an Atox1-like N-terminal domain (DI) and two of which, C244 and C246, reside in a short unstructured polypeptide chain at the C-terminus (DIII). The wild-type (WT) protein shows an extended X-ray absorption fine structure (EXAFS) spectrum characteristic of cluster formation, but it is not known how such a cluster is formed. Cys to Ser mutagenesis was used to investigate the Cu binding in more detail. Single Cys to Ser mutations, as represented by C22S and C244S, did little to affect the metal binding ratios of hCCS. Both mutants still showed approximately 2 Cu(I) ions and 1 Zn ion per protein. The double mutants C22/24S and C244/246S, on the other hand, showed Cu binding stoichiometries close to 1:1. The Zn-EXAFS of WT CCS showed a 3-4 histidine ligand environment that is consistent with Zn binding in the SOD-like domain II of CCS. The Zn environment remained unchanged between wild type and all of the mutant CCS proteins. Single Cys to Ser mutations displayed lower activity than WT protein, although close to full activity could be rescued by increasing the CCS:SOD ratios to 8:1 in the assay mixture. The structure of the Cu centers of the single mutants as revealed by EXAFS was also similar to that of WT protein, with clear indications of a Cu cluster. On the other hand, the double mutants showed a greater degree of perturbation. The DI C22/25S mutant was 70% active and formed a cluster with a more intense Cu-Cu interaction. The DIII C244/246S mutant retained only a fraction (16%) of activity and did not form a cluster. The results suggest the formation of a DIII-DIII cluster within a dimeric or tetrameric protein and further suggest that this cluster may be an important element of the copper transfer machinery.     

Introduction of a [4Fe-4S (S-cys)4]+1,+2 iron-sulfur center into a four-alpha helix protein using design parameters from the domain of the Fx cluster in the Photosystem I reaction center.

We describe the insertion of an iron-sulfur center into a designed four alpha-helix model protein. The model protein was re-engineered by introducing four cysteine ligands required for the coordination of the mulinucleate cluster into positions in the main-chain directly analogous to the domain predicted to ligand the interpeptide [4Fe-4S (S-cys)4] cluster, Fx, from PsaA and PsaB of the Photosystem I reaction center. This was achieved by inserting the sequence, CDGPGRGGTC, which is conserved in PsaA and PsaB, into interhelical loops 1 and 3 of the four alpha-helix model. The holoprotein was characterized spectroscopically after insertion of the iron-sulfur center in vitro. EPR spectra confirmed the cluster is a [4Fe-4S] type, indicating that the cysteine thiolate ligands were positioned as designed. The midpoint potential of the iron-sulfur center in the model holoprotein was determined via redox titration and shown to be -422 mV (pH 8.3, n = 1). The results support proposals advanced for the structure of the domain of the [4Fe-4S] Fx cluster in Photosystem I based upon sequence predictions and molecular modeling. We suggest that the lower potential of the Fx cluster is most likely due to factors in the protein environment of Fx rather than the identity of the residues proximal to the coordinating ligands.     

The CuA domain of Thermus thermophilus ba3-type cytochrome c oxidase at 1.6 A resolution.

The structure of the CuA-containing, extracellular domain of Thermus thermophilus ba3-type cytochrome c oxidase has been determined to 1.6 A resolution using multiple X-ray wavelength anomalous dispersion (MAD). The Cu2S2 cluster forms a planar rhombus with a copper-copper distance of 2.51 +/- 0.03 A. X-ray absorption fine-structure (EXAFS) studies show that this distance is unchanged by crystallization. The CuA center is asymmetrical; one copper is tetrahedrally coordinated to two bridging cysteine thiolates, one histidine nitrogen and one methionine sulfur, while the other is trigonally coordinated by the two cysteine thiolates and a histidine nitrogen. Combined sequence-structure alignment of amino acid sequences reveals conserved interactions between cytochrome c oxidase subunits I and II.     

Direct metal transfer between periplasmic proteins identifies a bacterial copper chaperone

Transition metals require exquisite handling within cells to ensure that cells are not harmed by an excess of free metal species. In gram-negative bacteria, copper is required in only small amounts in the periplasm, not in the cytoplasm, so a key aspect of protection under excess metal conditions is to export copper from the periplasm. Additional protection could be conferred by a periplasmic chaperone to limit the free metal species prior to export. Using isothermal titration calorimetry, we have demonstrated that two periplasmic proteins, CusF and CusB, of the Escherichia coli Cu(I)/Ag(I) efflux system undergo a metal-dependent interaction. Through the development of a novel X-ray absorption spectroscopy approach using selenomethionine labeling to distinguish the metal sites of the two proteins, we have demonstrated transfer of Cu(I) occurs between CusF and CusB. The interaction between these proteins is highly specific, as a homologue of CusF with a 51% identical sequence and a similar affinity for metal, did not function in metal transfer. These experiments establish a metallochaperone activity for CusF in the periplasm of gram-negative bacteria, serving to protect the periplasm from metal-mediated damage.     

Analogous copper(I) coordination in metallothionein from yeast and the separate domains of the mammalian protein

The three-dimensional structures of both vertebrate Cu12-metallothionein (class 1) and yeast Cu8-thionein (class 2) are still unknown. The different copper:protein stoichiometry compared with that of the (ZnCd)7-metallothioneins was expected to alter the metal-thiolate cluster structure considerably. In order to avoid possible domain interactions in the hepatic rat metallothionein, separate chemically synthesized alpha- and beta-domains were used rather than the apoprotein. Apo yeast thionein, and the alpha- and beta-domains of rat liver metallothionein-2 were reconstituted by Cu(I) titration. Reconstitution steps were monitored using spectroscopic methods including luminescence emission and circular dichroism. Upon UV irradiation a linear increase in intensity of the orange-red luminescence was observed near 600 nm up to 6 Cu eq using either compound regardless of the different cysteine sulfer content (yeast thionein 12S, alpha-domain 11S, beta-domain 9S). The characteristic dichroic properties of the yeast copper-protein between 240 and 400 nm were in good agreement with those of the respective class 1 metallothionein domains. All observed Cotton bands were of similar shape and appeared in the same wavelength regions. However, the molar ellipticities were less pronounced in the alpha- and beta-fragments employed. There appears to be a striking similarity between the oligonuclear Cu(I) binding centers in all metallothionein species.     

Expression, purification and copper-binding studies of the first metal-binding domain of Menkes protein.

The cDNA, coding for the first metal-binding domain (MBD1) of Menkes protein, was cloned into the T7-system based vector, pCA. The T7 lysozyme-encoding plasmid, pLysS, is shown to be crucial for expression, suggesting that the protein is toxic to the cells. Adding copper to the growth medium did not affect the plasmid stability. MBD1 is purified in two steps with a typical yield of 12 mg.L-1. Menkes protein, a P-type ATPase, contains a sequence GMXCXSC that is repeated six times, at the N-terminus. The paired cysteine residues are involved in metal binding. MBD1 has only two cysteine residues, which can exist as free thiol groups (reduced), as a disulphide bond (oxidized) or bound to a metal ion [e.g. Cu(I)-MBD1]. These three MBD1 forms have been investigated using CD. No major spectral change was seen between the different MBD1 forms, indicating that the folding is not changed upon metal binding. A copper-bound MBD1 was also studied by EPR, and the lack of an EPR signal suggests that the oxidation state of copper bound to MBD1 is Cu(I). Cu(I) binding studies were performed by equilibrium dialysis and revealed a stoichiometry of 1 : 1 and an apparent Kd = 46 microM. Oxidized MBD1, however, is not able to bind copper. Different copper complexes were investigated for their ability to reconstitute apo-MBD1. Given the same total copper concentration CuCl43- was superior to Cu(I)-thiourea (structural analogue of metallothionein) and Cu(I)-glutathione (used at fivefold higher copper concentration) although the latter two were able to partially reconstitute apo-MBD1. Cu(II) was not able to reconstitute apo-MBD1, presumably due to Cu(II)-induced oxidation of the thiol groups. Based on our results, glutathione and/or metallothionein are likely candidates for the in vivo incorporation of copper to Menkes protein.     

Copper transport and its defect in Wilson disease: characterization of the copper-binding domain of Wilson disease ATPase

Copper is an essential trace element which forms an integral component of many enzymes. While trace amounts of copper are needed to sustain life, excess copper is extremely toxic. An attempt is made here to present the current understanding of the normal transport of copper in relation to the absorption, intracellular transport and toxicity. Wilson disease is a genetic disorder of copper transport resulting in the accumulation of copper in organs such as liver and brain which leads to progressive hepatic and neurological damage. The gene responsible for Wilson disease (ATP7B) is predicted to encode a putative copper-transporting P-type ATPase. An important feature of this ATPase is the presence of a large N-terminal domain that contains six repeats of a copper-binding motif which is thought to be responsible for binding this metal prior to its transport across the membrane. We have cloned, expressed and purified the N-terminal domain (approximately 70 kD) of Wilson disease ATPase. Metal-binding properties of the domain showed the protein to bind several metals besides copper; however, copper has a higher affinity for the domain. The copper is bound to the domain in Cu(I) form with a copper: protein ratio of 6.5:1. X-ray absorption studies strongly suggest Cu(I) atoms are ligated to cysteine residues. Circular dichroism spectral analyses suggest both secondary and tertiary structural changes upon copper binding to the domain. Copper-binding studies suggest some degree of cooperativity in binding of copper. These studies as well as detailed structural information of the copper-binding domain will be crucial in determining the specific role played by the copper-transporting ATPase in the homeostatic control of copper in the body and how the transport of copper is interrupted by mutations in the ATPase gene.     

The influence of reducing agent and 1,10-phenanthroline concentration on DNA cleavage by phenanthroline + copper.

Copper in the presence of excess 1,10-phenanthroline, a reducing agent, and molecular oxygen causes cleavage of DNA with a preference for T-3',5'-A-steps, particularly in TAT triplets. The active molecular species is commonly thought to be the bis-(1,10-phenanthroline)Cu(I) complex, (Phen)2Cu(I), regardless of the reducing agent type. We have found that (Phen)2Cu(I) is not the predominant copper complex when 3-mercaptopropionic acid (MPA) or 2-mercaptoethanol are used as the reducing agents, but (Phen)2Cu(I) predominates when ascorbate is used as the reducing agent. Substitution of ascorbate for thiol significantly enhances the rate of DNA cleavage by 1,10-phenanthroline + copper, without altering the sequence selectivity. We show that (Phen)2Cu(I) is the complex responsible for DNA cleavage, regardless of reducing agent, and that 1,10-phenanthroline and MPA compete for copper coordination sites. DNA cleavage in the presence of ascorbate also occurs under conditions where the mono-(1,10-phenanthroline)Cu(I) complex predominates (1:1 phenanthroline:copper ratio), but preferential cleavage was observed at a CCGG sequence and not at TAT sequences. The second phenanthroline ring of the (Phen)2Cu(I) complex appears essential for determining the T-3',5'-A sequence preferences of phenanthroline + copper when phenanthroline is in excess.     

Copper speciation in the alpha and beta domains of recombinant human metallothionein by electrospray ionization mass spectrometry.

ESI-MS data are reported for Cu(I) binding to the metal-free and cadmium-alpha and beta domains of recombinant human metallothionein. These data provide information on the stoichiometric ratios of copper and cadmium that bind to the 11 thiolate sulfurs in the alpha fragment and the nine thiolate sulfurs in the beta fragment. The data show the effects of the existing three-dimensional structure on the formation of different Cu(I)-thiolate clusters. Charge-state spectra are reported for a range of Cu(I) binding at low and neutral pH to the isolated alpha and beta domains. There is an uneven distribution of charge states that show that changes in the three-dimensional structure take place as a function of Cu(I) loading. Metallation of the alpha domain at low pH takes place in a series of steps with the Cu7 species dominating until at higher levels of Cu(I) the clusters become unstable resulting in increased concentrations of the metal-free being detected. We interpret this behavior as being the result of the expansion of the Cu-S domain structure to accommodate digonal co-ordination for the increased Cu(I) loading. This larger structure is unstable in the mass spectrometer and demetallation takes place. Metallation of the beta domain at low pH proceeds in steps that involve initial formation of a Cu5S9 cluster, followed by the Cu6S9 at higher concentrations of Cu(I). The charge state spectra indicate a significant change in exposure of protonatable amino acids between Cu5S9 and Cu6S9 clusters, which indicates a change in peptide conformation when the Cu6S9 cluster forms. Metallation at neutral pH follows this same trend, namely, a much greater range of copper species is found during titrations of the Cd4S11-alpha fragment compared with the number of species that form when Cu(I) is added to Cd3S9-beta. The mass spectral data indicate that at neutral pH, the presence of the tetrahedral geometry of the Cd(II) facilitates formation of mixed trigonal and digonal geometries for the incoming Cu(I) so that the most prominent species in the beta fragment is Cd1Cu5S9 which transforms into Cu7S9 at higher concentrations of Cu(I), and finally to Cu9S9 at saturation, all species involving a number of Cu(I) in digonal geometries. The observation that the metallation patterns of the alpha and beta clusters follow different pathways at both low and neutral pH's, suggests that the structures in the two domains are quite different, in agreement with previous proposals     

Solution structure of the N-terminal domain of a potential copper-translocating P-type ATPase from Bacillus subtilis in the apo and Cu(I) loaded states

A putative partner of the already characterized CopZ from Bacillus subtilis was found, both proteins being encoded by genes located in the same operon. This new protein is highly homologous to eukaryotic and prokaryotic P-type ATPases such as CopA, Ccc2 and Menkes proteins. The N-terminal region of this protein contains two soluble domains constituted by amino acid residues 1 to 72 and 73 to 147, respectively, which were expressed both separately and together. In both cases only the 73-147 domain is folded and is stable both in the copper(I)-free and in the copper(I)-bound forms. The folded and unfolded state is monitored through the chemical shift dispersion of 15N-HSQC spectra. In the absence of any structural characterization of CopA-type proteins, we determined the structure of the 73-147 domain in the 1-151 construct in the apo state through 1H, 15N and 13C NMR spectroscopies. The structure of the Cu(I)-loaded 73-147 domain has been also determined in the construct 73-151. About 1300 meaningful NOEs and 90 dihedral angles were used to obtain structures at high resolution both for the Cu(I)-bound and the Cu(I)-free states (backbone RMSD to the mean 0.35(+/-0.06) A and 0.39(+/-0.07) A, respectively). The structural assessment shows that the structures are accurate. The protein has the typical betaalpha(betabeta)alphabeta folding with a cysteine in the C-terminal part of helix alpha1 and the other cysteine in loop 1. The structures are similar to other proteins involved in copper homeostasis. Particularly, between BsCopA and BsCopZ, only the charges located around loop 1 are reversed for BsCopA and BsCopZ, thus suggesting that the two proteins could interact one with the other. The variability in conformation displayed by the N-terminal cysteine of the CXXC motif in a number of structures of copper transporting proteins suggests that this may be the cysteine which binds first to the copper(I) carried by the partner protein.     

A distinct Cu4-thiolate cluster of human metallothionein-3 is located in the N-terminal domain

Metallothionein-3 (MT-3), also known as neuronal growth inhibitory factor, is a metalloprotein expressed almost exclusively in the brain. Isolated MT-3 contains four Cu(I) and three Zn(II) ions organized in homometallic metal-thiolate clusters located in two independent protein domains. In this work a Cu(I) binding to metal-free MT-3 has been studied, aiming at the better understanding of the domain specificity for this metal ion. The cluster formation was followed by electronic absorption, circular dichroism, and by luminescence spectroscopy at room temperature and 77 K. The stepwise incorporation of Cu(I) into recombinant human apo-MT-3 revealed the cooperative formation of two Cu(4)S(9) clusters in succession, formed in both protein domains, i.e. Cu(4)- and Cu(8)-MT-3. Further binding of four Cu(I) caused an expansion of these Cu(I) cores, leading to fully metal-loaded Cu(12)-MT-3 containing Cu(6)S(9) and Cu(6)S(11) clusters in the beta- and alpha-domains of the protein, respectively. The location of the preferentially formed Cu(4) cluster in the protein was established by immunochemistry. Using domain-specific antibodies, in combination with limited tryptic digestion of a partially metal-occupied Cu(4)-MT-3, we could demonstrate that the Cu(4)S(9) cluster is located in the N-terminal beta-domain of the protein that contains a total of nine cysteine ligands.     

Characterization and structure of a Zn2+ and [2Fe-2S]-containing copper chaperone from Archaeoglobus fulgidus

Bacterial CopZ proteins deliver copper to P1B-type Cu+-ATPases that are homologous to the human Wilson and Menkes disease proteins. The genome of the hyperthermophile Archaeoglobus fulgidus encodes a putative CopZ copper chaperone that contains an unusual cysteine-rich N-terminal domain of 130 amino acids in addition to a C-terminal copper binding domain with a conserved CXXC motif. The N-terminal domain (CopZ-NT) is homologous to proteins found only in extremophiles and is the only such protein that is fused to a copper chaperone. Surprisingly, optical, electron paramagnetic resonance, and x-ray absorption spectroscopic data indicate the presence of a [2Fe-2S] cluster in CopZ-NT. The intact CopZ protein binds two copper ions, one in each domain. The 1.8 A resolution crystal structure of CopZ-NT reveals that the [2Fe-2S] cluster is housed within a novel fold and that the protein also binds a zinc ion at a four-cysteine site. CopZ can deliver Cu+ to the A. fulgidus CopA N-terminal metal binding domain and is capable of reducing Cu2+ to Cu+. This unique fusion of a redox-active domain with a CXXC-containing copper chaperone domain is relevant to the evolution of copper homeostatic mechanisms and suggests new models for copper trafficking.     

Biochemical characterization of CopA,the Escherichia coli Cu(I)-translocating P-type ATPase

Escherichia coli CopA is a copper ion-translocating P-type ATPase that confers copper resistance. CopA formed a phosphorylated intermediate with [gamma-(32)P]ATP. Phosphorylation was inhibited by vanadate and sensitive to KOH and hydroxylamine, consistent with acylphosphate formation on conserved Asp-523. Phosphorylation required a monovalent cation, either Cu(I) or Ag(I). Divalent cations Cu(II), Zn(II), or Co(II) could not substitute, signifying that the substrate of this copper-translocating P-type ATPase is Cu(I) and not Cu(II). CopA purified from dodecylmaltoside-solubilized membranes similarly exhibited Cu(I)/Ag(I)-stimulated ATPase activity, with a K(m) for ATP of 0.5 mm. CopA has two N-terminal Cys(X)(2)Cys sequences, Gly-Leu-Ser-Cys(14)-Gly-His-Cys(17), and Gly-Met-Ser-Cys(110)-Ala-Ser-Cys(113), and a Cys(479)-Pro-Cys(481) motif in membrane-spanning segment six. The requirement of these cysteine residues was investigated by the effect of mutations and deletions. Mutants with substitutions of the N-terminal cysteines or deletion of the first Cys-(X)(2)-Cys motif formed acylphosphate intermediates. From the copper dependence of phosphoenzyme formation, the mutants appear to have 2-3 fold higher affinity for Cu(I) than wild type CopA. In contrast, substitutions in Cys(479) or Cys(481) resulted in loss of copper resistance, transport and phosphoenzyme formation. These results imply that the cysteine residues of the Cys-Pro-Cys motif (but not the N-terminal cysteine residues) are required for CopA function.     

Escherichia coli mechanisms of copper homeostasis in a changing environment

Escherichia coli is equipped with multiple systems to ensure safe copper handling under varying environmental conditions. The Cu(I)-translocating P-type ATPase CopA, the central component in copper homeostasis, is responsible for removing excess Cu(I) from the cytoplasm. The multi-copper oxidase CueO and the multi-component copper transport system CusCFBA appear to safeguard the periplasmic space from copper-induced toxicity. Some strains of E. coli can survive in copper-rich environments that would normally overwhelm the chromosomally encoded copper homeostatic systems. Such strains possess additional plasmid-encoded genes that confer copper resistance. The pco determinant encodes genes that detoxify copper in the periplasm, although the mechanism is still unknown. Genes involved in copper homeostasis are regulated by MerR-like activators responsive to cytoplasmic Cu(I) or two-component systems sensing periplasmic Cu(I). Pathways of copper uptake and intracellular copper handling are still not identified in E. coli.     

Copper(I) interaction with model peptides of WD6 and TM6 domains of Wilson ATPase: regulatory and mechanistic implications

With the aim to investigate the mechanism of Cu(I) transport by Wilson ATPase (ATP7B), we have studied the interaction of the peptides 2K10p (CH(3)CO-Lys-Gly-Met-Thr-Cys-Ala-Ser-Cys-Val-His-Asn-Lys-CONH(2)), and 2K8p (CH(3)CO-Lys-Leu-Cys-Ile-Ala-Cys-Pro-Cys-Ser-Lys-CONH(2)), part of the sixth metal binding domain (WD6) and the sixth transmembrane segment (TM6) of Wilson ATPase, respectively, by means of CD, NMR spectroscopy and homology modeling. In addition, the interaction of Cu(I) with the 2K8p mutants 1s (CH(3)CO-Lys-Leu-Ser-Ile-Ala-Cys-Pro-Cys-Ser-Lys-CONH(2)), 2s (CH(3)CO-Lys-Leu-Cys-Ile-Ala-Ser-Pro-Cys-Ser-Lys-CONH(2)) and 3s (CH(3)CO-Lys-Leu-Cys-Ile-Ala-Cys-Pro-Ser-Ser-Lys-CONH(2)), containing two cysteines in various positions, have been studied with the same methods, in order to understand the role of each cysteine in copper binding. Our studies show that the three cysteine thiolates present in the 2K8p peptide sequence act mainly as bridging ligands for Cu(I) binding, and dithiothreitol acts as an important ligand in Cu(I) ligation by 2K10p and the 2K8p mutants. Formation of oligomeric species has been evidenced for all peptides except 2s. Shift of the equilibrium between the various oligomeric species has been accomplished by reducing the Cu(I):peptide ratio. Significant shifts of proline protons upon interaction with Cu(I) have been observed for all proline containing peptides implying a possible role of proline in facilitating Cu(I) binding. These findings have been further discussed with respect to the molecular basis of copper trafficking and intermolecular interactions.