Cyanobacterial metallochaperone inhibits deleterious side reactions of copper

Edited by François M.M. Morel, Princeton University, Princeton, NJ, and approved November 15, 2011 (received for review October 28, 2011)
December 22, 2011
109 (1) 95-100


Copper metallochaperones supply copper to cupro-proteins through copper-mediated protein-protein-interactions and it has been hypothesized that metallochaperones thereby inhibit copper from causing damage en route. Evidence is presented in support of this latter role for cyanobacterial metallochaperone, Atx1. In cyanobacteria Atx1 contributes towards the supply of copper to plastocyanin inside thylakoids but it is shown here that in copper-replete medium, copper can reach plastocyanin without Atx1. Unlike metallochaperone-independent copper-supply to superoxide dismutase in eukaryotes, glutathione is not essential for Atx1-independent supply to plastocyanin: Double mutants missing atx1 and gshB (encoding glutathione synthetase) accumulate the same number of atoms of copper per cell in the plastocyanin pool as wild type. Critically, Δatx1ΔgshB are hypersensitive to elevated copper relative to wild type cells and also relative to ΔgshB single mutants with evidence that hypersensitivity arises due to the mislocation of copper to sites for other metals including iron and zinc. The zinc site on the amino-terminal domain (ZiaAN) of the P1-type zinc-transporting ATPase is especially similar to the copper site of the Atx1 target PacSN, and ZiaAN will bind Cu(I) more tightly than zinc. An NMR model of a substituted-ZiaAN-Cu(I)-Atx1 heterodimer has been generated making it possible to visualize a juxtaposition of residues surrounding the ZiaAN zinc site, including Asp18, which normally repulse Atx1. Equivalent repulsion between bacterial copper metallochaperones and the amino-terminal regions of P1-type ATPases for metals other than Cu(I) is conserved, again consistent with a role for copper metallochaperones to withhold copper from binding sites for other metals.
Copper chaperones deliver copper to the apo-forms of cupro-proteins or the compartments where they acquire metal (15). This conclusion is evident from the phenotypes of copper-chaperone mutants and in vitro studies of protein interactions (5, 6). Copper delivery by metallochaperones can sustain copper supply even if all copper ions are tightly bound and buffered in the cytosol (7, 8). In turn, tight buffering of copper is thought to keep these competitive ions out of binding sites that should be occupied by other metals (9). These observations have encouraged the notion that one selective advantage of copper chaperones is to prevent copper ions from inflicting damage while en route to vital destinations (10). However, copper sensitivity has not been reported in mutants of any of the three major eukaryotic copper-chaperone routes: to the trans-Golgi via Atx1, to superoxide dismutase via CCS (Copper Chaperone for Superoxide dismutase), or to cytochrome oxidase in mitochondria via Cox17, Sco1, and Cox11. Thus, a dogma that delivery of copper to enzymes via copper chaperones avoids copper-mediated cell damage is not supported by evidence. Some cytosolic bacterial copper chaperones are components of copper resistance determinants but, with the exception of Atx1 from cyanobacteria, these have not been linked to copper supply to any known cupro-proteins (1113). Here we provide evidence that a copper chaperone which can support the maturation of cupro-proteins can also inhibit deleterious side reactions of copper.
Cyanobacteria such as Synechocystis PCC 6803 (Synechocystis hereafter) require copper in thylakoid compartments which house copper-requiring enzymes plastocyanin and cytochrome oxidase. Thylakoid membranes are discrete from plasma membranes with no evidence of any membraneous interconnections implying that copper must traverse the cytosol to reach this location (1416). This statement is consistent with the observation that mutants deficient in the copper-transporting ATPases PacS and CtaA have impaired photosynthetic electron transport via plastocyanin, impaired cytochrome oxidase activity in isolated membranes, and altered abundance of transcripts encoding cytochrome c6 [an alternative heme-based electron carrier used in some cyanobacteria and green algae under copper deficiency (17 and references therein)] (18). All of these phenotypes imply a loss of inward copper supply to thylakoids and hence to plastocyanin and cytochrome oxidase, and here we confirm a loss of copper plastocyanin in the copper transport mutants. Phenotypes of Atx1 mutants grown in low copper medium also establish a role in trafficking copper to thylakoids (13), but here we show that when cells are cultured in standard BG11 media (which is rich in copper compared to natural environments) Atx1 is not essential for this function. Further, glutathione is not obligatory for the accumulation of copper plastocyanin even when Atx1 is absent. However, unlike either single mutant, the Δatx1ΔgshB double mutants are hypersensitive to elevated copper formally establishing that Atx1 can act to inhibit deleterious side reactions of copper. It is hypothesized that these side reactions include the mislocation of copper to exchangeable binding sites that should be occupied by other metals and a phenotype diagnostic of copper inactivation of the iron-sulfur clusters of a group of dehydratase enzymes in Escherichia coli (19), is also now observed for Δatx1ΔgshB Synechocystis. Metal sensors are one group of metallo-proteins whose metal occupancy can be monitored in vivo by following expression from their target gene promoters. The effects of copper on zinc sensing by Synechocystis ZiaR and Zur have been determined in vitro and correlated with expression of their target genes ziaA and znuA respectively in mutants missing the copper chaperone, as a read-out of copper occupancy of exchangeable zinc sites in vivo.
One zinc site that is highly similar to the copper sites that are the targets for Atx1 is located on the soluble amino-terminal domain of the zinc-transporting ATPase ZiaA. We previously established that the ZiaAN site binds copper in preference to zinc in vitro but speculated that it fails to gain access to copper in vivo because it does not form detectable interactions with the copper chaperone (20, 21). In contrast, interaction between Atx1 and the amino-terminal domain of the copper-transporting ATPase PacSN has been observed in vivo by bacterial two-hybrid assays, and in vitro by solution NMR enabling a structural model of the docked heterodimer to be generated (13, 22). Neither assay detects complexes between Atx1 and ZiaAN (20, 23). A structural model of ZiaAN recently confirmed a ferredoxin-like fold similar to that found in other P1-type ATPases, including PacSN, and copper chaperones (2226). The metal-binding sites of ZiaAN and PacSN include the motif Cys-Xaa2-Cys but there are differences in the residues surrounding these ligands. Switching several of these residues switched the specificity of the bacterial two-hybrid interactions (23). Here we have generated a mutant form of ZiaAN (ZiaANsub) that presents an interacting surface sufficiently similar to that of PacSN to allow the determination of a solution structure of a docked heterodimer with the copper chaperone. Databases have then been searched for evidence that analogous repulsion between copper chaperones and transporters for divalent metals is conserved.
In summary, Atx1 is required for normal activity of thylakoid cupro-proteins plastocyanin and cytochrome oxidase in copper limiting medium (13), but here we show that in copper-replete medium copper can be supplied to plastocyanin via the cytosol (requiring PacS and CtaA) but without requiring Atx1 or glutathione. However, normal copper supply via the copper chaperone reduces mislocation of copper to binding sites for other metals with evidence that these aberrant sites include (as examples) the iron-sulfur clusters of dehydratases required for branched chain amino acid biosynthesis, the exchangeable zinc site of the zinc-sensor Zur, and potentially the amino-terminal zinc site of zinc-exporter ZiaA.


Less Copper Is Found in Plastocyanin-Pools of ΔpacS or ΔctaA.

To test whether the P-type ATPases PacS and CtaA support copper supply to plastocyanin in cells grown in copper-replete BG11 medium, whole cell extracts were prepared under rigorously anaerobic conditions, fractionated by ion exchange and size exclusion chromatography, and fractions analyzed for copper by inductively coupled plasma mass spectrometry (ICP-MS). Three predominant copper complexes were detected in extracts from wild type cells (Fig. 1A), one of which was previously shown to be copper-plastocyanin (27). In ΔctaA mutants extra copper atoms accumulate in a high Mr complex designated Cu-P1 (Fig. 1 A and B). This multiprotein complex is not found in periplasm extracts (28), and is also present in the related cyanobacterium Synechococcus PCC 7942 (SI Appendix). Cu-P1 is a cytoplasmic protein complex which obtains copper from a route which is independent of both ATPases, and moreover CtaA, but not PacS, competes with Cu-P1 for copper (Fig. 1 A and B). In contrast, the copper content of the plastocyanin pool declines in mutants missing either ATPase (Fig. 1A), with negligible copper-plastocyanin in ΔctaA mutants (Fig. 1B). This dependency on the P1-type ATPases argues that copper must pass to plastocyanin via the cytosol. This statement remains true regardless of the direction of transport by either PacS or CtaA and noting that alternative models have been presented to explain the actions of P1-type ATPases that had been proposed to import copper (29).
Fig. 1.
Accumulation of copper-plastocyanin requires CtaA and PacS, but not Atx1 or glutathione, in standard copper-replete culture medium. (A), profiles of copper complexes in cell extracts from wild type and mutant cells, extracted and separated under anaerobic conditions using two-dimensional liquid chromatography based on relative molecular mass (Mr) and charge (pI). (B), number of atoms of copper per cell bound to plastocyanin or the P1-complex in wild type and mutant cells calculated from the volumes under the respective peaks (in A). Data are the means of three independent replicates with standard deviation (SD) of triplicates.

Copper Can Bypass Cyanobacterial Atx1 and Glutathione in Standard Medium.

In copper-replete cells the number of copper atoms in the plastocyanin pool does not decline in mutants missing the copper chaperone (Fig. 1 A and B). Copper must be able to pass via the cytosol without being carried by Atx1. Glutathione is thought to buffer metals and copper complexes are detected in Synechocystis cell extracts which comigrate with glutathione (compare glutathione elution profile, in SI Appendix: Fig. S1, with copper profiles in Fig. 1A). In ΔgshB mutants, confirmed to lack glutathione (SI Appendix: Fig. S1), the corresponding low Mr copper complex is also absent (Fig. 1A): It is noted that the copper-glutathione complex may exist in vivo, or form in vitro post cell lysis. Importantly, glutathione is required for copper-chaperone-independent activation of some forms of copper-zinc superoxide dismutase (30, 31), which may reflect a role in redox chemistry or copper delivery. Double mutants deficient in both glutathione and Atx1 were generated but again the number of atoms of copper in the plastocyanin pool remained the same as in wild type when cells are grown in standard copper-replete medium (Fig. 1 A and B). Thus, copper can reach plastocyanin in Δatx1ΔgshB but if the (hypothetical) role of copper chaperones is also to keep copper out of adventitious binding sites then such deleterious side reactions should be greater in these cells.

Mutants Missing Atx1 and Glutathione Are Hypersensitive to Copper.

Single and double mutants in atx1 and gshB show similar growth rates to wild type in normal BG11 culture medium which contains 0.3 μM copper. However, growth of Δatx1ΔgshB double mutants is inhibited by an addition of 0.5 μM copper while both single mutants (importantly including ΔgshB) show normal growth rates in copper-supplemented medium (Fig. 2 A and B; note logarithmic y-axes). Thus, the copper chaperone (and also glutathione) inhibits deleterious side reactions of copper. These side reactions could include redox reactions and/or the substitution of copper in proteins that should bind other metals. Copper toxicity in E. coli is due to the replacement of iron in iron-sulfur clusters of dehydratases required for branched chain amino acid biosynthesis (19), not DNA damage mediated by copper-catalyzed production of reactive oxygen species (32). Copper toxicity in E. coli is thus overcome by supplementation with branched chain amino acids (19) as compared to control cells supplemented with alanine, and the same phenotype is observed here (Fig. 2 C and D).
Fig. 2.
Atx1 mutants are copper sensitive in a ΔgshB background. Logarithmically growing wild type (closed circles), Δatx1 (closed squares), ΔgshB (open squares), and Δatx1ΔgshB (open circles) cells were inoculated to an OD595 nm of 0.03 in (A), BG11 medium or (B), in medium supplemented with copper. Δatx1ΔgshB were inoculated in (C), BG11 medium supplemented with equivalent (total amino acid) concentrations of alanine (closed circles) or isoleucine, leucine and valine, ILV (open circles) or (D), supplemented with copper alone (open squares), copper plus alanine (open triangles) or copper plus ILV (open diamonds). Data points are mean values from three separate cultures with SD of triplicates (some too small to show) and the experiments were repeated on two further occasions to obtain similar trends.

Zinc Homeostasis Is Aberrant in Δatx1ΔgshB Mutants.

Excess (or unchaperoned) copper atoms are predicted to aberrantly bind to a variety of sites for other metals but dehydratases are the most vulnerable: the first to give a growth phenotype under standard culture conditions (19; Fig. 2). In a previous attempt to detect the mislocation of copper to the exchangeable zinc sites of the zinc-sensor ZiaR in Δatx1 cells, a loss of repression of its ziaA target was detected in mutants missing the copper chaperone (33). However, copper (supplied as cuprous ions under anaerobic conditions) is ineffective at inhibiting ZiaR DNA binding (33), and zinc remains allosterically effective in the presence of equimolar copper (SI Appendix: Fig. S2). The DNA-binding site of a second zinc sensor, Synechocystis Zur, has now been identified (SI Appendix: Fig. S3), the recombinant protein purified to homogeneity (SI Appendix) and the exchangeable zinc site of Zur (the protein also has a structural zinc site; SI Appendix) binds copper (cuprous ions added under anaerobic conditions) in preference to zinc (Fig. 3 A and B). Critically, the DNA affinity of copper-Zur is an order of magnitude weaker than zinc-Zur (Fig. 3 C and D), even in the presence of equimolar zinc and copper (Fig. 3D). Copper inhibited the mechanism of zinc sensing by Zur not ZiaR in vitro, and RT-PCR shows loss of Zur-mediated repression of znuA but derepression of ziaA in Δatx1ΔgshB mutants (Fig. 3D inset); the former should normally occur in low zinc, the latter in high zinc and thus zinc sensing is aberrant in the mutants.
Fig. 3.
Mis-regulation of zinc homeostasis in Δatx1ΔgshB cells. (A), Copper (open circles) comigrates with Zur (closed circles) containing ca. one equivalent of zinc (open triangles) in a structural zinc finger (Zn1Zur) while a second equivalent of zinc remains unbound. (B), Apo-subtracted difference spectra of Zn1Zur show copper dependent changes in the region below 300 nm (the deduced exchangeable site contains a Cys) after addition of 1 equivalent of copper (solid line), followed by 1 equivalent of zinc (dashed line) or after coincident addition of 1 equivalent of both metals (dotted line). (c), Titration of hexachlorofluorescein-labeled znu operator-promoter with Zn2Zur (closed circles) or Zn1Zur treated with EDTA (open circles). (D), As in (C) but using copper-Zn1Zur (closed triangles) or Zn1Zur incubated with equimolar surplus copper and zinc (open triangles). In vitro assays were performed under rigorously anaerobic conditions in an anaerobic chamber or with gas-tight syringes. Inset shows RT-PCR analysis of the abundance of rps1, znuA (Zur-regulated) and ziaA (ZiaR-regulated) transcripts (all products are reverse transcriptase dependent) in total RNA from wild type (WT) and Δatx1ΔgshB (ΔΔ) cells cultured for 48 h with no metal supplement or maximum permissive concentrations of copper or zinc.
Impaired zinc-Zur-repression of zinc import will cause enhanced zinc uptake which will be detected by ZiaR, explaining elevated expression of ziaA previously detected by both RT-PCR and by reporter gene assays in Δatx1 cells (33), and now also detected in the double mutants (Fig. 3D inset). These data are fully consistent with the mislocation of copper to exchangeable zinc sites in cells missing the copper chaperone. The double mutants are hypersensitive to zinc (16 μM) in BG11 medium containing normal basal levels of copper (0.3 μM) but this is overcome if micro-nutrient levels of copper are removed from the media, consistent with antagonism between the two metals (potentially at zinc export by ZiaA) and again this phenotype is more pronounced in Δatx1ΔgshB than in ΔgshB (SI Appendix: Fig. S4).

In Vitro Interaction Studies Between Atx1 and ZiaAN.

In common with the exchangeable zinc site of Zur, the exchangeable zinc site of the amino-terminal region of the zinc exporter ZiaA, ZiaAN, also has a tighter affinity for Cu(I) than zinc (20, 34). Unlike the related domain of the copper transporter PacSN, ZiaAN does not form detectable interactions with Atx1 in vivo or in vitro and this is thought to inhibit ZiaAN from acquiring Cu(I) in vivo (13, 20, 23). We previously switched four residues in the region of the ZiaAN metal-binding motif to the analogous residues from PacSN and obtained evidence of a gain of protein interaction (23). To further investigate the hypothesis that divalent metal-transporting ATPases have evolved to repulse copper chaperones we switched up to eight residues in the region of the ZiaAN metal-binding motif to the analogous residues from PacSN (SI Appendix). Metal-mediated protein-protein-complex formation between Atx1 and the ZiaAN mutants was monitored by NMR. Correlation times for molecular tumbling as estimated from the ratio of R2 and R1 relaxation rates for amide 15N nuclei measured at 500 MHz for apo-ZiaANsub, apo-Atx1, and Atx1-Cu(I)-ZiaANsub, establish that the mutant ATPase domain now forms heterodimers with Cu(I)-Atx1. By making one side of the ZiaAN surface increasingly similar to PacSN the proportion of Atx1-heterodimer increases. These complexes were not detected in the absence of Cu(I) (SI Appendix).
The NMR solution structure of the Atx1-Cu(I)-ZiaANsub complex was then calculated and the heterodimer interface analyzed (Fig. 4 A and B). Two salt bridges occur between Glu13 (Atx1) and Arg18 (ZiaANsub) plus Glu26 (Atx1) and Arg28 (ZiaANsub) at the opposite edges of the protein interface and stabilize the complex. These interactions are quite stable even when the complex, after solvation, is subjected to molecular dynamics calculations. In contrast, a hypothetical model generated by substituting the ZiaANsub mutant in the Atx1-Cu(I)-ZiaANsub complex with a structural model of wild type ZiaAN, shows that repulsion would occur between Glu13 (Atx1) and Asp18 (ZiaAN), and obviously no stabilizing interaction would occur between Glu26 (Atx1) and Gly28 (ZiaAN) (Fig. 4B). In summary, characterization of a gain-of-interaction mutant has enabled us to orientate the two proteins and hence visualize why the amino-terminal region of wild type ZiaAN does not form a complex with Atx1 analogous to PacSN-Cu(I)-Atx1.
Fig. 4.
Swapping residues at the ZiaAN zinc site gains interaction with the copper chaperone. (A), Electrostatic surface of the Atx1-Cu(I)-ZiaANsub adduct. Positively charged, negatively charged, and neutral amino acids are represented in blue, red and white respectively with Cys-thiols in yellow. (B), Average structure of the Atx1-Cu(I)-ZiaANsub complex after MD simulation and model of the hypothetical Atx1-Cu(I)-ZiaAN WT adduct. The key interface residues mutated in ZiaANsub are in magenta, and the side chains of some residues forming stabilizing contacts are shown. (C), Repulsive residues on loops 1 (N) and loop 5 or the proximal end of helix α2 (C) of representative and structurally characterized bacterial copper chaperones and sets of ATPases from the same organism (white represents any residues other than Asp, red, or Glu, pink).

Chaperone-Repulsive Negative Charge Is Conserved in P1-Type ATPases for Other Metals.

Sequence databases have been searched (SI Appendix) for evidence that features of ZiaAN that repulse Atx1 are common in ATPases that have been deduced to transport metals other than copper, notably the presence of negative charge in the region of the metal-binding site. In all sequences (with one intriguing exception) where the repulsive negative charge analogous to Asp18 (ZiaAN) was missing from loop 1 it was alternatively present on proximal loop 5 as either an Asp or Glu (SI Appendix: Fig. S5). However, in all cases these negatively charged residues are thought to provide an additional ligand for other divalent metals as is the case for ZntA from E. coli and CadA from Listeria monocytogenes (35, 36), but the restricted choice of ligand at this position could argue that this has been selected to coincidentally provide negative charge repulsive to Atx1. Notably, structurally characterized bacterial copper chaperones other than Atx1 from Synechocystis are missing the complementary repulsive negative charge on loop 1 analogous to Glu13 of cyanobacterial Atx1 however they alternatively possess conserved negative charge on loop 5 or at the end of helix α2 (Fig. 4C, SI Appendix: Fig. S5). In other bacterial copper chaperones loop 5 plus the end of helix α2 are proximal to the metal site, analogous to loop 5 of the amino-terminal regions of ATPases, but in Atx1 the carboxyl-terminal region is atypically truncated and unstructured consistent with selection for additional negative charge on loop 1 (25, 26, 37). A database search for copper chaperones in the genomes of organisms possessing predicted P1-type ATPases for other divalent metals reveals that negative charge on loop 5 or the end of helix α2 is universally present (except cyanobacterial Atx1 as noted above). Importantly, in the copper chaperones these negatively charged residues do not act as metal-ligands but still the proteins conserve negative charge in the region of the metal site which will repulse the amino-terminal regions of divalent metal-transporting P1-type ATPases (SI Appendix: Fig. S5).


Cyanobacteria are unusual among bacteria in having cytoplasmic compartments (thylakoids) plus known cytoplasmic copper enzymes (10). The contribution of Atx1 in copper supply to these enzymes, at least in copper-deficient BG11 medium (13), represents a bacterial model with analogy to the copper chaperones which support the maturation of eukaryotic cupro-proteins. However, it is now evident that Atx1 is not needed for copper to reach plastocyanin provided the medium is sufficiently copper-rich, even in mutants missing glutathione (Fig. 1). It was originally hypothesized that CtaA imports copper into the cytosol at the plasma membrane and PacS exports copper from the cytosol into thylakoids (18). However, there is no biochemical proof that any P1-type ATPases can import copper and hence it is alternatively proposed that both PacS and CtaA export copper from the cytosol (29, 38). Regardless of the direction of transport, because the accumulation of copper in plastocyanin depends upon CtaA and PacS, copper must enter the cytosol either to supply ATPase substrate, or as product, while en route to plastocyanin in cells grown in copper-replete BG11 medium (Fig. 1). This observation questions how copper traverses the cytosol when Atx1 and glutathione are absent and why this is disadvantageous. Toxicity to excess copper in E. coli has been correlated with copper substitution for a less competitive metal, notably iron in iron-sulfur clusters (19). In Δatx1ΔgshB mutants it is proposed that such copper substitutions are more prevalent and contribute towards the copper hypersensitivity of this strain (Fig. 2). It is envisaged that such copper substitutions occur in a wide range of proteins with data suggesting that these include the iron-sulfur clusters of dehydratases (Fig. 2) and exchangeable zinc sites (Fig. 3, SI Appendix: Fig. S4). Zinc sensing by Zur rather than ZiaR is inhibited by copper in vitro enabling expression of target genes, znuA and ziaA, to be used as probes of maloccupancy of zinc sites with copper; both transcripts were elevated in Δatx1ΔgshB. Aberrant expression of znuA will increase zinc uptake and cause ZiaR-mediated derepression of ziaA (Fig. 3).
The amino-terminal regions of P1-type ATPases undergo intramolecular interactions with a cytosolic actuator domain and these interactions are inhibited by metal binding to Cys-Xaa2-Cys motifs (39). A favored model is that the amino-terminal metal-binding regions modulate the rate of transport as a function of metal binding (40, 41). Metal-dependent interaction between copper chaperones and amino-terminal regions of copper-transporting ATPases thus serves to enhance copper transport as the chaperone population becomes increasingly saturated with copper. Whether this mechanism requires metal transfer between the chaperone and the ATPase, or whether the transient complex is the active species remains to be established. In either model, copper-dependent docking of a copper chaperone to a zinc-transporting ATPase would modulate the transporter in response to the wrong metal and indeed Atx1-interactions are detected both in vivo and in vitro with PacSN but not ZiaAN (13, 20, 22, 23). Here we provide compelling gain-of-interaction data by introducing residues that enable Atx1-interaction with PacSN, into ZiaAN, modelling the resulting complex and then visualizing a hypothetical, analogous encounter complex between wild type ZiaAN and Atx1 (Fig. 4). At least one of the substituted residues normally repulses Atx1 and repulsive negative charge is a conserved feature of metal transporters for divalent metals and copper chaperones (Fig. 4C, SI Appendix: Fig. S5). These observations argue that electrostatic repulsion between these two proteins reflects a selective advantage of a copper chaperone that restrains copper from binding sites for other metals.
The recent structure of a P1-type ATPase has revealed a positively charged platform region surrounding the proposed entry site for copper (42). Equivalent charge is conserved in other copper transporters but also in transporters for divalent metals including ZiaA (SI Appendix: Fig. S6). Thus, this platform region would be attractive to the opposite charge surrounding the metal site of copper chaperones, consistent with copper chaperones donating metal directly to the platform (41), but this questions why copper chaperones would not also (aberrantly) interact with positively charged platform regions of transporters for divalent metals. One explanation is that the apo-state of the amino-terminal regions of P1-type ATPases also occlude access to the platform.
Many metallo-proteins bind a wrong metal more tightly than the correct metal. Withholding the wrong metals from proteins may be a greater cellular challenge than supplying the correct element (43). Copper ions are highly competitive, with cuprous ions being especially competitive for binding sites containing thiolate ligands (44), and aberrant binding of copper to proteins has been linked to disease, notably age-related neuronal disorders (cited in ref. 21). All of the evidence here argues that a copper chaperone serves to reduce such mislocation of this competitive metal.

Materials and Methods

General Reagents, Bacterial Cultures, and Growth Assays.

Reagents and chemicals were sourced from standard commercial suppliers. Escherichia coli strains DH5α, JM101, and BL21λDE3 were grown in Luria-Bertani medium (45). To determine copper tolerance of wild type, Δatx1, ΔgshB, and Δatx1ΔgshB strains of Synechocystis, logarithmically growing cells were first subcultured into fresh BG11 medium every 4 d for a minimum of 12 d to standardize growth rates, then inoculated at an OD595 nm of 0.03 in BG11 medium with or without copper supplementation (0.5 μM CuSO4) and growth monitored by measuring OD595 nm daily for 4 d. Assays were triplicated then the experiment repeated twice more to obtain similar trends. The effects of amino acids on growth of Δatx1ΔgshB were performed as described above however, following subculturing to standardize growth rates cells were inoculated at an OD595 nm of 0.03 in standard BG11 medium supplemented with L-alanine (1.5 mM) or a mixture of L-isoleucine, L-leucine, and L-valine (each at 0.5 mM) with or without supplementation with 38 μM CuSO4.

Production of ΔgshB and Δatx1ΔgshB Mutants of Synechocystis.

The Δatx1 Synechocystis mutant strain was produced previously (13). To produce ΔgshB and Δatx1ΔgshB strains, the gene slr1238 was amplified by PCR and subcloned into pGEM-T. A section of slr1238 was removed by restriction digestion with BamHI and then ligated to the kanR gene (released from the plasmid pUC4K by BamHI restriction digestion). Wild type and Δatx1 Synechocystis cells were transformed to kanamycin resistance as described previously (18), and integration and segregation confirmed by PCR.

Analysis of Copper Complexes in Synechocystis Cell Extracts.

Synechocystis whole cell extracts were prepared in an anaerobic chamber as previously (27), with the addition of an ultracentrifuge step to clarify the lysate. Samples containing 30 mg protein were fractionated by anion exchange and high-pressure size-exclusion chromatography (under nitrogen) as described previously (27) and analyzed for metal by ICP-MS. Glutathione concentrations were determined using standard enzymatic assays (Sigma).

Mutagenesis, Production, and Purification of Recombinant ZiaAN Plus Atx1.

The expression construct, pET-ZiaAN, plus purification protocols for ZiaAN and Atx1 were previously described (13, 23). The primers used for sequential rounds of mutagenesis are described in SI Appendix.

Analysis of Copper Binding to Recombinant Zur.

Metal-binding analyses of recombinant Zur (purified as described in SI Appendix) were performed under rigorously anaerobic conditions as described previously (33). To analyze metal binding to Zur by size-exclusion chromatography, zinc and Cu(I) were added to 10 μM of protein in an anaerobic chamber and bound and free metal ions resolved on Sephadex G25 matrix (GE Healthcare) with fractions analyzed for protein content by Bradford assay and metal content by ICP-MS. UV-Visible analyses of metal binding to Zur were performed under anaerobic conditions in buffer 400 mM KCl, 100 mM NaCl, 10 mM Hepes (pH 7.8) as described previously (33).

Fluorescence Anisotropy Analysis of the Effects of Zinc and Cu(I) on DNA Binding.

Fluorescently labeled, complimentary oligonucleotides of sequence 5′-GACAATAAACGATAATCATTATCATTGCATAAT-3′ (containing a Zur binding motif identified by bioinformatics and gel-shift assays; SI Appendix: Fig. S3) were prepared as described previously (46). Zur was prepared in buffer 400 mM KCl, 100 mM NaCl, 10 mM Hepes (pH 7.8) in the presence of 1.5 equivalents of zinc and/or copper (additional to a structural zinc ion) or 2 mM EDTA and titrated under anaerobic conditions with 10 nM of annealed DNA in buffer 130 mM KCl, 30 mM NaCl, 10 mM Hepes, (pH 7.8) in the presence of 3.5 μM of zinc and/or Cu(I). Changes in anisotropy were measured at 25 °C as described previously (33).

Isolation of RNA and Reverse Transcriptase-PCR.

Total RNA was isolated from logarithmically growing cells as described previously (47). Where cultures were treated with supplementary ZnSO4 (16 μM) or CuSO4 (1 μM), cells at an OD595 nm of 0.1 were incubated for 48 h under standard conditions following addition of metal. Preparation of RNA and RT-PCR analyses of ziaA expression were as described previously (33), while primers for znuA were 5′-TAGTTCGAGTCAGCGTACTG-3′, 5′-CTGTCGTTTCACCAAGGTAG-3′. Products were analyzed on 1% (wt/vol) agarose gels.

NMR Titration of Proteins.

Cu(I) was added to mixtures containing ZiaAN or one of its variants, and Atx1, with one partner 15N-labeled (SI Appendix). The chemical shift changes observed upon formation of the Atx1-Cu(I)-ZiaANsub metal-mediated complex, together with the available solution structures of the Atx1 [Cu(I) form] and of ZiaANsub (apo form), were used as input data to calculate a model of the complex using the program HADDOCK (48).

Molecular Dynamics (MD) Simulations of Protein Adduct(s).

The simulations were performed using the AMBER 10 suite of programs (49). MD simulations were run for the Atx1-Cu(I)-ZiaANsub protein adduct obtained with HADDOCK. The protein complex was solvated using a standard water box. To make the whole system neutral, five Na+ ions were added. The protein complex was subjected to a short minimization; the minimized structure was then subjected to MD calculations, after increasing the temperature to 300 K and then equilibrated for 500 ps at the same temperature. Ptraj was used for calculating the root mean square deviation (rmsd). Calculations were run using the AMBER Portal on the We-NMR website.

Data Availability

Data deposition: The atomic coordinates of ZiaANsub and Atx1-Cu-ZiaANsub have been deposited in the Protein Data Bank, (PDB ID code 2LDI) and the BioMagResBank, (accession no. 17668).


This work was supported by Biotechnology and Biological Sciences Research Council Grants BBS/B/02576 and BB/E001688/1, Basic Research Investment Fund (FIBR) proteomica (contract number RBRN07BMCT) and by the Biological NMR funded by the European commission (BioNMR EC) contract 261863 and the Worldwide e-Infrastructure for NMR and structural biology funded by the European commission (We-NMR EC) contract 261572.

Supporting Information

Supporting Appendix (PDF)
Supporting Information


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Information & Authors


Published in

Go to Proceedings of the National Academy of Sciences
Proceedings of the National Academy of Sciences
Vol. 109 | No. 1
January 3, 2012
PubMed: 22198771


Data Availability

Data deposition: The atomic coordinates of ZiaANsub and Atx1-Cu-ZiaANsub have been deposited in the Protein Data Bank, (PDB ID code 2LDI) and the BioMagResBank, (accession no. 17668).

Submission history

Published online: December 22, 2011
Published in issue: January 3, 2012


  1. copper chaperone
  2. P1 type ATPase
  3. zinc sensors
  4. Zur
  5. synechocystis PCC 6803


This work was supported by Biotechnology and Biological Sciences Research Council Grants BBS/B/02576 and BB/E001688/1, Basic Research Investment Fund (FIBR) proteomica (contract number RBRN07BMCT) and by the Biological NMR funded by the European commission (BioNMR EC) contract 261863 and the Worldwide e-Infrastructure for NMR and structural biology funded by the European commission (We-NMR EC) contract 261572.


This article is a PNAS Direct Submission.



Steve Tottey
Fraunhofer USA Center for Molecular Biotechnology, Newark, DE 19711
Carl J. Patterson
Department of Chemistry, Biophysical Sciences Institute, School of Biological and Biomedical Sciences, University of Durham, Durham DH1 3LE, United Kingdom;
Lucia Banci
CERM (Centro di Ricerca di Risonanze Magnetiche) and Department of Chemistry, University of Florence, 50019 Sesto Fiorentino (FI), Italy;
Ivano Bertini
CERM (Centro di Ricerca di Risonanze Magnetiche) and Department of Chemistry, University of Florence, 50019 Sesto Fiorentino (FI), Italy;
Isabella C. Felli
CERM (Centro di Ricerca di Risonanze Magnetiche) and Department of Chemistry, University of Florence, 50019 Sesto Fiorentino (FI), Italy;
Anna Pavelkova
CERM (Centro di Ricerca di Risonanze Magnetiche) and Department of Chemistry, University of Florence, 50019 Sesto Fiorentino (FI), Italy;
Samantha J. Dainty
Department of Chemistry, Biophysical Sciences Institute, School of Biological and Biomedical Sciences, University of Durham, Durham DH1 3LE, United Kingdom;
Rafael Pernil
Department of Chemistry, Biophysical Sciences Institute, School of Biological and Biomedical Sciences, University of Durham, Durham DH1 3LE, United Kingdom;
Kevin J. Waldron
Cell and Molecular Biosciences, Medical School, University of Newcastle, Newcastle upon Tyne NE2 4HH, United Kingdom; and
Andrew W. Foster
Department of Chemistry, Biophysical Sciences Institute, School of Biological and Biomedical Sciences, University of Durham, Durham DH1 3LE, United Kingdom;
Cell and Molecular Biosciences, Medical School, University of Newcastle, Newcastle upon Tyne NE2 4HH, United Kingdom; and
Nigel J. Robinson1 [email protected]
Department of Chemistry, Biophysical Sciences Institute, School of Biological and Biomedical Sciences, University of Durham, Durham DH1 3LE, United Kingdom;


To whom correspondence should be addressed. E-mail: [email protected].
Author contributions: S.T., C.J.P., L.B., I.B., I.C.F., A.P., S.J.D., R.P., K.J.W., A.W.F., and N.J.R. designed research; S.T., C.J.P., A.P., S.J.D., R.P., K.J.W., and A.W.F. performed research; S.T., C.J.P., L.B., I.B., I.C.F., A.P., S.J.D., R.P., K.J.W., A.W.F., and N.J.R. analyzed data; S.T., C.J.P., and A.P. made equal contributions; and S.T., C.J.P., L.B., I.B., I.C.F., A.P., S.J.D., R.P., K.J.W., A.W.F., and N.J.R. wrote the paper.

Competing Interests

The authors declare no conflict of interest.

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    Cyanobacterial metallochaperone inhibits deleterious side reactions of copper
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