Impact of cofactor on stability of bacterial (CopZ) and human (Atox1) copper chaperones

Here, we present the first characterization of in vitro unfolding and thermodynamic stability of two copper chaperone proteins: Bacillus subtilis CopZ and Homo sapiens Atox1. We find that the unfolding reactions for apo- and Cu(I)-forms of CopZ and Atox1, induced by the chemical denaturant, guanidine hydrochloride (GuHCl), and by thermal perturbation are reversible two-state reactions. For both proteins, the unfolding midpoints shift to higher GuHCl concentrations and the thermodynamic stability is increased in the presence of Cu(I). Despite the same overall fold, apo-CopZ exhibits much lower thermal stability than apo-Atox1. Although the thermal stability of both proteins is increased in the presence of copper, the stabilizing effect is largest for the less stable variant. Divergent energetic properties of the apo- and holo-forms may be linked to conformational changes that facilitate copper transfer to the target.     

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.     

Structural insight into the distinct properties of copper transport by the Helicobacter pylori CopP protein

Helicobacter pylori CopP (HpCopP) is a putative copper binding regulatory protein composed of 66 amino acid residues. The small HpCopP protein is homologous to CopZ, encoded by the E. hirae and B. subtilis cop operons. To clarify the role of HpCopP in copper metabolism in H. pylori, we studied the structural and copper binding characteristics by NMR spectroscopy. Based on the resonance assignments, the tertiary structure of HpCopP was determined. Unlike the betaalphabetabetaalphabeta fold of the homologous CopZ, HpCopP adopts the betaalphabetabetaalpha fold. The superposition with structures of other bacterial copper binding proteins showed that the global structure of HpCopP follows the general topology of the family, regardless of absence of the C-terminal beta-strand. The Cu(I) binding property of HpCopP was well conserved like CopZs: the structural changes due to Cu(I) and Ag(I) bindings were primarily restricted to the metal binding motif (CXXC motif). On the other hand, the Cu(II) binding property of CopP was different with that of CopZ: in the absence of reducing agent, Cu(II) ion oxidized a mutant HpCopP, resulting in disulfide bond formation in the CXXC motif. The Cu(II) ion binding property was evaluated using the mutant HpCopP, in which two amino acids were artificially introduced at the C-terminus, since the reduced state of the CXXC motif was more stabile in the mutant HpCopP without a reducing agent. Here, the structure and copper binding property of HpCopP are discussed in detail.     

X-ray absorption and NMR spectroscopic studies of CopZ,a copper chaperone in Bacillus subtilis: the coordination properties of the copper ion

XAS studies have been performed, under various experimental conditions, on a copper(I)-transporting protein, CopZ, of Bacillus subtilis. The copper(I) ion, reduced with dithiothreitol, is three-coordinate with three sulfur donor atoms, two of which presumably provided by the protein and one by dithiothreitol. If a molar excess of acetate (15 mM; 5:1 respect to CopZ) or citrate (6 mM; 2:1 respect to CopZ) is present in solution, the EXAFS spectra suggest the presence of a dimeric form involving a close contact between Cu(I) ions from two molecules, where Cu is still three-coordinate. (1)H and (15)N NMR data provide further structural details. If copper reduction is accomplished with ascorbate, the data indicate that one oxygen of ascorbate enters in the first-coordination sphere of copper, together with two sulfur atoms, in a dimeric form of the protein. These results are instructive and have been discussed with respect to the molecular basis of copper trafficking.     

Structure and metal binding studies of the second copper binding domain of the Menkes ATPase

Biological utilisation of copper requires that the metal, in its ionic forms, be meticulously transported, inserted into enzymes and regulatory proteins, and excess be excreted. To understand the trafficking process, it is crucial that the structures of the proteins involved in the varied processes be resolved. To investigate copper binding to a family of structurally related copper-binding proteins, we have characterised the second Menkes N-terminal domain (MNKr2). The structure, determined using 1H and 15N heteronuclear NMR, of the reduced form of MNKr2 has revealed two alpha-helices lying over a single beta-sheet and shows that the binding site, a Cys(X)2Cys pair, is located on an exposed loop. 1H-15N HSQC experiments demonstrate that binding of Cu(I) causes changes that are localised to conserved residues adjacent to the metal binding site. Residues in this area are important to the delivery of copper by the structurally related Cu(I) chaperones. Complementary site-directed mutagenesis of the adjacent residues has been used to probe the structural roles of conserved residues.     

Distinct characteristics of Ag+ and Cd2+ binding to CopZ from Bacillus subtilis

The chaperone CopZ together with the P-type ATPase transporter CopA constitute a copper-detoxification system in Bacillus subtilis that is commonly found in bacteria and higher cells. Previous studies of the regulation of the copZA operon showed that expression is significantly upregulated in response to elevated concentrations of environmental silver and cadmium, as well as copper. Here, we have used spectroscopic and bioanalytical methods to investigate in detail the capacity of CopZ to bind these metal ions (as Ag(+) and Cd(2+)). We demonstrate that Ag(+) binding mimics closely that of Cu(+): Ag(+)-mediated dimerisation of the protein occurs, and distinct Ag(+)-bound species are formed at higher Ag(+) loadings. Cd(2+) also binds to CopZ, but exhibits significantly different behaviour. Cd(2+)-mediated dimerisation is only observed at low loadings, such that at 0.5 and one Cd(2+) per CopZ the protein is present mainly in a monomeric form; and multinuclear higher-order forms of Cd(2+)-CopZ are not observed. Competition binding studies reveal that Ag(+) binds with an affinity very similar to that of Cu(+), while Cd(2+) binding is significantly weaker. These data provide support for the proposal that CopZ may be involved in the detoxification of silver and cadmium, in addition to copper.     

Copper transfer from the Cu(I) chaperone, CopZ, to the repressor, Zn(II)CopY: metal coordination environments and protein interactions

Extracellular copper regulates the DNA binding activity of the CopY repressor of Enterococcus hirae and thereby controls expression of the copper homeostatic genes encoded by the cop operon. CopY has a CxCxxxxCxC metal binding motif. CopZ, a copper chaperone belonging to a family of metallochaperones characterized by a MxCxxC metal binding motif, transfers copper to CopY. The copper binding stoichiometries of CopZ and CopY were determined by in vitro metal reconstitutions. The stoichiometries were found to be one copper(I) per CopZ and two copper(I) per CopY monomer. X-ray absorption studies suggested a mixture of two- and three-coordinate copper in Cu(I)CopZ, but a purely three-coordinate copper coordination with a Cu-Cu interaction for Cu(I)2CopY. The latter coordination is consistent with the formation of a compact binuclear Cu(I)-thiolate core in the CxCxxxxCxC binding motif of CopY. Displacement of zinc, by copper, from CopY was monitored with 2,4-pyridylazoresorcinol. Two copper(I) ions were required to release the single zinc(II) ion bound per CopY monomer. The specificity of copper transfer between CopZ and CopY was dependent on electrostatic interactions. Relative copper binding affinities of the proteins were investigated using the chelator, diethyldithiocarbamic acid (DDC). These data suggest that CopY has a higher affinity for copper than CopZ. However, this affinity difference is not the sole factor in the copper exchange; a charge-based interaction between the two proteins is required for the transfer reaction to proceed. Gain-of-function mutation of a CopZ homologue demonstrated the necessity of four lysine residues on the chaperone for the interaction with CopY. Taken together, these results suggest a mechanism for copper exchange between CopZ and CopY.     

Copper trafficking: the solution structure of Bacillus subtilis CopZ.

A sequence with a high homology (39% residue identity) with that of the copper-transport CopZ protein from Enterococcus hirae and with the same MXCXXC metal-binding motif has been identified in the genome of Bacillus subtilis, and the corresponding protein has been expressed. The protein, constituted by 73 amino acids, does bind copper(I) under reducing conditions and fully folded in both copper-bound and copper-free forms under the present experimental conditions. The solution structure of the copper-bound form was determined through NMR spectroscopy on an 15N-labeled sample. A total of 1508 meaningful nuclear Overhauser effects, 38 dihedral phi angles, and 48 dihedral psi angles were used in the structural calculations, which lead to a family of 30 conformers with an average rmsd to the mean structure of 0.32 +/- 0.06 A for the backbone and of 0.85 +/- 0.07 A for the heavy atoms. NMR data on the apoprotein also show that, also in this form, the protein is in a folded state and essentially maintains the complete secondary structure. Some disorder is observed in the loop devoted to copper binding. These results are compared with those reported for CopZ from E. hirae whose structure is well-defined only in the apo form. The different behaviors of copper-loaded E. hirae and B. subtilis are tentatively accounted for on the basis of the presence of dithiothreitol used in the latter case, which would stabilize the monomeric form. The comparison is extended to other similar proteins, with particular attention to the copper-binding loop. The nature and the location of conserved residues around the metal-binding site are discussed with respect to their relevance for the metal-binding process. Proposals for the role of CopZ are also presented.     

Understanding copper trafficking in bacteria: interaction between the copper transport protein CopZ and the N-terminal domain of the copper ATPase CopA from Bacillus subtilis

In this paper the interaction of cytoplasmic CopZ and the N-terminal domain of the CopA ATPase from Bacillus subtilis has been studied by NMR through (15)N-(1)H HSQC experiments in order to understand the role of the two proteins in the whole copper trafficking mechanism of the bacteria. It appears that the two proteins interact in a fashion similar to that of the yeast homologue proteins [Arnesano, F., Banci, L., Bertini, I., Cantini, F., Ciofi-Baffoni, S., Huffman, D. L., and O'Halloran, T. V. (2001) J. Biol. Chem. 276, 41365-41376], although the surface potentials are reversed. A structural model for the interaction is proposed. (15)N mobility studies on the free proteins and on their complex are also reported. From these data, it appears that copper is largely transferred from CopZ to CopA, thus suggesting their possible involvement in a detoxification process. Comparing functional data of homologous proteins of other bacteria, it can be concluded that this class of proteins is involved in copper homeostasis but the specific roles are species dependent.     

Solution structures of the reduced and Cu(I) bound forms of the first metal binding sequence of ATP7A associated with Menkes disease

The coding sequence for the first N-terminal copper binding motif of the human Menkes disease protein (MNK1; residues 2-79) was synthesized, cloned, and expressed in bacteria for biochemical and structural studies. MNK1 adopts the betaalphabetabetaalphabeta fold common to all the metal binding sequences (MBS) found in other metal transport systems (e.g., the yeast copper chaperone for superoxide dismutase CCS, the yeast copper chaperone ATX1 bound to Hg(II), and most recently Cu(I), the bacterial copper binding protein, CopZ, and the bacterial Hg(II) binding protein MerP), although substantial differences were found in the metal binding loop. Similar to ATX1, MNK1 binds Cu(I) in a distorted linear bicoordinate geometry. As with MerP, MNK1 has a high affinity for both Hg(II) and Cu(I), although it displays a marked preference for Cu(I). In addition, we found that F71 is a key residue in the compact folding of MNK1, and its mutation to alanine results in an unfolded structure. The homologous residue in MerP has also been mutated with similar results. Finally, to understand the relationship between protein folding and metal affinity and specificity, we expressed a chimeric MBS with the MNK1 protein carrying the binding motif of MerP (CAAC-MNK1); this chimeric protein showed differences in structure and the dynamics of the binding site that may account for metal specificity.     

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.     

Solution structure of apo CopZ from Bacillus subtilis: further analysis of the changes associated with the presence of copper

The solution structure of apo CopZ from Bacillus subtilis has been determined with the aim of investigating the changes in the hydrophobic interactions around the M-X-C-X-X-C copper(I) binding motif upon metal binding. The methionine of this motif (Met 11 in CopZ) points toward the solvent in apo CopZ, whereas its sulfur atom is close to the metal ion in the metal-loaded protein, though probably not at binding distance. This change is associated with the weakening of the interaction between Leu 37 and Cys 16, present in the apo form, and the formation of an interaction between Met 11 and Tyr 65. Loops 1, 3, and 5 are affected by metal binding. Comparison with the structure of other homologous proteins confirms that often metal binding affects a hydrophobic patch around the metal site, possibly for optimizing and tuning the hydrophobic interactions with the partners. It is also shown that copper(I) exchanges among apo CopZ molecules in slow exchange on the NMR time scale, whereas it is known that such exchange between partner molecules (i.e., metallochaperones and metal pumps) is fast.     

High Cu(I) and low proton affinities of the CXXC motif of Bacillus subtilis CopZ

CopZ, an Atx1-like copper chaperone from the bacterium Bacillus subtilis, functions as part of a complex cellular machinery for Cu(I) trafficking and detoxification, in which it interacts specifically with the transmembrane Cu(I)-transporter CopA. Here we demonstrate that the cysteine residues of the MXCXXC Cu(I)-binding motif of CopZ have low proton affinities, with both exhibiting pK(a) values of 6 or below. Chelator competition experiments demonstrated that the protein binds Cu(I) with extremely high affinity, with a small but significant pH-dependence over the range pH 6.5-8.0. From these data, a pH-corrected formation constant, beta(2)= approximately 6 x 10(22) M(-2), was determined. Rapid exchange of Cu(I) between CopZ and the Cu(I)-chelator BCS (bathocuproine disulfonate) indicated that the mechanism of exchange does not involve simple dissociation of Cu(I) from CopZ (or BCS), but instead proceeds via the formation of a transient Cu(I)-mediated protein-chelator complex. Such a mechanism has similarities to the Cu(I)-exchange pathway that occurs between components of copper-trafficking pathways.     

The Enterococcus hirae copper chaperone CopZ delivers copper(I) to the CopY repressor

Expression of the cop operon which effects copper homeostasis in Enterococcus hirae is controlled by the copper responsive repressor CopY. Purified Zn(II)CopY binds to a synthetic cop promoter fragment in vitro. Here we show that the 8 kDa protein CopZ acts as a copper chaperone by specifically delivering copper(I) to Zn(II)CopY and releasing CopY from the DNA. As shown by gel filtration and luminescence spectroscopy, two copper(I) are thereby quantitatively transferred from Cu(I)CopZ to Zn(II)CopY, with displacement of the zinc(II) and transfer of copper from a non-luminescent, exposed, binding site in CopZ to a luminescent, solvent shielded, binding site in CopY.     

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.     

Solution structure of long neurotoxin NTX-1 from the venom of Naja naja oxiana by 2D-NMR spectroscopy.

The NMR solution structures of NTX-1 (PDB code 1W6B and BMRB 6288), a long neurotoxin isolated from the venom of Naja naja oxiana, and the molecular dynamics simulation of these structures are reported. Calculations are based on 1114 NOEs, 19 hydrogen bonds, 19 dihedral angle restraints and secondary chemical shifts derived from 1H to 13C HSQC spectrum. Similar to other long neurotoxins, the three-finger like structure shows a double and a triple stranded beta-sheet as well as some flexible regions, particularly at the tip of loop II and the C-terminal tail. The solution NMR and molecular dynamics simulated structures are in good agreement with root mean square deviation values of 0.23 and 1 A for residues involved in beta-sheet regions, respectively. The overall fold in the NMR structure is similar to that of the X-ray crystallography, although some differences exist in loop I and the tip of loop II. The most functionally important residues are located at the tip of loop II and it appears that the mobility and the local structure in this region modulate the binding of NTX-1 and other long neurotoxins to the nicotinic acetylcholine receptor.     

The stress response protein Gls24 is induced by copper and interacts with the CopZ copper chaperone of Enterococcus hirae

Intracellular copper routing in Enterococcus hirae is accomplished by the CopZ copper chaperone. Under copper stress, CopZ donates Cu⁺ to the CopY repressor, thereby releasing its bound zinc and abolishing repressor–DNA interaction. This in turn induces the expression of the cop operon, which encodes CopY and CopZ, in addition to two copper ATPases, CopA and CopB. To gain further insight into the function of CopZ, the yeast two-hybrid system was used to screen for proteins interacting with the copper chaperone. This led to the identification of Gls24, a member of a family of stress response proteins. Gls24 is part of an operon containing eight genes. The operon was induced by a range of stress conditions, but most notably by copper. Gls24 was overexpressed and purified, and was shown by surface plasmon resonance analysis to also interact with CopZ in vitro . Circular dichroism measurements revealed that Gls24 is partially unstructured. The current findings establish a novel link between Gls24 and copper homeostasis.     

Metal binding domains 3 and 4 of the Wilson disease protein: solution structure and interaction with the copper(I) chaperone HAH1

The Wilson disease protein or ATP7B is a P 1B-type ATPase involved in human copper homeostasis. The extended N-terminus of ATP7B protrudes into the cytosol and contains six Cu(I) binding domains. This report presents the NMR structure of the polypeptide consisting of soluble Cu(I) binding domains 3 and 4. The two domains exhibit ferredoxin-like folds, are linked by a flexible loop, and act independently of one another. Domains 3 and 4 tend to aggregate in a concentration-dependent manner involving nonspecific intermolecular interactions. Both domains can be loaded with Cu(I) when provided as an acetonitrile complex or by the chaperone HAH1. HAH1 forms a 70% complex with domain 4 that is in fast exchange with the free protein in solution. The ability of HAH1 to form a complex only with some domains of ATP7B is an interesting property of this class of proteins and may have a signaling role in the function of the ATPases.