Energetics of copper trafficking between the Atx1 metallochaperone and the intracellular copper transporter, Ccc2

The Atx1 metallochaperone protein is a cytoplasmic Cu(I) receptor that functions in intracellular copper trafficking pathways in plants, microbes, and humans. A key physiological partner of the Saccharomyces cerevisiae Atx1 is Ccc2, a cation transporting P-type ATPase located in secretory vesicles. Here, we show that Atx1 donates its metal ion cargo to the first N-terminal Atx1-like domain of Ccc2 in a direct and reversible manner. The thermodynamic gradient for metal transfer is shallow (K(exchange) = 1.4 +/- 0.2), establishing that vectorial delivery of copper by Atx1 is not based on a higher copper affinity of the target domain. Instead, Atx1 allows rapid metal transfer to its partner. This equilibrium is unaffected by a 50-fold excess of the Cu(I) competitor, glutathione, indicating that Atx1 also protects Cu(I) from nonspecific reactions. Mechanistically, we propose that a low activation barrier for transfer between partners results from complementary electrostatic forces that ultimately orient the metal-binding loops of Atx1 and Ccc2 for formation of copper-bridged intermediates. These thermodynamic and kinetic considerations suggest that copper trafficking proteins overcome the extraordinary copper chelation capacity of the eukaryotic cytoplasm by catalyzing the rate of copper transfer between physiological partners. In this sense, metallochaperones work like enzymes, carefully tailoring energetic barriers along specific reaction pathways but not others.     

A copper metallochaperone for photosynthesis and respiration reveals metal-specific targets, interaction with an importer, and alternative sites for copper acquisition.

A bacterial two-hybrid assay revealed interaction between a protein now designated bacterial Atx1 and amino-terminal domains of copper-transporting ATPases CtaA (cellular import) and PacS (thylakoid import) but not the related zinc (ZiaA) or cobalt (CoaT) transporters from the same organism (Synechocystis PCC 6803). The specificity of metallochaperone interactions coincides with metal specificity. After reconstitution in a N(2) atmosphere, bacterial Atx1 bound 1 mol of copper mol(-1), and apoPacS(N) acquired copper from copper-Atx1. Copper was displaced from Atx1 by p-(hydroxymercuri)phenylsulfonate, indicative of thiol ligands, and two cysteine residues were obligatory for two-hybrid interaction with PacS(N). This organism contains compartments (thylakoids) where the copper proteins plastocyanin and cytochrome oxidase reside. In copper super-supplemented mutants, photooxidation of cytochrome c(6) was greater in Deltaatx1DeltactaA than in DeltactaA, showing that Atx1 contributes to efficient switching from iron in cytochrome c(6) to copper in plastocyanin for photosynthetic electron transport. Cytochrome oxidase activity was also less in membranes purified from low [copper]-grown Deltaatx1 or DeltapacS, compared with wild-type, but the double mutant Deltaatx1DeltapacS was non-additive, consistent with Atx1 acting via PacS. Conversely, activity in Deltaatx1DeltactaA was less than in either respective single mutant, revealing that Atx1 can function without the major copper importer and consistent with a role in recycling endogenous copper.     

Characterization of the binding interface between the copper chaperone Atx1 and the first cytosolic domain of Ccc2 ATPase

The interaction of the copper chaperone Atx1 and the first cytosolic domain of Ccc2 ATPase, Ccc2a, was investigated by NMR in solution. In particular, a solution of Cu(I)-15NAtx1 was titrated with apo-Ccc2a, and, vice versa, a solution of Cu(I)-15NCcc2a was titrated with apo-Atx1. By following the 15N and 1H chemical shifts, a new species is detected in both experiments. This species is the same in both titrations and is in fast exchange with the parent species on the NMR time scale. Nuclear relaxation data are consistent with the formation of an adduct. Judging from the nuclear Overhauser effect spectroscopy patterns, the structure of Cu(I)-15NCcc2a in the presence of apo-Atx1 is not significantly altered, whereas Cu(I)-15NAtx1 in the presence of apo-Ccc2a experiences some changes with respect to both the apoproteins and the Cu(I)-loaded proteins. The structure of the Cu(I)-15NAtx1 moiety in the adduct was obtained from 1137 nuclear Overhauser effects to a final root mean square deviation to the mean structure of 0.76 +/- 0.13 A for the backbone and 1.11 +/- 0.11 A for the heavy atoms. 15N and 1H chemical shifts suggest the regions of interaction that, together with independent information, allow a structural model of the adduct to be proposed. The apo form of Atx1 displays significant mobility in loops 1 and 5, the N-terminal part of helix alpha1, and the C-terminal part of helix alpha2 on the ms-micros time scale. These regions correspond to the metal binding site. Such mobility is largely reduced in the free Cu(I)-Atx1 and in the adduct with apo-Ccc2a. The analogous mobility of Ccc2a in both Cu(I) and apo forms is reduced with respect to Atx1. Such an adduct is relevant as a structural and kinetic model for copper transfer from Atx1 to Ccc2a in physiological conditions.     

Interplay between glutathione,Atx1 and copper. 1. Copper(I) glutathionate induced dimerization of Atx1

Copper is both an essential element as a catalytic cofactor and a toxic element because of its redox properties. Once in the cell, Cu(I) binds to glutathione (GSH) and various thiol-rich proteins that sequester and/or exchange copper with other intracellular components. Among them, the Cu(I) chaperone Atx1 is known to deliver Cu(I) to Ccc2, the Golgi Cu–ATPase, in yeast. However, the mechanism for Cu(I) incorporation into Atx1 has not yet been unraveled. We investigated here a possible role of GSH in Cu(I) binding to Atx1. Yeast Atx1 was expressed in Escherichia coli and purified to study its ability to bind Cu(I). We found that with an excess of GSH [at least two GSH/Cu(I)], Atx1 formed a Cu(I)-bridged dimer of high affinity for Cu(I), containing two Cu(I) and two GSH, whereas no dimer was observed in the absence of GSH. The stability constants (log β) of the Cu(I) complexes measured at pH 6 were 15–16 and 49–50 for CuAtx1 and Cu₂^I(GS⁻)₂(Atx1)₂, respectively. Hence, these results suggest that in vivo the high GSH concentration favors Atx1 dimerization and that Cu₂^I(GS⁻)₂(Atx1)₂ is the major conformation of Atx1 in the cytosol.     

Copper chaperones,intracellular copper trafficking proteins. Function,structure, and mechanism of action

This review summarizes findings on a new family of small cytoplasmic proteins called copper chaperones. The copper chaperones bind and deliver copper ions to intracellular compartments and insert the copper into the active sites of specific partners, copper-dependent enzymes. Three types of copper chaperones have been found in eukaryotes. Their three-dimensional structures have been determined, intracellular target proteins identified, and mechanisms of action have been revealed. The Atx1 copper chaperone binds Cu(I) and interacts directly with the copper-binding domains of a P-type ATPase copper transporter, its physiological partner. The copper chaperone CCS delivers Cu(I) to Cu,Zn-superoxide dismutase 1. Cox17 and Cox11 proteins serve as copper chaperones for cytochrome c oxidase, a copper-dependent enzyme.     

Multiple protein domains contribute to the action of the copper chaperone for superoxide dismutase.

The copper chaperone for superoxide dismutase (SOD1) inserts the catalytic metal cofactor into SOD1 by an unknown mechanism. We demonstrate here that this process involves the cooperation of three distinct regions of the copper chaperone for SOD1 (CCS): an amino-terminal Domain I homologous to the Atx1p metallochaperone, a central portion (Domain II) homologous to SOD1, and a short carboxyl-terminal peptide unique to CCS molecules (Domain III). These regions fold into distinct polypeptide domains as revealed through proteolysis protection studies. The biological roles of the yeast CCS domains were examined in yeast cells. Surprisingly, Domain I was found to be necessary only under conditions of strict copper limitation. Domain I and Atx1p were not interchangeable in vivo, underscoring the specificity of the corresponding metallochaperones. A putative copper site in Domain II was found to be irrelevant to yeast CCS activity, but SOD1 activation invariably required a CXC in Domain III that binds copper. Copper binding to purified yeast CCS induced allosteric conformational changes in Domain III and also enhanced homodimer formation of the polypeptide. Our results are consistent with a model whereby Domain I recruits cellular copper, Domain II facilitates target recognition, and Domain III, perhaps in concert with Domain I, mediates copper insertion into apo-SOD1.     

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.     

NMR structural analysis of the soluble domain of ZiaA-ATPase and the basis of selective interactions with copper metallochaperone Atx1

A Cu(I) metallochaperone, Atx1, interacts with the amino-terminal domain of a Cu(I)-transporting ATPase, PacS_N, but not with a domain of related Zn-transporting ATPase, ZiaA_N in Synechocystis PCC 6803. This is thought to prevent ZiaA_N from acquiring Cu(I), which it binds more tightly than Zn. Solution structures of Atx1, PacS_N, and the heterodimer were previously described. Here we report solution structural studies of the ZiaA_N soluble domain. Apo-ZiaA_N has a typical ferredoxin-like fold followed by an atypical 34 residues of unstructured polypeptide containing a His₇ motif. ZiaA_N competes with the metallochromic indicator 4-(2-pyridylazo)resorcinol for 1 equiv of Zn, which can be displaced by thiol-modifying p-mercuriphenylsulfonic acid, establishing that a high-affinity site involves thiols of the CXXC motif within the ferredoxin-like fold. A single equivalent of Zn affects nuclear magnetic resonance signals arising from the CXXC motif as well as all seven His residues. The presence of NMR-line broadening in both sites implies that Zn₁-ZiaA_N undergoes exchange phenomena, consistent with CXXC-bound Zn coincidentally sampling various His ligands. These Zn-dependent dynamic changes could either aid metal transfer or alter intramolecular interactions. No formation of Atx1–Cu(I)–ZiaA_N heterodimers was observed, and in the presence of equimolar ZiaA_N and PacS_N, only Atx1–Cu(I)–PacS_N complexes were detected. Residues flanking the CXXC motif of PacS_N (R₁₃-ASS₂₀) differ in charge and bulk from those of ZiaA_N (D₁₈-KLK₂₅) and make contacts in the Atx1–Cu(I)–PacS_N complex. Crucially, swapping these residues flanking the CXXC motifs of ZiaA_N and PacS_N reciprocally swaps partner choice by Atx1. These few residues of the two ATPases have diverged during evolution to bias Atx1 interactions in favor of PacS_N rather than ZiaA_N.     

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.     

RNA sequence and the nature of the CuA-binding site in cytochrome c oxidase.

For cytochrome c oxidase subunit II (COXII), DNA and protein sequences suggest that Met-207 (bovine numbering) is conserved in all species except plants. Sequencing of plant mitochondrial COXII mRNAs now indicates that Met-207 is also conserved among plants as a result of a C-to-U type of RNA editing. Considering the strict evolutionary conservation of Met-207 and the homology of COXII to type I (blue) copper proteins and nitrous oxide reductase, we propose a model in which Met-207 is associated with the CuA-binding site (along with Cys-196, Cys-200 and His-204) and plays a role in determining its reduction potential and stability.     

Solution structure of the yeast copper transporter domain Ccc2a in the apo and Cu(I)-loaded states

Ccc2 is an intracellular copper transporter in Saccharomyces cerevisiae and is a physiological target of the copper chaperone Atx1. Here we describe the solution structure of the first N-terminal MTCXXC metal-binding domain, Ccc2a, both in the presence and absence of Cu(I). For Cu(I)-Ccc2a, 1944 meaningful nuclear Overhauser effects were used to obtain a family of 35 structures with root mean square deviation to the average structure of 0.36 +/- 0.06 A for the backbone and 0.79 +/- 0.05 A for the heavy atoms. For apo-Ccc2a, 1970 meaningful nuclear Overhauser effects have been used with 35 (3)J(HNHalpha) to obtain a family of 35 structures with root mean square deviation to the average structure of 0.38 +/- 0.06 A for the backbone and 0.82 +/- 0.07 A for the heavy atoms. The protein exhibits a betaalphabetabetaalphabeta, ferrodoxin-like fold similar to that of its target Atx1 and that of a human counterpart, the fourth metal-binding domain of the Menkes protein. The overall fold remains unchanged upon copper loading, but the copper-binding site itself becomes less disordered. The helical context of the copper-binding site, and the copper-induced conformational changes in Ccc2a differ from those in Atx1. Ccc2a presents a conserved acidic surface which complements the basic surface of Atx1 and a hydrophobic surface. These results open new mechanistic aspects of copper transporter domains with physiological copper donor and acceptor proteins.     

Evidence that conserved residues Cys-62 and Cys-154 within the Azotobacter vinelandii nitrogenase MoFe protein α-subunit are essential for nitrogenase activity but conserved residues His-83 and Cys-88 are not

Metallocluster extrusion requirements, interspecies MoFe-protein primary sequence comparisons and comparison of the primary sequences of the MoFe-protein subunits with each other have been used to assign potential P-cluster (Fe-S cluster) domains within the MoFe protein. In each alpha-beta unit of the MoFe protein, alpha-subunit domains, which include potential Fe-S cluster ligands Cys-62, His-83, Cys-88 and Cys-154, and beta-subunit domains, which include potential Fe-S cluster ligands Cys-70, His-90, Cys-95 and Cys-153, are proposed to comprise nearly equivalent P-cluster environments located adjacent to each other in the native protein. As an approach to test this model and to probe the functional properties of the P clusters, amino acid residue substitutions were placed at the alpha-subunit Cys-62, His-83, Cys-88 and Cys-154 positions by site-directed mutagenesis of the Azotobacter vinelandii nifD gene. The diazotrophic growth rates, MoFe-protein acetylene-reduction activities, and whole-cell S = 3/2 electron paramagnetic resonance spectra of these mutants were examined. Results of these experiments show that MoFe-protein alpha-subunit residues, Cys-62 and Cys-154, are probably essential for MoFe-protein activity but that His-83 and Cys-88 residues are not. These results indicate either that His-83 and Cys-88 do not provide essential P-cluster ligands or that a new cluster-ligand arrangement is formed in their absence.     

Crystal structure of Klebsiella aerogenes UreE, a nickel-binding metallochaperone for urease activation

UreE is proposed to be a metallochaperone that delivers nickel ions to urease during activation of this bacterial virulence factor. Wild-type Klebsiella aerogenes UreE binds approximately six nickel ions per homodimer, whereas H144*UreE (a functional C-terminal truncated variant) was previously reported to bind two. We determined the structure of H144*UreE by multi-wavelength anomalous diffraction and refined it to 1.5 A resolution. The present structure reveals an Hsp40-like peptide-binding domain, an Atx1-like metal-binding domain, and a flexible C terminus. Three metal-binding sites per dimer, defined by structural analysis of Cu-H144*UreE, are on the opposite face of the Atx1-like domain than observed in the copper metallochaperone. One metal bridges the two subunits via the pair of His-96 residues, whereas the other two sites involve metal coordination by His-110 and His-112 within each subunit. In contrast to the copper metallochaperone mechanism involving thiol ligand exchanges between structurally similar chaperones and target proteins, we propose that the Hsp40-like module interacts with urease apoprotein and/or other urease accessory proteins, while the Atx1-like domain delivers histidyl-bound nickel to the urease active site.     

Mitochondrial copper metabolism in yeast: interaction between Sco1p and Cox2p

Yeast mitochondrial Sco1p is required for the formation of a functional cytochrome c oxidase (COX). It was suggested that Sco1p aids copper delivery to the catalytic center of COX. Here we show by affinity chromatography and coimmunoprecipitation that Sco1p interacts with subunit Cox2p. In addition we provide evidence that Sco1p can form homomeric complexes. Both homomer formation and binding of Cox2p are neither dependent on the presence of copper nor affected by mutations of His-239, Cys-148 or Cys-152. These amino acids, which are conserved among the members of the Sco1p family, have been suggested to act in the reduction of the cysteines in the copper binding center of Cox2p and are discussed as ligands for copper.     

X-ray absorption spectroscopy of the copper chaperone HAH1 reveals a linear two-coordinate Cu(I) center capable of adduct formation with exogenous thiols and phosphines

The human copper chaperone HAH1 transports copper to the Menkes and Wilson proteins, which are copper-translocating P-type ATPases located in the trans-Golgi apparatus and believed to provide copper for important enzymes such as ceruloplasmin, tyrosinase, and peptidylglycine monooxygenase. Although a substantial amount of structural data exist for HAH1 and its yeast and bacterial homologues, details of the copper coordination remain unclear and suggest the presence of two protein-derived cysteine ligands and a third exogenous thiol ligand. Here we report the preparation and reconstitution of HAH1 with Cu(I) using a protocol that minimizes the use of thiol reagents believed to be the source of the third ligand. We show by x-ray absorption spectroscopy that this reconstitution protocol generates an occupied Cu(I) binding site with linear biscysteinate coordination geometry, as evidenced by (i) an intense edge absorption centered at 8982.5 eV, with energy and intensity identical to the rigorously linear two-coordinate model complex bis-2,3,5,6-tetramethylbenzene thiolate Cu(I) and (ii) an EXAFS spectrum that could be fit to two Cu-S interactions at 2.16 A, a distance typical of digonal Cu(I) coordination. Binding of exogenous ligands (GSH, dithiothreitol, and tris-(2-carboxyethyl)-phosphine) to the Cu(I) was investigated. When GSH or dithiothreitol was added to the chaperone during the reconstitution procedure, the resulting Cu(I)- HAH1 remained two-coordinate, whereas the addition of the phosphine during reconstitution elicited a three-coordinate species. When the exogenous ligands were titrated into the Cu(I)-HAH1, all formed three-coordinate adducts but with differing affinities. Thus, GSH and dithiothreitol showed weaker binding, with estimated KD values in the range 10-25 mm, whereas tris-(2-carboxyethyl)-phosphine showed stronger affinity, with a KD value of <5 mm. The implications of these findings for mechanisms of copper transport are discussed.     

Structural model of the CopA copper ATPase of Enterococcus hirae based on chemical cross-linking

The CopA copper ATPase of Enterococcus hirae belongs to the family of heavy metal pumping CPx-type ATPases and shares 43% sequence similarity with the human Menkes and Wilson copper ATPases. Due to a lack of suitable protein crystals, only partial three-dimensional structures have so far been obtained for this family of ion pumps. We present a structural model of CopA derived by combining topological information obtained by intramolecular cross-linking with molecular modeling. Purified CopA was cross-linked with different bivalent reagents, followed by tryptic digestion and identification of cross-linked peptides by mass spectrometry. The structural proximity of tryptic fragments provided information about the structural arrangement of the hydrophilic protein domains, which was integrated into a three-dimensional model of CopA. Comparative modeling of CopA was guided by the sequence similarity to the calcium ATPase of the sarcoplasmic reticulum, Serca1, for which detailed structures are available. In addition, known partial structures of CPx-ATPase homologous to CopA were used as modeling templates. A docking approach was used to predict the orientation of the heavy metal binding domain of CopA relative to the core structure, which was verified by distance constraints derived from cross-links. The overall structural model of CopA resembles the Serca1 structure, but reveals distinctive features of CPx-type ATPases. A prominent feature is the positioning of the heavy metal binding domain. It features an orientation of the Cu binding ligands which is appropriate for the interaction with Cu-loaded metallochaperones in solution. Moreover, a novel model of the architecture of the intramembranous Cu binding sites could be derived.     

A 2.0-A structure of the blue copper protein (cupredoxin) from Alcaligenes faecalis S-6

The structure of a blue copper protein, cupredoxin, from the potent denitrifying bacterium Alcaligenes faecalis S-6, has been determined and refined against 2 A x-ray diffraction data. The agreement between observed and calculated structure factors is 0.159, and estimated errors in coordinates are 0.09-0.15 A. The protein folds in a beta sandwich similar to plastocyanin and azurin and includes features such as a "kink" and a "tyrosine loop" which have been noted previously for these proteins as well as immunoglobulins. The copper is bound by four ligands, in a distorted tetrahedral arrangement, with Cu-S gamma = 2.07 A (Cys-78), Cu-N delta 1 = 2.10 and 2.21 for His-40 and His-81, and Cu-S delta = 2.69 A (Met-86). Two of the ligands are further oriented by hydrogen bonds either to other side chains (Asn-9 to His-40), backbone atoms (NH...S) or a water molecule (to His-40). The methionine ligand has no extra constraints. The C-terminal loop containing three of the ligands is hydrogen-bonded to the strand containing His-40 by hydrogen bonds between the conserved residues Thr-79 and Asn-41. The pronounced dichroism of the crystal is a result of the orientation of the normal to the C beta-S gamma-Cu plane parallel to the crystallographic 6-fold axis.     

Atx1-like chaperones and their cognate P-type ATPases: copper-binding and transfer

Copper is an essential yet toxic metal ion. To satisfy cellular requirements, while, at the same time, minimizing toxicity, complex systems of copper trafficking have evolved in all cell types. The best conserved and most widely distributed of these involve Atx1-like chaperones and P_1B-type ATPase transporters. Here, we discuss current understanding of how these chaperones bind Cu(I) and transfer it to the Atx1-like N-terminal domains of their cognate transporter.     

Cd2+- or Hg2+-binding proteins can replace the Cu+-chaperone Atx1 in delivering Cu+ to the secretory pathway in yeast

Copper delivery to Ccc2--the Golgi Cu+-ATPase--was investigated in vivo, replacing the Cu+-chaperone Atx1 by various structural homologues in an atx1-Delta yeast strain. Various proteins, displaying the same ferredoxin-like fold and (M/L)(T/S)CXXC metal-binding motif as Atx1 and known as Cu+-, Cd2+- or Hg2+-binding proteins were able to replace Atx1. Therefore, regardless of their original function, these proteins could all bind copper and transfer it to Ccc2, suggesting that Ccc2 is opportunistic and can interact with many different proteins to gain Cu+. The possible role of electrostatic potential surfaces in the docking of Ccc2 with these Atx1-homologues is discussed.     

Crystal structure of the Atx1 metallochaperone protein at 1.02 A resolution.

BACKGROUND: Metallochaperone proteins function in the trafficking and delivery of essential, yet potentially toxic, metal ions to distinct locations and particular proteins in eukaryotic cells. The Atx1 protein shuttles copper to the transport ATPase Ccc2 in yeast cells. Molecular mechanisms for copper delivery by Atx1 and similar human chaperones have been proposed, but detailed structural characterization is necessary to elucidate how Atx1 binds metal ions and how it might interact with Ccc2 to facilitate metal ion transfer. RESULTS: The 1.02 A resolution X-ray structure of the Hg(II) form of Atx1 (HgAtx1) reveals the overall secondary structure, the location of the metal-binding site, the detailed coordination geometry for Hg(II), and specific amino acid residues that may be important in interactions with Ccc2. Metal ion transfer experiments establish that HgAtx1 is a functional model for the Cu(I) form of Atx1 (CuAtx1). The metal-binding loop is flexible, changing conformation to form a disulfide bond in the oxidized apo form, the structure of which has been solved to 1.20 A resolution. CONCLUSIONS: The Atx1 structure represents the first structure of a metallochaperone protein, and is one of the largest unknown structures solved by direct methods. The structural features of the metal-binding site support the proposed Atx1 mechanism in which facile metal ion transfer occurs between metal-binding sites of the diffusible copper-donor and membrane-tethered copper-acceptor proteins. The Atx1 structural motif represents a prototypical metal ion trafficking unit that is likely to be employed in a variety of organisms for different metal ions.