Investigating the Role of Zinc and Copper Binding Motifs of Trafficking Sites in the Cyanobacterium Synechocystis PCC 6803

Although zinc and copper are required by proteins with very different functions, these metals can be delivered to cellular locations by homologous metal transporters within the same organism, as demonstrated by the cyanobacterial (Synechocystis PCC 6803) zinc exporter ZiaA and thylakoidal copper importer PacS. The N-terminal metal-binding domains of these transporters (ZiaAN and PacSN, respectively) have related ferredoxin folds also found in the metallochaperone Atx1, which delivers copper to PacS, but differ in the residues found in their M/IXCXXC metal-binding motifs. To investigate the role of the nonconserved residues in this region on metal binding, the sequence from ZiaAN has been introduced into Atx1 and PacSN, and the motifs of Atx1 and PacSN swapped. The motif sequence can tune Cu(I) affinity only approximately 3-fold. However, the introduction of the ZiaAN motif (MDCTSC) dramatically increases the Zn(II) affinity of both Atx1 and PacSN by up to 2 orders of magnitude. The Atx1 mutant with the ZiaAN motif crystallizes as a side-to-side homodimer very similar to that found for [Cu(I)2–Atx1]2 (Badarau et al. Biochemistry2010, 49, 779820726513). In a crystal structure of the PacSN mutant possessing the ZiaAN motif (PacSNZiaAN), the Asp residue from the metal-binding motif coordinates Zn(II). This demonstrates that the increased Zn(II) affinity of this variant and the high Zn(II) affinity of ZiaAN are due to the ability of the carboxylate to ligate this metal ion. Comparison of the Zn(II) sites in PacSNZiaAN structures provides additional insight into Zn(II) trafficking in cyanobacteria.

Z inc and copper are essential metal ions for most organisms but exhibit a number of important differences. The biological functions of copper primarily utilize its redox activity, which contributes to the toxicity of this metal. 1 Redox-inactive zinc is more abundant in biological systems and is required by many more proteins. 2 The tight binding of zinc and copper to biological metal sites means that the intracellular availability of both has to be carefully controlled. 3 Copper trafficking generally involves metallochaperones that deliver the metal to specific targets by ligand-exchange reactions. 3,4 No zinc metallochaperone is currently known, 5 although metallothioneins involved in zinc storage have been proposed to also act as a direct source of zinc for target proteins. 6,7 Zinc export from the cytosol of the cyanobacterium Synechocystis PCC 6803 occurs via the P-type ATPase ZiaA. 8 In the same organism, the P-type ATPase PacS, along with the copper metallochaperone Atx1, traffic copper to the thylakoids for photosynthesis and respiration. 9,10 ZiaA and PacS each possess a single N-terminal metal-binding domain (MBD; ZiaA N and PacS N respectively) structurally similar to Atx1 in having a M/IXCXXC metal-binding motif anchored on a ferredoxin (βαββαβ) fold ( Figure 1). 11−13 ZiaA N is unusual in having an unstructured C-terminal extension that contains seven His residues that are involved in Zn(II) binding. 13 We have recently shown that the Zn(II) affinity of ZiaA N is up to 2 orders of magnitude higher than those of PacS N and Atx1. 14 The Cu(I) affinities of copper and zinc trafficking proteins in Synechocystis all fall within approximately 1 order of magnitude at pH 7.0. 14 However, the Cu(I) affinity of Synechocystis Atx1 is almost 10-fold greater than that of PacS N (and ZiaA N ), and this Atx1 can dimerize in the presence of Cu(I), 11,12 which enhances its Cu(I) affinity. 14 The Cu(I) affinities of the trafficking sites are at least 6 orders of magnitude greater than their Zn(II) affinities, 14 consistent with theoretical studies, 15 yet Atx1 has been proposed to be able to bind zinc in vivo. 16 The non-Cys residues in the M/IXCXXC motifs of metaltrafficking proteins have been implicated in metal binding and transfer. 17−22 This includes contributing to Zn(II) coordination in a Zn(II)-transporting ATPase form Escherichia coli (E. coli), facilitating the rate and extent of dissociation of the Atx1− Cu(I)−BCA complex [BCA (bicinchoninic acid) is a tight Cu(I) ligand used as a copper-transfer partner mimic], influencing the flexibility of this part of the protein, and forming potentially important hydrogen-bonds, particularly with the Cys ligands. In this work, we use the Synechocystis system to help understand the importance of the non-Cys residues in M/IXCXXC motifs on the binding of zinc and copper. We have grafted the motif from ZiaA N onto both Atx1 (Atx1 ZiaA N ) and PacS N (PacS N ZiaA N ) and have swapped these regions between Atx1 and PacS N , giving Atx1 PacS N and PacS N Atx1 , respectively ( Figure 1C). These mutations have a limited effect (maximum ∼3-fold) on Cu(I) affinity. However, introducing the ZiaA N loop has a dramatic influence on the Zn(II) affinities of both Atx1 and PacS N . In its crystal structure, Zn(II)−Atx1 ZiaA N forms a side-to-side dimer with the monomers bridged by a single Zn(II) ion. The introduced Asp11 residue on the MDC 12 TSC 15 motif is involved in an intermolecular hydrogen bond with Ser14 from the adjacent molecule. In a crystal structure of Zn(II)−PacS N ZiaA N , the metal ion is coordinated by the carboxylate of the corresponding Asp residue, which must be the cause of the enhanced Zn(II) affinity. The comparison of Zn(II)−PacS N ZiaA N crystal structures provide additional insight into potential intermediate sites formed during Zn(II) trafficking in Synechocystis.

■ MATERIALS AND METHODS
Site-Directed Mutagenesis. The Atx1 ZiaA N , Atx1 PacS N , PacS N ZiaA N , and PacS N Atx1 mutants ( Figure 1C) were generated using QuikChange mutagenesis (Stratagene) with pETATX1 (encoding full length Atx1) 10 and pETPACS71 (encoding PacS N , which constitutes the first 71 amino acids of PacS) 12 as templates and the primers given in Table S1 in the Supporting Information. Both strands of all DNA constructs were confirmed by sequencing.
Protein Purification, Reduction, Quantification, and Analysis. Proteins (including His61Tyr Atx1 12 ) were purified, reduced and quantified as previously reported, 12,14,23 and the Atx1 ZiaA N , Atx1 PacS N , PacS N ZiaA N , and PacS N Atx1 variants were verified by mass spectrometry. Far-UV (185−250 nm) circular dichroism (CD) spectra 14 and analytical gel filtration chromatography 12 were performed as described previously. The dimerization constant (K dim ) for Cu(I)−Atx1 PacS N was determined by gel filtration as described previously. 12 Zinc Titrations and the Determination of Zn(II) and Cu(I) Affinities. Titrations of Zn(II) into apo-proteins were performed in 25 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (Hepes) pH 7.4 plus 100 mM NaCl and monitored for changes in absorbance at 240 nm on a λ35 UV/vis spectrophotometer (Perkin-Elmer). 14 Zn(II) affinities were measured using the competitive chelator RhodZin-3 in 25 mM Hepes pH 7.4 plus 100 mM NaCl. 14 Data were fit to a model (eq 1) considering a single species (ZnP, where P is the apo-protein) for Atx1 ZiaA N and PacS N ZiaA N to obtain the Zn(II) affinity of the apo-protein (K Zn ) and two species (ZnP and ZnP 2 , see eq 2) for His61Tyr Atx1, Atx1 PacS N , and PacS N Atx1 to determine both K Zn and the affinity of the apo-protein for zincprotein (K Zn2 ). Below are eqs 1 and 2: [L] [ZnL] [L]  Protein Crystallization, X-ray Data Collection, Structure Determination, and Refinement. Atx1 ZiaA N (25 mg/ mL) loaded with 1 equiv of Zn(II) in 20 mM Hepes pH 7.0 and 100 mM NaCl was crystallized anaerobically from 1.6 M trisodium citrate pH 6.5 using the hanging drop method of vapor diffusion (1 μL protein and 0.5 mL well solution). PacS N ZiaA N (10 mg/mL) loaded with 1 equiv of Zn(II) in 20 mM Hepes pH 7.0 plus 35 mM NaCl was crystallized from 0.1 M Hepes pH 7.5 plus 10% (w/v) PEG 4000 and 5% (w/v) isopropanol (condition 1) using the sitting drop method (250 nL protein and 100 μL well solution). A second crystal form of Zn(II)−PacS N ZiaA N was obtained from 20% (w/v) PEG 3350 plus 0.2 M NaF (condition 2). All crystals were frozen using Nparatone oil as the cryoprotectant. Diffraction data were collected at 100 K on beamline I02 at the Diamond Light Source (Didcot, U.K.). The identity of the metal was confirmed by calculation of an anomalous difference Fourier map using additional data sets collected both above [high-energy remote (hrm)] and below [low-energy remote (lrm)] the zinc K-edge  (peak) to confirm the presence and absence, respectively, of the anomalous signal (Table 1). Data were processed and integrated using iMOSFLM and scaled with Scala. 24,25 The structures were solved by molecular replacement using Molrep 26 and 2XMT (Atx1) and 2XMW (PacS N ) as the search models. 12 Final models were produced with iterative cycles of refinement (Refmac5) and model-building using COOT. 27 Figure S1 in the Supporting Information) demonstrate fully folded proteins and domains in all cases.
Oligomerization State of the Apo and Metal-Loaded Proteins. For most proteins studied, the addition of Zn(II) causes a decrease in absorbance at 240 nm ( Figure 2 and Figure  S2 in the Supporting Information) in the UV/vis spectrum [the absorbance at 240 nm does initially increase upon the addition of Zn(II) to apo-PacS N Atx1 (Supporting Information Figure  S2B)]. This seems counterintuitive considering that the formation of Zn(II)−S bonds is normally accompanied by an increase in absorbance at this wavelength due to the appearance of S(Cys)→Zn(II) ligand to metal charge transfer bands. 29 However, a number of factors can give rise to changes in absorbance at 240 nm, including the protonation state of the Cys ligands, and the observed effects are probably the result of several contributing factors. Regardless of the absolute values, the change in absorbance at this wavelength upon Zn(II) addition does give insight into the stoichiometry of the complexes formed. The Zn(II) titrations (Figure 2 and Figure  S2 in the Supporting Information) show an inflection point at ∼0.5 equiv, followed by a plateau after 1 equiv, for all proteins except Atx1 ZiaA N , for which the decrease in absorbance at 240 nm is linear up to 1 equiv (Figure 2A). These data indicate the formation of a single Zn(II)-form (ZnP) for Atx1 ZiaA N , as previously seen for ZiaA N , 14 and two Zn(II)-loaded species (ZnP and ZnP 2 ) for PacS N ZiaA N , His61Tyr Atx1, Atx1 PacS N , and PacS N Atx1 , as found for wild type (WT) Atx1 and PacS N . 14 Consistent with this, most of these proteins elute as dimers from a gel filtration column when loaded with 0.5 equiv of Zn(II) (Figure 3 and Figure S3 and Table S3 in the Supporting Information), whereas Atx1 ZiaA N elutes as a monomer, most probably as a mixture of apo-and Zn(II)-protein ( Figure 3A and Table S3 in the Supporting Information). The elution volume of PacS N ZiaA N loaded with 0.5 equiv of Zn(II) decreases (apparent molecular weight increases) with increasing protein concentration, consistent with a relatively weak ZnP 2 dimer ( Figure 3B and Table S3 in the Supporting Information). Atx1 ZiaA N and PacS N ZiaA N elute as monomers when loaded with 1 equiv of Zn(II) ( Figure 3A,B and Table S3 in the Supporting Information). All of the other proteins have a greater tendency to dimerize at the relatively high protein concentrations used for the gel filtration experiments (90−200 μM) and are recovered with approximately 0.5 Zn(II) equiv ( Figure S3 and Table S3 in the Supporting Information).
We have previously shown that although WT apo-Atx1 is a monomer, when the protein is loaded with 1 equiv of Cu(I) [Cu(I)−Atx1], it elutes from a gel filtration column as a dimer. However, the elution volume increases upon lowering the  The simultaneous fit of the data for Atx1 PacS N to eq 2 gives a K Zn of (2.5 ± 0.1) × 10 8 M −1 and K Zn2 of (3.6 ± 0.4) × 10 5 M −1 and for Atx1 ZiaA N to eq 1 gives a K Zn of (2.5 ± 0.1) × 10 10 M −1 .
protein concentration below 100 μM, indicative of dimer dissociation in this concentration range, and an equilibrium constant (K dim ) of (5 ± 2) × 10 5 M −1 has been determined. 12 The mutation of His61 into a Tyr, the residue in the corresponding position on loop 5 in PacS N and ZiaA N ( Figure  1), results in monomeric Cu(I)−Atx1 and a His residue at this key location, whose side chain hydrogen bonds with the Cys ligands (Figure 1), favors dimerization. All of the apo-variants loaded with 1 equiv of Cu(I) elute as monomers on a gelfiltration column (Figure 3 and Figure S3 and Table S3 in the Supporting Information) except for Cu(I)−Atx1 PacS N , which elutes as a dimer ( Figure S3A and Table S3 in the Supporting Information). A dimerization constant of (6 ± 1) × 10 4 M −1 was determined for Cu(I)−Atx1 PacS N ( Figure S4 in the Supporting Information), ∼10-fold lower than that of WT Atx1. 12 This lowered dimerization constant for Atx1 PacS N and the absence of dimer formation for Atx1 ZiaA N demonstrate that residues in the native IACEAC motif of Atx1 contribute to the stability of the dimer formed in the presence of Cu(I).
Zinc(II) and Copper(I) Affinities. Introducing the metalbinding motif of Atx1 into PacS N has almost no effect on Zn(II) affinity (K Zn ), whereas replacing the sequence of Atx1 with that of PacS N decreases the Zn(II) affinity 3-fold ( Figure  4A and Table 2). The His61Tyr Atx1 mutation decreases the Zn(II) affinity by a similar amount (Table 2), and this loop 5 residue is therefore not involved in Zn(II) binding. The affinity of apo-protein for Zn(II)-protein (K Zn2 ) is enhanced 2-to 3fold in Atx1 PacS N and PacS N Atx1 but is almost unaltered by the His61Tyr Atx1 mutation ( Table 2). The largest changes in Zn(II) affinity (up to 40-fold) result from introducing the ZiaA N sequence into PacS N and Atx1 ( Figure 4B and Table 2). Atx1 ZiaA N has the highest Zn(II) affinity of all the proteins studied, ∼2and 15-fold tighter than those of ZiaA N and PacS N ZiaA N , respectively ( Table 2). The Cu(I) affinity (K Cu ) of monomeric WT Atx1 is approximately an order of magnitude greater than those of WT PacS N and ZiaA N at pH 7 (Table 2). 14 Some of this difference is due to the presence of His61 on loop 5 of Atx1 because replacement with a Tyr, the residue found in this position in both PacS N and ZiaA N , results in a ∼2.5-fold decrease in Cu(I) affinity (Table 2). 14 The introduction of the metal-binding motif of PacS N has almost no effect on Cu(I) affinity of Atx1, but K Cu decreases ∼2to 3-fold in Atx1 ZiaA N ( Figure 5 and Table 2). The introduction of the Atx1 loop into PacS N (in PacS N Atx1 ) does increase the Cu(I) affinity ∼3-fold (Table 2), whereas the Cu(I) affinity of PacS N ZiaA N is very similar to that of PacS N ( Table 2). The non-Cys residues in the loop can influence the Cu(I) affinity by a similar amount as that seen upon mutating the loop 5 residue. These mutations all change the second-coordination sphere, which has a small effect on the Cu(I) affinity of copper-trafficking proteins, with the most important contribution being from residues that can influence the pK a values of the Cys ligands. 23,30 Crystal Structures of Zn(II)−Atx1 ZiaA N and Zn(II)− PacS N ZiaA N . To gain insight into the structural causes of the large changes in Zn(II) affinity, Zn(II)−Atx1 ZiaA N and Zn(II)− PacS N ZiaA N have been crystallized. Atx1 ZiaA N loaded with 1 equiv of Zn(II) crystallizes as a Zn(II)-bridged dimer (Figure 6), an arrangement that is probably relevant for all the ZnP 2 forms that we have observed in solution (Table S3 in the Supporting  Information). This arrangement (contact area ∼450 Å 2 ) is remarkably similar to that of the side-to-side Cu(I) 2 −Atx1   Figure 7A). The bond angles range from 104 to 120°, consistent with tetrahedral coordination.
In the alternate crystal form (condition 2), the asymmetric unit contains a dimer with the monomer-monomer interface distal from the metal site. Zn(II)−PacS N ZiaA N is a monomer in solution ( Figure 3B and Table S3 in the Supporting  Information), and this crystallographic dimer is therefore an artifact. The metal site structure in this form of Zn(II)− PacS N ZiaAN is similar to that found in the condition 1 crystal structure except that Asp13 is replaced by a water ligand with a Zn(II)−O distance of ∼2.1 Å ( Figure 7B). The carboxylate group of Asp13 points away from the metal site, is solvent exposed, and is not involved in any interactions.
Attempts to crystallize ZiaA N have been unsuccessful, and NMR studies could not determine the structure of the high affinity Zn(II) site. 13 NMR has also been used to investigate the MBD of the related Zn(II)-exporting ATPase from E. coli (ZntA). 18 In this case a (Cys) 2 Asp Zn(II) site has been suggested, but because of the limitations of NMR data, neither the precise Zn(II) coordination number nor the geometry of the site could be resolved. This NMR study also suggested the possibility of a water ligand completing the coordination environment because of the solvent exposure of the Zn(II) site. Surprisingly, a recent NMR study of cyclic peptides that mimic Cu(I)-and Zn(II)-binding CXXC motifs (MTCSGCSRPG and MDCSGCSRPG, respectively) has found that the Asp residue (underlined) coordinates Cu(I) but not Zn(II). 33 The crystal structures of Zn(II)−PacS N ZiaA N are the first of a Zn(II) site bound by a CXXC motif involved in zinc transport. These structures provide strong evidence that the Asp residue preceding the CXXC motif binds Zn(II) in a monodentate fashion and that a water ligand can occupy the fourth coordination position of a tetrahedral site in the MBDs of Zn(II)-transporting proteins.
Insight into Zn(II) Trafficking Provided by the M/IXCXXC Motif Variants. The sequence of the metalbinding motif has a much more significant influence on Zn(II) than Cu(I) affinities, and introducing the ZiaA N sequence into Atx1 and PacS N increases the Zn(II) affinity by up to ∼40-fold. This is due to the side chain of Asp13 coordinating Zn(II), as seen in a Zn(II)−PacS N ZiaA N crystal structure ( Figure 7A), which must also be the cause of the higher Zn(II) affinity of ZiaA N . The two crystal forms of PacS N ZiaA N have Zn(II) sites with Cys 2 His coordination, with either the carboxylate of Asp13 or a water molecule as the fourth ligand. A carboxylate group has a lower affinity for Zn(II) than Cys and His, 34 consistent with replacement of Asp13 and not the other ligands by water. The coordination of Zn(II) by Asp18 in ZiaA N tunes its Zn(II) affinity so that it is tighter than those of the Cu(I)-trafficking proteins (with two Cys ligands) but below that of the Zn(II) sensor (His 2 Asp 2 coordination for SmtB from the cyanobacterium Synechococcus). 35,36 It has been suggested that Asp18 prevents ZiaA N from forming a stable complex with Cu(I)− Atx1. 37 A negatively charged residue close to the CXXC motif appears to be conserved in ATPases for metals (divalent) other than copper, and repulsion has been suggested as a common mechanism to prevent the binding of Cu(I). The presence of an Asp adjacent to the first Cys of the CXXC motif has almost no effect on Cu(I) affinity, as it is not required for coordination but is important for Zn(II) binding. With Zn(II) bound to ZiaA N , the negative charge of Asp13 will no longer contribute to repulsing Atx1. As observed in our crystal structures, this Asp can readily dissociate, which may occur as Zn(II) is subsequently trafficked, allowing the negative charge to help prevent unwanted interactions (vide infra). The increase in Zn(II) affinity due to the introduction of an additional Zn(II) ligand appears to be sufficient to allow the MBDs of zinc and   water ligand in (B), and Asp13 has moved away from the Zn(II) ion. The anomalous density for zinc is shown (orange mesh) contoured at 5σ. copper transporters that have very similar structures 12,13 to discriminate between these metals. It is therefore likely that the Zn(II) affinity of ZiaA N (10 −10 M) is in the range of physiological free zinc concentrations in Synechocystis, although this has not been determined. This is supported by the observation that in E. coli up-regulation of ZntA expression occurs at nanomolar levels of intracellular free Zn(II), 38 which matches the Zn(II) affinities of both the MBD and the transmembrane site of ZntA (∼10 8 M −1 ). 39 The His residue (His48) involved in Zn(II) coordination in the PacS N ZiaA N structures belongs to an adjacent molecule. The recruitment of this His ligand only occurs at the high protein concentrations required for crystallization [Zn(II)−PacS N ZiaA N is monomeric in dilute solution ( Figure 3B and Table S3 in the Supporting Information)] and is probably replaced by a water ligand in solution. However, in ZiaA N , His residues from the unstructured C-terminus interact in solution with Zn(II) bound to the CXXC site. 13 These interactions were proposed to either aid metal transfer or alter intramolecular interactions. The observation that the side chain of Asp13 can be replaced by water when a His ligand is present highlights the fluxionality of this Zn(II) site, which will assist Zn(II)-trafficking. Our structures suggest two possible intermediates involving one of the His residues from the unstructured region of ZiaA N , which could be important for zinc transfer. The coordination of Zn(II) by two His residues from the C-terminal region of ZiaA N (as well as by two Cys residues) would result in loss of the Asp ligand, enabling it to maintain a repulsive interaction with Atx1, to potentially hinder Cu(I) binding.
When the CXXC site of Atx1 binds 1 equiv of Zn(II), an exposed Zn(II) site with Cys 2 (H 2 O) 2 coordination will be present, which will be susceptible to ligand exchange reactions. This form may play a role in Zn(II) trafficking as it has been suggested that Atx1 can bind zinc in Synechocystis. 16 The unsaturated nature of such a Zn(II) site also makes it prone to coordinating additional ligands, such as Asp18 in ZiaA N . Two additional Cys ligands can also be recruited from a second protein molecule, as seen in the crystal structure of Zn(II)− Atx1 ZiaA N (Figure 6), and as indicated for other proteins in this work, and also WT Atx1, 14,31 by studies in solution (Figures 2  and 3, and Figures S2 and S3 and Table S3 in the Supporting Information). These tetrathiolate sites are reminiscent of Zn(II) structural sites, 7 and their buried nature suggests they have limited functionality for Zn(II) trafficking but may play a role in storing the metal. However, given the fact that these dimers are relatively weak, there is likely fast dimer-monomer exchange so that Zn(II) can be easily accessed for the supply of endogenous Zn(II)-binding proteins. We have recently shown 31 that heterodimers between partner proteins (e.g., Atx1 and PacS N ) are formed in the presence of Zn(II) and are more stable than the corresponding homodimers, and may have a role in regulating the activity of copper-transporting ATPases.

■ CONCLUSIONS
The Zn(II) affinity of the MBD of a cyanobacterial zinc transporter is greatly enhanced by the presence of an Asp in the metal-binding motif due to the ability of the carboxylate group of this residue to coordinate the metal. The Zn(II) site in the MBD seems highly fluxional, which must be important for trafficking this metal and for other potential roles that the ligands, and particularly the Asp residue, may need to perform. The residues in the M/IXCXXC metal-binding motif of copper and zinc trafficking proteins have little influence on Cu(I) affinity.

* S Supporting Information
Figures showing CD spectra, Zn(II) titrations, gel filtration profiles and the dependence on protein concentration of the gel filtration elution volume for Cu(I)−Atx1 PacS N , and tables showing primers, mass analyses, and gel filtration data. This material is available free of charge via the Internet at http:// pubs.acs.org.

Accession Codes
The coordinates and structure factors have been deposited in the Protein Data Bank under the accession codes 4A47, 4A48, and 484J.