A core mutation affecting the folding properties of a soluble domain of the ATPase protein CopA from Bacillus subtilis

The two N-terminal domains of the P-type copper ATPase, CopAa and CopAb, from Bacillus subtilis differ in their folding capabilities in vitro. Whereas CopAb has the typical betaalphabetabetaalphabeta structure and is a rigid protein, CopAa is found to be largely unfolded. A sequence analysis of the two and of orthologue homologous proteins indicates that Ser46 in CopAa may destabilise the hydrophobic core, as also confirmed through a bioinformatic energy study. CopAb has a Val in the corresponding position. The S46V and S46A mutants are found to be folded, although the latter displays multiple conformations. S46VCopAa, in both apo and copper(I) loaded forms, has very similar structural and dynamic properties with respect to CopAb, besides a different length of strand beta2 and beta4. It is intriguing that the oxygen of Thr16 is found close, though at longer than bonding distance, to copper in both domains, as it also occurs in a human orthologue domain. This study contributes to understanding the behaviour of proteins that do not properly fold in vitro. A possible biological significance of the peculiar folding behaviour of this domain is discussed.     

Cu(I)- and proton-binding properties of the first N-terminal soluble domain of Bacillus subtilis CopA

CopA, a P-type ATPase transporter involved in copper detoxification in Bacillus subtilis, contains two soluble Atx1-like domains separated by a short linker at its N-terminus, an arrangement that occurs widely in copper transporters from both prokaryotes and eukaryotes. Both domains were previously found to bind Cu(I) with very high affinity. Above a level of 1 Cu(I) per CopAab, dimerization occurred, leading to a highly luminescent multinuclear Cu(I) species [Singleton C & Le Brun NE (2009) Dalton Trans, 688-696]. To try to understand the contributions of each domain to the complex Cu(I)-binding behaviour of this and related proteins, we purified a wild-type form of the first domain (CopAa). In isolation, the domain bound Cu(I) with very high affinity (K = ～ 1 × 10(18) m(-1) ) and underwent Cu(I)-mediated protein association, resulting in a mixture of dimer and tetramer species. Addition of further Cu(I) up to 1 Cu(I) per CopAa monomer led to a weakly luminescent species, whereas further additions [2 Cu(I) per CopAa monomer] resulted in protein unfolding. Analysis of the MTCAAC binding motif Cys residue acid-base properties revealed pK(a) values of 5.7 and 7.3, consistent with the pH dependence of Cu(I) binding, and with the proposal that low proton affinity is associated with high Cu(I) affinity. Finally, Cu(I) exchange between CopAa and the chelator bathocuproine sulfonate revealed rapid exchange in both directions, demonstrating an interaction between the protein and the chelator that catalyses metal ion transfer. Overall, CopAa exhibits similarities to CopAab in terms of affinity and complexity of Cu(I) binding, but the details of Cu(I) binding are distinct.     

Structural basis for the function of the N-terminal domain of the ATPase CopA from Bacillus subtilis

The solution structure of the N-terminal region (151 amino acids) of a copper ATPase, CopA, from Bacillus subtilis, is reported here. It consists of two domains, CopAa and CopAb, linked by two amino acids. It is found that the two domains, which had already been separately characterized, interact one to the other through a hydrogen bond network and a few hydrophobic interactions, forming a single rigid body. The two metal binding sites are far from one another, and the short link between the domains prevents them from interacting. This and the surface electrostatic potential suggest that each domain receives copper from the copper chaperone, CopZ, independently and transfers it to the membrane binding site of CopA. The affinity constants of silver(I) and copper(I) are similar for the two sites as monitored by NMR. Because the present construct "domain-short link-domain" is shared also by the last two domains of the eukaryotic copper ATPases and several residues at the interface between the two domains are conserved, the conclusions of the present study have general validity for the understanding of the function of copper ATPases.     

Copper alters aggregation behavior of prion protein and induces novel interactions between its N- and C-terminal regions

Copper is reported to promote and prevent aggregation of prion protein. Conformational and functional consequences of Cu(2+)-binding to prion protein (PrP) are not well understood largely because most of the Cu(2+)-binding studies have been performed on fragments and truncated variants of the prion protein. In this context, we set out to investigate the conformational consequences of Cu(2+)-binding to full-length prion protein (PrP) by isothermal calorimetry, NMR, and small angle x-ray scattering. In this study, we report altered aggregation behavior of full-length PrP upon binding to Cu(2+). At physiological temperature, Cu(2+) did not promote aggregation suggesting that Cu(2+) may not play a role in the aggregation of PrP at physiological temperature (37 °C). However, Cu(2+)-bound PrP aggregated at lower temperatures. This temperature-dependent process is reversible. Our results show two novel intra-protein interactions upon Cu(2+)-binding. The N-terminal region (residues 90-120 that contain the site His-96/His-111) becomes proximal to helix-1 (residues 144-147) and its nearby loop region (residues 139-143), which may be important in preventing amyloid fibril formation in the presence of Cu(2+). In addition, we observed another novel interaction between the N-terminal region comprising the octapeptide repeats (residues 60-91) and helix-2 (residues 174-185) of PrP. Small angle x-ray scattering studies of full-length PrP show significant compactness upon Cu(2+)-binding. Our results demonstrate novel long range inter-domain interactions of the N- and C-terminal regions of PrP upon Cu(2+)-binding, which might have physiological significance.     

Structure and Cu(I)-binding properties of the N-terminal soluble domains of Bacillus subtilis CopA

CopA, a P-type ATPase from Bacillus subtilis, plays a major role in the resistance of the cell to copper by effecting the export of the metal across the cytoplasmic membrane. The N-terminus of the protein features two soluble domains (a and b), that each contain a Cu(I)-binding motif, MTCAAC. We have generated a stable form of the wild-type two-domain protein, CopAab, and determined its solution structure. This was found to be similar to that reported previously for a higher stability S46V variant, with minor differences mostly confined to the Ser(46)-containing beta3-strand of domain a. Chemical-shift analysis demonstrated that the two Cu(I)-binding motifs, located at different ends of the protein molecule, are both able to participate in Cu(I) binding and that Cu(I) is in rapid exchange between protein molecules. Surprisingly, UV-visible and fluorescence spectroscopy indicate very different modes of Cu(I) binding below and above a level of 1 Cu(I) per protein, consistent with a major structural change occurring above 1 Cu(I) per CopAab. Analytical equilibrium centrifugation and gel filtration results show that this is a result of Cu(I)-mediated dimerization of the protein. The resulting species is highly luminescent, indicating the presence of a solvent-shielded Cu(I) cluster.     

A NMR study of the interaction of a three-domain construct of ATP7A with copper(I) and copper(I)-HAH1: the interplay of domains

ATP7A is a P-type ATPase involved in copper(I) homeostasis in humans. It possesses a long N-terminal tail protruding into the cytosol and containing six copper(I)-binding domains, which are individually folded and capable of binding one copper(I) ion. ATP7A receives copper from a soluble protein, the metallochaperone HAH1. The exact role and interplay of the six soluble domains is still quite unclear, as it has been extensively demonstrated that they are strongly redundant with respect to copper(I) transport in vivo. In the present work, a three-domain (fourth to sixth, MNK456) construct has been investigated in solution by NMR, in the absence and presence of copper(I). In addition, the interaction of MNK456 with copper(I)-HAH1 has been studied. It is proposed that the fourth domain is the preferential site for the initial interaction with the partner. A significant dependence of the overall domain dynamics on the metallation state and on the presence of HAH1 is observed. This dependence could constitute the molecular mechanism to trigger copper(I) translocation and/or ATP7A relocalization from the trans-Golgi network to the plasmatic membrane.     

Interaction of the two soluble metal-binding domains of yeast Ccc2 with copper(I)-Atx1

Yeast Ccc2 is a P-type ATPase responsible for transport of copper(I) from the cytosol to the trans-Golgi network. It possesses a soluble cytosolic N-terminal region containing two copper(I)-binding domains. Homologous eukaryotic copper-transporting ATPases have from one to six domains. We have expressed a fragment encompassing residues 1-150 of Ccc2, which corresponds to the two domains, and found that the second domain was substantially less structured than the first. The first domain could bind copper(I) and interact with the partner protein Atx1 at variance with the second. Similar results are found in ATPases from other organisms and may represent a general feature, whose biochemical implications are not yet fully appreciated.     

Cu(I) affinities of the domain 1 and 3 sites in the human metallochaperone for Cu,Zn-superoxide dismutase

The delivery of copper by the human metallochaperone CCS is a key step in the activation of Cu,Zn-superoxide dismutase (SOD1). CCS is a three-domain protein with Cu(I)-binding CXXC and CXC motifs in domains 1 and 3, respectively. A detailed analysis of the binding of copper to CCS, including variants in which the Cys residues from domains 1 and 3 have been mutated to Ser, and also using separate domain 1 and 3 constructs, demonstrates that CCS is able to bind 1 equiv of Cu(I) in both of these domains. The Cu(I) affinity of domain 1 is approximately 5 × 10(17) M(-1) at pH 7.5, while that of domain 3 is at least 1 order of magnitude weaker. The CXXC site will therefore be preferentially loaded with Cu(I), suggesting that domain 1 plays a role in the acquisition of the metal. The delivery of copper to the target occurs via domain 3 whose structural flexibility and ability to be transiently metalated during copper delivery appear to be more important than the Cu(I) affinity of its CXC motif. The Cu(I) affinity of domain 1 of CCS is comparable to that of HAH1, another cytosolic copper metallochaperone. CCS and HAH1 readily exchange Cu(I), providing a mechanism whereby cross-talk can occur between copper trafficking pathways.     

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.     

Copper transfer to the N-terminal domain of the Wilson disease protein (ATP7B): X-ray absorption spectroscopy of reconstituted and chaperone-loaded metal binding domains and their interaction with exogenous ligands

The copper-transporting ATPases are 165-175 kDa membrane proteins, composed of 8 transmembrane segments and two large cytosolic domains, the N-terminal copper-binding domain and the catalytic ATP-hydrolyzing domain. In ATP7B, the Wilson disease protein, the N-terminal domain is made up of six metal-binding sub-domains containing the MXCXXC motif which is known to coordinate copper via the two cysteine residues. We have expressed the N-terminal domain of ATP7B as a soluble C-terminal fusion with the maltose binding protein. This expression system produces a protein which can be reconstituted with copper without recourse to the harsh denaturing conditions or low pH reported by other laboratories. Here we describe the reconstitution of the metal binding domains (MBD) with Cu(I) using a number of different protocols, including copper loading via the chaperone, Atox1. X-ray absorption spectra have been obtained on all these derivatives, and their ability to bind exogenous ligands has been assessed. The results establish that the metal-binding domains bind Cu(I) predominantly in a bis cysteinate environment, and are able to bind exogenous ligands such as DTT in a similar fashion to Atox1. We have further observed that exogenous ligand binding induces the formation of a Cu-Cu interaction which may signal a conformational change of the N-terminal domain.     

The Four Hydrophobic Residues on the Hsp70 Inter-Domain Linker Have Two Distinct Roles

The ubiquitous molecular chaperone 70-kDa heat shock proteins (Hsp70) play key roles in maintaining protein homeostasis. Hsp70s contain two functional domains: a nucleotide binding domain and a substrate binding domain. The two domains are connected by a highly conserved inter-domain linker, and allosteric coupling between the two domains is critical for chaperone function. The auxiliary chaperone 40-kDa heat shock proteins (Hsp40) facilitate all the biological processes associated with Hsp70s by stimulating the ATPase activity of Hsp70s. Although an overall essential role of the inter-domain linker in both allosteric coupling and Hsp40 interaction has been suggested, the molecular mechanisms remain largely unknown. Previously, we reported a crystal structure of a full-length Hsp70 homolog, in which the inter-domain linker forms a well-ordered β strand. Four highly conserved hydrophobic residues reside on the inter-domain linker. In DnaK, a well-studied Hsp70, these residues are V389, L390, L391, and L392. In this study, we biochemically dissected their roles. The inward-facing side chains of V389 and L391 form extensive hydrophobic contacts with the nucleotide binding domain, suggesting their essential roles in coupling the two functional domains, a hypothesis confirmed by mutational analysis. On the other hand, L390 and L392 face outward on the surface. Mutation of either abolishes DnaK's in vivo function, yet intrinsic biochemical properties remain largely intact. In contrast, Hsp40 interaction is severely compromised. Thus, for the first time, we separated the two essential roles of the highly conserved Hsp70 inter-domain linker: coupling the two functional domains through V389 and L391 and mediating the interaction with Hsp40 through L390 and L392.     

Binding of copper(I) by the Wilson disease protein and its copper chaperone

The Wilson disease protein (WND) is a transport ATPase involved in copper delivery to the secretory pathway. Mutations in WND and its homolog, the Menkes protein, lead to genetic disorders of copper metabolism. The WND and Menkes proteins are distinguished from other P-type ATPases by the presence of six soluble N-terminal metal-binding domains containing a conserved CXXC metal-binding motif. The exact roles of these domains are not well established, but possible functions include exchanging copper with the metallochaperone Atox1 and mediating copper-responsive cellular relocalization. Although all six domains can bind copper, genetic and biochemical studies indicate that the domains are not functionally equivalent. One way the domains could be tuned to perform different functions is by having different affinities for Cu(I). We have used isothermal titration calorimetry to measure the association constant (K(a)) and stoichiometry (n) values of Cu(I) binding to the WND metal-binding domains and to their metallochaperone Atox1. The association constants for both the chaperone and target domains are approximately 10(5) to 10(6) m(-1), suggesting that the handling of copper by Atox1 and copper transfer between Atox1 and WND are under kinetic rather than thermodynamic control. Although some differences in both n and K(a) values are observed for variant proteins containing less than the full complement of six metal-binding domains, the data for domains 1-6 were best fitted with a single site model. Thus, the individual functions of the six WND metal-binding domains are not conferred by different Cu(I) affinities but instead by fold and electrostatic surface properties.     

Copper transfer studies between the N-terminal copper binding domains one and four of human Wilson protein

Human Wilson protein functions in the secretory pathway to insert copper ultimately into the multicopper oxidase ceruloplasmin and also plays a role in the excretion of excess copper to the bile. This copper-transporting P-type ATPase possesses six N-terminal cytosolic copper-binding domains contained within an approximately 72 amino acid consensus motif and the first four of these domains, denoted WLN1-4, are implicated in copper acquisition from the metallochaperone HAH1, whereas the domains closest to the membrane portion of the enzyme, WLN5-6, are essential for copper transport across the membrane. In order to test our hypothesis that copper transfer occurs between domains in the N-terminus of Wilson protein, we expressed and purified to homogeneity copper-binding domains 1, 3, 4, 5-6, and 6, denoted by WLN1, WLN3, WLN4, WLN5-6, and WLN6, respectively. Since we determined WLN1 and WLN4 to have the highest and lowest isoelectric points (6.77 and 3.85, respectively) and thus are readily separated via ion exchange chromatography, we developed a copper transfer assay between these domains. We anaerobically incubated either Cu(I)-WLN1 with apo-WLN4 or apo-WLN1 with Cu(I)-WLN4, then separated these domains and quantified the amount of copper that migrates from one domain to another by ICP-MS. Regardless of whether we start with Cu(I)-WLN1 or Cu(I)-WLN4 as the initial copper donor, we demonstrate facile copper transfer between WLN1 and WLN4, thereby demonstrating the feasibility of copper transfer between these domains in vivo.     

An atomic-level investigation of the disease-causing A629P mutant of the Menkes protein, ATP7A

Menkes disease is a fatal disease that can be induced by various mutations in the ATP7A gene, leading to unpaired uptake of dietary copper. The ATP7A gene encodes a copper(I)-translocating ATPase. Here the disease-causing A629P mutation, which occurs in the last of the six copper(I)-binding soluble domains of the ATPase (hereafter MNK6), was investigated. To understand why this apparently minor amino acid replacement is pathogenic, the solution structures and dynamics on various time-scales of wild-type and A629P-MNK6 were determined both in the apo- and copper(I)-loaded forms. The interaction in vitro with the physiological ATP7A copper(I)-donor (HAH1) was additionally studied. The A629P mutation makes the protein beta-sheet more solvent accessible, possibly resulting in an enhanced susceptibility of ATP7A to proteolytic cleavage and/or in reduced capability of copper(I)-translocation. A small reduction of the affinity for copper(I) is also observed. Both effects could concur to pathogenicity.     

Cu(I) binding and transfer by the N terminus of the Wilson disease protein

Wilson and Menkes diseases are genetic disorders of copper metabolism caused by mutations in the Wilson (WND) and Menkes (MNK) copper-transporting P1B-type ATPases. The N termini of these ATPases consist of six metal binding domains (MBDs). The MBDs interact with the copper chaperone Atox1 and are believed to play roles in catalysis and in copper-mediated cellular relocalization of WND and MNK. Although all six MBDs have similar folds and bind one Cu(I) ion via a conserved CXXC motif, biochemical and genetic data suggest that they have distinct functions. Most studies aimed at characterizing the MBDs have employed smaller polypeptides consisting of one or two domains. The role of each MBD is probably defined by its environment within the six-domain N terminus, however. To study the properties of the individual domains within the context of the intact Wilson N terminus (N-WND), a series of variants in which five of the six metal binding CXXC motifs are mutated to SXXS was generated. For each variant, the Cu(I) binding affinity and the ability to exchange Cu(I) with Atox1 were investigated. The results indicate that Atox1 can deliver Cu(I) to and remove Cu(I) from each MBD, that each MBD has stronger Cu(I) retention properties than Atox1, and that all of the MBDs as well as Atox1 have similar K(Cu) values of (2.2-6.3) x 10(10) m(-1). Therefore, the specific role of each MBD is not conferred by its position within the intact N-WND but may be related to interactions with other domains and partner proteins.     

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.     

An NMR study of the interaction between the human copper(I) chaperone and the second and fifth metal-binding domains of the Menkes protein

The interaction between the human copper(I) chaperone, HAH1, and one of its two physiological partners, the Menkes disease protein (ATP7A), was investigated in solution using heteronuclear NMR. The study was carried out through titrations involving HAH1 and either the second or the fifth soluble domains of ATP7A (MNK2 and MNK5, respectively), in the presence of copper(I). The copper-transfer properties of MNK2 and MNK5 are similar, and differ significantly from those previously observed for the yeast homologous system. In particular, no stable adduct is formed between either of the MNK domains and HAH1. The copper(I) transfer reaction is slow on the time scale of the NMR chemical shift, and the equilibrium is significantly shifted towards the formation of copper(I)-MNK2/MNK5. The solution structures of both apo- and copper(I)-MNK5, which were not available, are also reported. The results are discussed in comparison with the data available in the literature for the interaction between HAH1 and its partners from other spectroscopic techniques.     

Solution structure and intermolecular interactions of the third metal-binding domain of ATP7A, the Menkes disease protein

The third metal-binding domain of the human Menkes protein (MNK3), a copper(I)-transporting ATPase, has been expressed in Escherichia coli and characterized in solution. The solution structure of MNK3, its copper(I)-binding properties, and its interaction with the physiological partner, HAH1, have been studied. MNK3 is the domain most dissimilar in structure from the other domains of the Menkes protein. This is reflected in a significant rearrangement of the last strand of the four-stranded beta-sheet when compared with the other known homologous proteins or protein domains. MNK3 is also peculiar with respect to its interaction with the copper(I) ion, as it was found to be a comparatively weak binder. Copper(I) transfer from metal-loaded HAH1 was observed experimentally, but the metal distribution was shifted toward binding by HAH1. This is at variance with what is observed for the other Menkes domains.     

Copper-dependent protein-protein interactions studied by yeast two-hybrid analysis

An important step in copper homeostasis is delivery of copper to a specific P-type ATPase in the Golgi apparatus (Ccc2 in yeast, ATP7A and ATP7B in humans) by a small copper chaperone protein (Atx1 in yeast, ATOX1 in humans). Atx1 and ATOX1 both contain an MXCXXC motif that is also present in Ccc2 (two motifs) and ATP7A/B (six motifs). Protein-protein interactions probably require coordination of one Cu(I) by cysteines from both MXCXXC motifs. We applied yeast two-hybrid analysis to screen systematically all possible interactions between MXCXXC-containing domains in these proteins. We demonstrate that ATOX1 and Atx1 preferentially interact with domains 2 and 4 of ATP7B and that Atx1 interacts with both Ccc2 domains. All combinations show a remarkable bell-shaped dependency on copper concentration that is maximal just below normal copper levels. Our results suggest that yeast two-hybrid analysis can be used to study the intracellular copper status of a cell.