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At least 19 recordsLinked to original sources

PubMed2001

Metal transporters that contribute copper to metallochaperones in Saccharomyces cerevisiae.

Copper metallochaperones represent a new family of soluble, low-molecular-weight proteins that function to deliver copper to specific sites within a cell. How the metallochaperones acquire their copper, however, is not known. In this study, we have conducted a survey of known metal ion transporters in bakers' yeast, Saccharomyces cerevisiae, to identify those that contribute copper to pathways involving the metallochaperones Atxlp and Lys7p. The results indicatethat, in addition to the well known Ctr1p and Ctr3p high-affinity copper transporters, the metallochaperones can acquire their copper through pathways involving the relatively non-specific divalent metal ion transporter Fet4p and the putative low-affinitycopper transporter Ctr2p. We have examined the localization of Ctr2p using an epitope tagged version of the protein and find that Ctr2p does not localize to the cell surface but may operate at the level of the vacuole to mobilize intracellular copper. Inaddition to Ctrlp, Ctr2p, Ctr3p and Fet4p, other metal transport systems can act as upstream donors of copper for the metallochaperones when copper availability in the medium is increased. Although the nature of these auxiliary systems is unknown, they do not appear to involve the yeast members of the Nramp family of divalent transporters, or uptake mechanisms that involve endocytosis. Since vastly different metal transporters located at either the cell surface or intracellular sites can all contribute copper to metallochaperones, it is unlikely that the metallochaperones directly interact with the metal transporters to obtain the metal.

Antiporters↗

PubMed2004

Ionic self-complementarity induces amyloid-like fibril formation in an isolated domain of a plant copper metallochaperone protein.

BACKGROUND: Arabidopsis thaliana copper metallochaperone CCH is a functional homologue of yeast antioxidant ATX1, involved in cytosolic copper transport. In higher plants, CCH has to be transported to specialised cells through plasmodesmata, being the only metallochaperone reported to date that leaves the cell where it is synthesised. CCH has two different domains, the N-terminal domain conserved among other copper-metallochaperones and a C-terminal domain absent in all the identified non-plant metallochaperones. The aim of the present study was the biochemical and biophysical characterisation of the C-terminal domain of the copper metallochaperone CCH. RESULTS: The conformational behaviour of the isolated C-domain in solution is complex and implies the adoption of mixed conformations in different environments. The ionic self-complementary peptide KTEAETKTEAKVDAKADVE, derived from the C-domain of CCH, adopts and extended conformation in solution with a high content in beta-sheet structure that induces a pH-dependent fibril formation. Freeze drying electron microscopy studies revealed the existence of well ordered amyloid-like fibrils in preparations from both the C-domain and its derivative peptide. CONCLUSION: A number of proteins related with copper homeostasis have a high tendency to form fibrils. The determinants for fibril formation, as well as the possible physiological role are not fully understood. Here we show that the plant exclusive C-domain of the copper metallochaperone CCH has conformational plasticity and forms fibrils at defined experimental conditions. The putative influence of these properties with plant copper delivery will be addressed in the future.

Amino Acid Sequence↗

PubMed2003

Analysis of the metal-binding selectivity of the metallochaperone CopZ from Enterococcus hirae by electrospray ionization mass spectrometry.

Metallochaperones are soluble proteins involved in metal transport and regulation in vivo. Copper metallochaperones belong to a structural family of metal binding domains displaying a ferredoxin-like fold (betaalphabetabetaalphabeta) and a consensus metal-binding motif MXCXXC. The metal-binding selectivities for this class of proteins are poorly documented so far. The present study focuses on the measurement of the selectivity of the copper metallochaperone CopZ from Enterococcus hirae for different metal ions using an experimental approach based on electrospray ionization mass spectrometry (ESI-MS). All the metal cations tested, i.e. Cu(I), Cu(II), Hg(II), Cd(II) and Co(II), form specific metal complexes with CopZ. The study of a chemically modified CopZ as well as variants of CopZ in the active site demonstrated that the complexes observed by ESI-MS, i.e. in the gas phase, corresponded to the complexes previously observed by other analytical methods in solution. Competition experiments allowed the classification of the metal ions by increasing affinities for CopZ as follows: Co << Cd < Hg < Cu. A dissociation constant in the range of 20 microM was determined for cobalt. The affinity of CopZ for the other metals tested was found to be higher, with dissociation constants smaller than micromolar.

Bacterial Proteins↗

PubMed2003

Thermotoga maritima IscU. Structural characterization and dynamics of a new class of metallochaperone.

Members of the IscU family of proteins are among the most conserved of all protein groups, extending across all three kingdoms of life. IscU serves as a scaffold for the assembly of intermediate iron-sulfur cluster centers and further mediates delivery to apo protein targets. Several proteins that mediate delivery of single metal ions to apo targets (termed metallochaperones) have recently been characterized structurally. Each displays a ferredoxin-like betaalphabetabetaalphabeta motif as a structural core. Assembly and delivery of a polynuclear iron-sulfur cluster is, however, a more complex pathway and presumably would demand a distinctive protein mediator. Here, we demonstrate Thermotoga maritima IscU (Tm IscU) to display unique structural and motional characteristics that distinguish it from other members of this class of proteins. In particular, IscU adopts a mobile, physiologically relevant, molten globule-like state that is vastly different from the previously identified ferredoxin-like fold that has thus far been characterized for other metallochaperones. The secondary structural content of Tm IscU is consistent with previous circular dichroism measurements on apo and holo protein, consisting of six alpha-helices and three beta-strands, the latter forming an anti-parallel beta-sheet. Extensive dynamics studies are consistent with a protein that has reasonably well defined secondary structural elements, but with a tertiary structure that is fluxional among widely different conformational arrangements. Analogous conformational flexibility does not exist in other structurally characterized metallochaperones; however, such a dynamic molecule may account for the lack of long-range NOEs, and allow both for the flexibility that is necessary for the multiple roles of Fe-S cluster assembly, and recognition and delivery of that cluster to a target protein. Additionally, the fluxionality of IscU is unique in that the protein appears to be more compact (based on 1H/2H exchange, R1, R2, and NOE data) but yet more fluid (lack of long-range NOEs) than typical molten globule proteins.

Amino Acid Motifs↗

PubMed2001

The metallochaperone Atox1 plays a critical role in perinatal copper homeostasis.

Copper plays a fundamental role in the biochemistry of all aerobic organisms. The delivery of this metal to specific intracellular targets is mediated by metallochaperones. To elucidate the role of the metallochaperone Atox1, we analyzed mice with a disruption of the Atox1 locus. Atox1(-/-) mice failed to thrive immediately after birth, with 45% of pups dying before weaning. Surviving animals exhibited growth failure, skin laxity, hypopigmentation, and seizures because of perinatal copper deficiency. Maternal Atox1 deficiency markedly increased the severity of Atox1(-/-) phenotype, resulting in increased perinatal mortality as well as severe growth retardation and congenital malformations among surviving Atox1(-/-) progeny. Furthermore, Atox1-deficient cells accumulated high levels of intracellular copper, and metabolic studies indicated that this defect was because of impaired cellular copper efflux. Taken together, these data reveal a direct role for Atox1 in trafficking of intracellular copper to the secretory pathway of mammalian cells and demonstrate that this metallochaperone plays a critical role in perinatal copper homeostasis.

Animals↗

PubMed1999

Structure-function analyses of the ATX1 metallochaperone.

Saccharomyces cerevisiae Atx1p represents a member of the family of metallochaperone molecules that escort copper to distinct intracellular targets. Atx1p specifically delivers copper to the Ccc2p copper transporter in the Golgi. Additionally, when overproduced, Atx1p substitutes for superoxide dismutase 1 in preventing oxidative damage; however the mechanistic overlap between these functions is unresolved. The crystal structure of Atx1p has been solved recently. By examining a surface electrostatic potential distribution, multiple conserved lysines are revealed on one face of Atx1p. An additional conserved lysine (Lys65) lies in close proximity to the metal binding site. Through site-directed mutagenesis, residues in the metal binding region including Lys65 were found to be necessary for both copper delivery to Ccc2p and for Atx1p antioxidant activity. Copper trafficking to Ccc2p also relied on the lysine-rich face of Atx1p. Surprisingly however, elimination of these lysines did not inhibit the antioxidant activity of Atx1p. We provide evidence that Atx1p does not suppress oxidative damage by a metallochaperone mechanism but may directly consume superoxide. Purified Cu-Atx1p reacts noncatalytically with superoxide anion in vitro. We conclude that the copper-trafficking and antioxidant functions of Atx1p arise from chemically and structurally distinct attributes of this metallochaperone.

Antioxidants↗

PubMed1999

A gain of superoxide dismutase (SOD) activity obtained with CCS, the copper metallochaperone for SOD1.

The incorporation of copper ions into the cytosolic superoxide dismutase (SOD1) is accomplished in vivo by the action of the copper metallochaperone CCS (copper chaperone for SOD1). Mammalian CCS is comprised of three distinct protein domains, with a central region exhibiting remarkable homology (approximately 50% identity) to SOD1 itself. Conserved in CCS are all the SOD1 zinc binding ligands and three of four histidine copper binding ligands. In CCS the fourth histidine is replaced by an aspartate (Asp(200)). Despite this conservation of sequence between SOD1 and CCS, CCS exhibited no detectable SOD activity. Surprisingly, however, a single D200H mutation, targeting the fourth potential copper ligand in CCS, granted significant superoxide scavenging activity to this metallochaperone that was readily detected with CCS expressed in yeast. This mutation did not inhibit the metallochaperone capacity of CCS, and in fact, D200H CCS appears to represent a bifunctional SOD that can self-activate itself with copper. The aspartate at CCS position 200 is well conserved among mammalian CCS molecules, and we propose that this residue has evolved to preclude deleterious reactions involving copper bound to CCS.

Amino Acid Sequence↗

PubMed2001

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.

Animals↗

PubMed2004

Solution structures of a cyanobacterial metallochaperone: insight into an atypical copper-binding motif.

The Atx1 copper metallochaperone from Synechocystis PCC 6803, ScAtx1, interacts with two P(1)-type copper ATPases to supply copper proteins within intracellular compartments, avoiding ATPases for other metals en route. Here we report NMR-derived solution structures for ScAtx1. The monomeric apo form has a betaalphabetabetaalpha fold with backbone motions largely restricted to loop 1 containing Cys-12 and Cys-15. The tumbling rate of Cu(I)ScAtx1 (0.1-0.8 mm) implies dimers. Experimental restraints are satisfied by symmetrical dimers with Cys-12 or His-61, but not Cys-15, invading the copper site of the opposing subunit. A full sequence of copper ligands from the cell surface to thylakoid compartments is proposed, considering in vitro homodimer liganding to mimic in vivo liganding in ScAtx1-ATPase heterodimers. A monomeric high resolution structure for Cu(I)ScAtx1, with Cys-12, Cys-15, and His-61 as ligands, is calculated without violations despite the rotational correlation time. (2)J(NH) couplings in the imidazole ring of His-61 establish coordination of N(epsilon2) to copper. His-61 is analogous to Lys-65 in eukaryotic metallochaperones, stabilizing Cu(I)S(2) complexes but by binding Cu(I) rather than compensating charge. Cys-Cys-His ligand sets are an emergent theme in some copper metallochaperones, although not in related Atx1, CopZ, or Hah1. Surface charge (Glu-13) close to the metal-binding site of ScAtx1 is likely to support interaction with complementary surfaces of copper-transporting ATPases (PacS-Arg-11 and CtaA-Lys-14) but to discourage interaction with zinc ATPase ZiaA and so inhibit aberrant formation of copper-ZiaA complexes.

Apoproteins↗

PubMed2004

Copper-mediated homo-dimerisation for the HAH1 metallochaperone.

The HAH1 metallochaperone is a key protein implicated in copper homeostasis in human cells. Using as solid-phase based assay completed with Biacore studies, we provided evidence that HAH1 forms homo-dimers in the presence of copper. Biacore analysis allowed us to determine the kinetic parameters of this interaction, characterised by an apparent affinity constant of 6muM. Moreover, we demonstrated that copper-loaded HAH1 interacts independently with each of the six individual metal-binding domains of the copper-translocating Menkes ATPase. Finally, the homo-dimerisation of the metallochaperone was confirmed in living cells by using fluorescence resonance energy transfer. Results have been discussed in the context of intracellular copper control.

Adenosine Triphosphatases↗

PubMed2002

Metallochaperone Atox1 transfers copper to the NH2-terminal domain of the Wilson's disease protein and regulates its catalytic activity.

Copper is essential for the growth and development of mammalian cells. The key role in the intracellular distribution of copper belongs to the recently discovered family of metallochaperones and to copper-transporting P-type ATPases. The mutations in the ATPase ATP7B, the Wilson's disease protein (WNDP), lead to intracellular accumulation of copper and severe hepatic and neurological abnormalities. Several of these mutations were shown to disrupt the protein-protein interactions between WNDP and the metallochaperone Atox1, suggesting that these interactions are important for normal copper homeostasis. To understand the functional consequences of the Atox1-WNDP interaction at the molecular level, we produced recombinant Atox1 and characterized its effects on WNDP. We demonstrate that Atox1 transfers copper to the purified amino-terminal domain of WNDP (N-WNDP) in a dose-dependent and saturable manner. A maximum of six copper atoms can be transferred to N-WNDP by the chaperone. Furthermore, the incubation of copper Atox1 with the full-length WNDP leads to the stimulation of the WNDP catalytic activity, providing strong evidence for the direct effect of Atox1 on the function of this transporter. Our data also suggest that Atox1 can regulate the copper occupancy of WNDP. The incubation with apo-Atox1 results in the removal of copper from the metalated N-WNDP and apparent down-regulation of WNDP activity. Interestingly, at least one copper atom remains tightly bound to N-WNDP even in the presence of excess apo-Atox1. We suggest that this incomplete reversibility reflects the functional non-equivalency of the metal-binding sites in WNDP and speculate about the intracellular consequences of the reversible Atox1-mediated copper transfer.

Adenosine Triphosphatases↗

PubMed2003

Metallochaperone for Cu,Zn-superoxide dismutase (CCS) protein but not mRNA is higher in organs from copper-deficient mice and rats.

Cu,Zn-superoxide dismutase (SOD1) is an abundant metalloenzyme important in scavenging superoxide ions. Cu-deficient rats and mice have lower SOD1 activity and protein, possibly because apo-SOD1 is degraded faster than holo-SOD1. SOD1 interacts with and requires its metallochaperone CCS for donating copper. We produced dietary Cu deficiency in rodents to determine if the reduction in SOD1 was related to the level of its specific metallochaperone CCS. CCS levels determined by immunoblot were 2- to 3-fold higher in liver, heart, kidney, and brain from male Cu-deficient rats and mice under a variety of conditions. CCS was also higher in livers of Cu-deficient dams. Interestingly, CCS levels in brain of Cu-deficient mice were also higher even though SOD1 activity and protein were not altered, suggesting that the rise in CCS is correlated with altered Cu status rather than a direct result of lower SOD1. A DNA probe specific for rat CCS detected a single transcript by Northern blot hybridization with liver RNA. CCS mRNA levels in mouse and rat liver were not altered by dietary treatment. These results suggest a posttranscriptional mechanism for higher CCS protein when Cu is limiting in the cell, perhaps due to slower protein turnover. Elevation in CCS level is one of the most dramatic alterations in Cu binding proteins accompanying Cu deficiency and may be useful to assess Cu status.

Animals↗

PubMed1999

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.

Acid Anhydride Hydrolases↗

PubMed2002

Metallochaperones: bind and deliver.

Metallochaperones deliver metal ions directly to target proteins via specific protein-protein interactions. Recent research has led to a molecular picture of how some metallochaperones bind metal ions, recognize their partner proteins, and accomplish metal ion transfer.

Metalloproteins↗

PubMed2001

Copper delivery by metallochaperone proteins.

Copper is an essential element in all living organisms, serving as a cofactor for many important proteins and enzymes. Metallochaperone proteins deliver copper ions to specific physiological partners by direct protein-protein interactions. The Atx1-like chaperones transfer copper to intracellular copper transporters, and the CCS chaperones shuttle copper to copper,zinc superoxide dismutase. Crystallographic studies of these two copper chaperone families have provided insights into metal binding and target recognition by metallochaperones and have led to detailed molecular models for the copper transfer mechanism.

Carrier Proteins↗

PubMed2004

A novel copper site in a cyanobacterial metallochaperone.

The thylakoid lumen of the cyanobacterium Synechocystis PCC 6803 is supplied with copper via two copper-transporting ATPases and a metallochaperone intermediary. We show that the copper site of this metallochaperone is unusual and consists of two cysteine residues and a histidine imidazole located on structurally dynamic loops. Substitution of this histidine residue enhances bacterial two-hybrid interaction with the cytosolic copper exporter, but not the copper importer, suggesting that the interacting surfaces are distinct, with implications for metal transfer.

ATP-Binding Cassette Transporters↗

PubMed2000

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.

Biological Transport↗

PubMed2002

Metallochaperones and metal-transporting ATPases: a comparative analysis of sequences and structures.

A comparative structural genomic analysis of a new class of metal-trafficking proteins can provide insights into the intracellular chemistry of reactive cofactors such as copper and zinc. Starting from the sequences of the metallochaperone Atx1 and from the first soluble domain of the copper-transporting ATPase Ccc2, both from yeast, a search on the available genomes was performed using a homology criterion and a metal-binding motif x'-x"-C-x'''-x''''-C. By limiting ourselves to 20% identity with any of the proteins found, several soluble copper-transport proteins were identified, as well as soluble domains of membrane-bound ATPases. Structural models were calculated using high-resolution solution structures as templates, and the models were validated using statistical and energy criteria. Residue conservation and substitution have been interpreted and discussed in terms of structure-function relationship. The potential energy surfaces have been analyzed in terms of protein-protein interactions. We find that metallochaperones and their physiological partner ATPases from several phylogenetic kingdoms recognize one another, via an interplay of electrostatics, hydrogen bonding, and hydrophobic interactions, in a manner that precisely orients the metal-binding side chains for rapid metal transfer between otherwise tight binding sites. Finally, other putative metal-transport proteins are mentioned that have low homology and/or a different metal-binding consensus motif and that appear to use similar structures for recognition and transfer. This analysis highlights the wealth and the complexity of the field.

Adenosine Triphosphatases↗