Characterization of a Cobalt-Specific P_1B-ATPase

Abstract

The P_1B-type ATPases are a ubiquitous family of P-type ATPases involved in the transport of transition metal ions. Divided into subclasses on the basis of sequence characteristics and substrate specificity, these integral membrane transporters play key roles in metal homeostasis, metal tolerance, and the biosynthesis of metalloproteins. The P_1B-4-ATPases have the simplest architecture of the five P_1B-ATPase families and have been suggested to play a role in Co²⁺ transport. A P_1B-4-ATPase from Sulfitobacter sp. NAS-14.1, designated sCoaT, has been cloned, expressed, and purified. Activity assays indicate that sCoaT is specific for Co²⁺. A single Co²⁺ binding site is present, and optical, electron paramagnetic resonance (EPR), and X-ray absorption (XAS) spectroscopic data are consistent with tetrahedral coordination by oxygen and nitrogen ligands, including a histidine and likely a water. Surprisingly, there is no evidence for coordination by sulfur. Mutation of a conserved cysteine residue, Cys 327, in the signature transmembrane SPC metal binding motif does not abolish ATP hydrolysis activity or affect the spectroscopic analysis, establishing that this residue is not involved in the initial Co²⁺ binding by sCoaT. In contrast, replacements of conserved transmembrane residues Ser 325, His 657, Glu 658, and Thr 661 with alanine abolish ATP hydrolysis activity and Co²⁺ binding, indicating that these residues are necessary for Co²⁺ transport. These data represent the first in vitro characterization of a P_1B-4-ATPase and its Co²⁺ binding site.

The P-type ATPases, found in a variety of species from bacteria to humans, are a family of integral transmembrane proteins that transport charged substrates across cell membranes using the energy provided by ATP hydrolysis (1, 2). P-type ATPases are divided into subclasses on the basis of substrate specificity. These subclasses include the P_1A-ATPases (K⁺), the P_1B-ATPases (heavy metals), the P₂-ATPases (Ca²⁺, Na⁺/K⁺, H⁺/K⁺), the P₃-ATPases (H⁺), the P₄-ATPases (phospholipids), and the P₅-ATPases (unknown substrate) (3–5). All P-type ATPases share a common architecture consisting of multiple transmembrane (TM) helices, soluble nucleotide binding and phosphorylation domains (N- and P-domains, referred to collectively as the ATP binding domain or ATPBD), and a soluble actuator domain (A-domain). All P-type ATPAses are believed to follow a Post-Albers mechanism in which catalytic phosphorylation of a conserved aspartic acid residue within an invariant DKTGT sequence in the P-domain causes conformational switching between an E1 high affinity cation binding state and an E2 lower affinity state (6–9) (Figure 1). Crystal structures are available for multiple conformational states of the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) (10), and some structures have also been determined of the Na⁺/K⁺-ATPase (11, 12), the H⁺-ATPase (13), and the Cu⁺-ATPase (14).

Figure 1.

The Post-Albers catalytic cycle, represented for a Co²⁺ ATPase. The cycle is defined by two conformational states (E1 and E2) that are interconverted by the covalent attachment and release of phosphate to and from a conserved aspartic acid residue. In the E1 state, which has high affinity for the substrate being transported, the binding site is accessible to the cytoplasm. Upon hydrolysis of ATP, a conformational change in the TM helices converts the binding site to being accessible to the periplasm (or other cellular compartment) and lowers the substrate affinity of E2. After the substrate is released, dephosphorylation occurs and the protein reverts to the E1 state.

The P_1B-type ATPases transport transition metal ions, including Zn²⁺/Cd²⁺/Pb²⁺ (15–17), Cu²⁺ (18), Cu⁺/Ag⁺ (19, 20), and Co²⁺ (21). Members of the P_1B subgroup are widely distributed across all domains of life, conferring heavy metal tolerance and playing an essential role in the distribution of metal micronutrients and the biosynthesis of metalloproteins (20, 22). In humans, mutations in the Cu⁺-ATPases ATP7A and ATP7B lead to Menkes syndrome and Wilson disease, respectively (23). The core architectural features of the P_1B-type ATPases include at least six TM helices and two large cytoplasmic loops housing the ATPBD (containing the N- and P-domains) and the A-domain (Figure 2). The C-terminal TM helices 4–6 contain residues that are proposed to form the metal binding site(s), and the identities and positions of these residues are believed to determine metal specificity (24). Mutagenesis data indicate that a three-residue cysteine-containing “signature” sequence motif in TM4 (CPC, CPH, SPC, PCP) is of particular importance for metal transport activity (25–29). On the basis of these signature sequence motifs and some experimental data, the P_1B-ATPases have been grouped into five substrate-specific subfamilies designated P_1B-1 through P_1B-5 (24, 30). The P_1B-1, P_1B-2, and P_1B-3 subfamilies contain two additional TM helices at the N-terminus. The subfamilies are further differentiated according to the presence or absence of N- and/or C-terminal soluble metal binding domains (MBDs), which likely regulate ATPase function (31–34).

Figure 2.

Architecture and key residues of P_1B-4-ATPases. Left: Overall topology with conserved residues in TM helices shown in approximate locations. Right: Noncontinuous alignment of TM helices 4 and 6 of sCoaT and LpCopA. The phosphorylation site in the P-domain is highlighted in light purple. Site I proposed metal-binding residues of LpCopA are colored in orange, with the corresponding proposed sCoaT metal-binding residues colored in blue. Site II residues of LpCopA are colored in purple along with conserved sCoaT residues, and nonconserved sCoaT residues are colored in red.

The P_1B-1-ATPases are specific for Cu⁺/Ag⁺, the P_1B-2-ATPases are Zn²⁺/Cd²⁺/Pb²⁺ transporters, and the P_1B-3 ATPases are specific for Cu²⁺. Representative members of these three families have been purified and characterized biochemically (17–19, 25, 35, 36). In addition, a crystal structure of the Legionella pneumophila CopA P_1B-1-ATPase (LpCopA) has been determined recently (14). Less is known about the P_1B-4- and P_1B-5-ATPases. While the P_1B-4-ATPases have been suggested to play a role in Co²⁺ transport (21), their substrate specificity has not been established. The substrate for the P_1B-5-ATPases is unknown (37). The P_1B-4-ATPases have the simplest architecture, with only six TM helices and no N- or C-terminal MBDs. A conserved SPC motif is located in TM4 (Figure 2). Genetic data obtained more than 10 years ago for the Synechocystis PCC6803 P_1B-4-ATPase CoaT indicate that it is linked to Co²⁺ tolerance (21). In Cupriavidus metallidurans CH34, the P_1B-4-ATPase CzcP is proposed to play a role in resistance to Zn²⁺, Cd²⁺, and Co²⁺ (38). The plant P_1B-4-ATPase HMA1 transports Ca²⁺ and expression of HMA1 in yeast confers tolerance to Zn²⁺, Cd²⁺, Co²⁺ and Cu⁺ (39, 40). Thus, the metal substrate of the P_1B-4-ATPases has not yet been clearly defined, and no biochemical characterization of a purified P_1B-4-ATPase has been reported. To further understand this subfamily of P_1B-ATPases, we have expressed, purified, and characterized a P_1B-4-ATPase from Sulfitobacter sp. NAS-14.1, designated sCoaT. Isolated sCoaT binds Co²⁺ and exhibits Co²⁺-induced ATPase activity. The Co²⁺ binding site has been probed by spectroscopy and the roles of conserved residues have been assessed by mutagenesis. These data provide new insight into the function and properties of the P_1B-4-ATPase subfamily.

MATERIALS AND METHODS

Homology Modeling of sCoaT Based on LpCopA

Homology modeling of sCoaT and LpCopA was performed using DeepView (http://www.expasy.org/spdbv/) (41). Chain B of the LpCopA structure (PDB accession code 3RFU) was used as the reference sequence onto which the sequence of sCoaT was mapped. Structural alignment was performed on the last four TM helices of both sCoaT and LpCopA (starting from TM helix 3 of sCoaT and TM helix 5 of LpCopA) to focus on the structural region that houses residues believed to determine metal ion specificity (24). The starting position of TM helices was determined using TMHMM 2.0 (42). In the resultant homology model, residues 227–682 of sCoaT are aligned with residues 291–733 of LpCopA.

Cloning and Expression of Wildtype sCoaT (WT-sCoaT)

The gene sequence encoding the P_1B-4-ATPase sCoaT was PCR amplified with PfuTurbo DNA polymerase (Stratagene) using Sulfitobacter sp. NAS-14.1 genomic DNA (ATCC) as a template and the following oligonucleotide primers (Integrated DNA Technologies): forward, 5′-CAC-CGT-GCG-GAA-AGT-CGT-TGC-CGA-T-3′; reverse, 5′-CTA-TGC-CGA-TTT-GAC-GCG-ATT-GTC-CTT-GA-3′. The primers were designed on the basis of PubMed entry GI:83956091 with the introduction of an overhang (underlined) for TOPO cloning. The amplified gene (2.04 kbp) was subcloned into the pET151/D-TOPO vector (Invitrogen), which introduces an N-terminal hexahistidine tag with a TEV cleavage site. This vector was transformed into E. coli Top10 chemically competent cells (Invitrogen) following the manufacturer’s protocol and spread on a Luria-Bertani (LB) agar plate containing 100μg/mL ampicillin. DNA sequencing (ACGT Inc.) of individual colonies confirmed the gene sequence.

For expression, E. coli BL21 Star (DE3) chemically competent cells were transformed with the plasmid encoding sCoaT. Overnight cultures were used to inoculate baffled flasks containing 1 L LB media supplemented with 100 mg ampicillin which were then grown at 37 °C with shaking. Protein expression was induced by addition of 1 mM IPTG at an OD₆₀₀ of 0.6, at which point the growth temperature was lowered to 18 °C. The cells were harvested after 16–18 h by centrifugation at 4800g for 10 min in a Sorvall SLC 4000 rotor at 4 °C, resuspended in 50 mM Tris, pH 7.5, 200 mM NaCl, 500 mM sucrose, and 1 mM PMSF, and stored at −80 °C.

Purification of WT-sCoaT

All purification procedures were carried out at 4 °C. Cells were lysed by sonication for 12 min using 15 s pulses with a 30 s rest period between each pulse. Lysed cells were centrifuged at 8000g for 30 min to remove cell debris. The supernatant was then ultracentrifuged at 163000g for 1 hr. Pelleted membranes were washed once with fresh buffer (50 mM Tris, pH 7.5, 500 mM NaCl, 500 mM sucrose, 1mM PMSF) and homogenized, then pelleted again by ultracentrifugation at 163000g for 45 min. The pelleted, washed membranes were resuspended and homogenized in 25 mM Tris, pH 7.0, 100 mM sucrose, 500mM NaCl, 1 mM PMSF and diluted to 3 mg/mL, with the concentration determined by the detergent compatible Lowry assay (Bio-Rad). For solubilization, n-dodecyl-β-D-maltopyranoside (DDM) was added dropwise to a final concentration of 1% and allowed to incubate for 1 h at 4 °C with gentle stirring. The suspension was cleared by centrifugation at 163000g for 45 min. The supernatant was then applied to a 10 mL Ni²⁺-loaded HiTrap Chelating HP column (GE Healthcare) and washed with four column volumes of 25 mM Tris, pH 7.5, 100 mM NaCl, 20% glycerol, 0.02% DDM (Buffer A) followed by a wash of six column volumes of Buffer A with 50 mM imidazole. The pure sCoaT protein was eluted with Buffer A containing 300 mM imidazole. Fractions were concentrated in an Amicon 15 mL spin concentrator with a 50 kDa cutoff filter (Millipore), and imidazole was removed by desalting on a HiPrep 26/10 Desalting column (GE Healthcare) equilibrated with Buffer A. The protein concentration was determined by the Pierce 660 nm protein assay, and protein expression and purification were assessed by 15% SDS-PAGE.

To remove the N-terminal hexahistidine tag, purified sCoaT was treated with TEV protease. Hexahistidyl-tagged TEV protease was added to purified sCoaT at a 1:2 molar ratio along with 1 mM DTT and incubated at 4 °C with gentle rocking for 16–18 h. The cleavage reaction mixture was then loaded onto a 10 mL Ni²⁺-loaded HiTrap Chelating HP column (GE Healthcare) and washed with five column volumes of Buffer A, during which cleaved sCoaT was eluted. Uncleaved sCoaT and hexahistydyl-tagged TEV protease remained bound to the column. Removal of the hexahistidine tag from sCoaT was confirmed by Western blotting followed by immunostaining with anti-hexahistidine antibody (Sigma Aldrich) and by N-terminal sequence analysis (University of Illinois Champaign-Urbana Protein Sciences Facility).

Cloning, Expression, and Purification of sCoaT Mutants

Mutations S325A, C327A, H657A, E658A, and T661A were introduced using the QuikChange II Site-Directed Mutagenesis kit (Stratagene). Plasmid DNA of pET151/WT-sCoaT was used as template DNA for mutagenesis. The entire sequences of the mutants were verified by automated DNA sequencing (ACGT Inc.). The pET151/mutant plasmids were transformed into E. coli BL21 Star (DE3) chemically competent cells, and growth and purification were conducted using the same procedures as for WT-sCoaT.

ATPase Activity Assay

The metal-dependent ATPase activity of purified sCoaT was measured by the pyruvate kinase-lactate dehydrogenase coupled spectroscopic assay (17, 43). In this assay, regeneration of ATP is coupled to the oxidation of NADH, and enzyme activity is determined by monitoring the decrease of the NADH absorption peak at 340 nm. For activity measurements, sCoaT was exchanged into a buffer containing 25 mM Tris, pH 7.5, 50 mM KCl, 10% glycerol, and 0.02% DDM. The ATPase activity assay mixture (1 mL) contained 50 mM HEPES-KOH, pH 7.5, 100 mM KCl, 0.1% asolectin, 0.1% DDM, 5 mM MgSO₄, 0.25 mM NADH, 1.25 mM phosphoenolpyruvate, 9 units of pyruvate kinase, 9 units of lactate dehydrogenase, and 10 μg sCoaT, with or without Co²⁺, Pb²⁺, Zn²⁺, Cd²⁺, Cu²⁺, Cu⁺ (generated from Cu²⁺ in the presence of excess dithiothreitol) and Ni²⁺. Insolubility of Ag⁺ under the assay conditions precluded testing Ag⁺-dependent activity. Assay mixtures were activated by the addition of 5 mM ATP and monitored for 3 min at 25 °C using a Perkin Elmer LAMBDA 1050 UV-Vis spectrophotometer (Keck Biophysics Facility, Northwestern University) at 340 nm.

Metal Loading and Analysis

Purified sCoaT was first exchanged into a buffer containing 25 mM Tris, pH 7.5, 50 mM NaCl, 10% glycerol, and 0.02% DDM using repeated dilution and concentration in an Amicon spin concentrator. Cobalt loading was performed by addition of 5 molar equivalents of CoCl₂ to the sCoaT solution followed by overnight (16–18 h) incubation at 4 °C with gentle rocking. Excess cobalt was removed using an EconoPac 10DG desalting column (Bio-Rad). Metal content was measured by inductively coupled plasma optical emission spectrometry (ICP-OES) using a Varian Vista MPX ICP-OES (Integrated Molecular Structure Education and Research Center, Northwestern University). All ICP-OES samples were prepared by digestion of protein in 5 mL of 5% TraceSelect nitric acid (Sigma Aldrich) in Chelexed water followed by filtration through a 22 μm sterile filter. Cobalt standards for calibration (Sigma Aldrich) were also prepared in 5% nitric acid. Reported ICP-OES results are the average of three replicate experiments performed for at least two independent protein sample preparations. Optical spectra were measured using a Perkin Elmer LAMBDA 1050 UV-Vis instrument.

Electron Paramagnetic Resonance (EPR) Spectroscopy

Samples for EPR spectroscopy were concentrated to 0.6–1mM Co²⁺ in 25 mM Tris, pH 7.5, 50 mM NaCl, 0.02% DDM, 50% glycerol. Deuterated samples were prepared in the same buffer using D₂O and d₈-glycerol (Sigma Aldrich). Samples were transferred to custom Q-band EPR tubes, frozen, and stored in liquid nitrogen. Q-band continuous wave (CW) EPR and Electron Nuclear Double Resonance (ENDOR) spectra were collected on custom-built instruments each equipped with a liquid helium immersion dewar at 2 K using 100 kHz field modulation and dispersion mode detection under rapid passage conditions (44). These spectrometers have been described previously (45). Q-band pulsed ENDOR spectra were collected at 2 K on custom-built instruments previously described (45–47) and employed the Davies (π – T – π/2 – τ – π – τ – echo) microwave pulse sequence, in which the RF pulse is applied during time period T (48). All pulse experiment data acquisition was performed with the SpecMan software package (49) (http://specman4epr.com) in conjunction with a Spin-Core PulseBlaster ESR_PRO 400 MHz word generator and Agilent Technologies Acquiris DP235 500MS/sec digitizer.

X-ray Absorption (XAS) Spectroscopy

Samples for XAS were prepared in 25 mM Tris, pH 7.5, 50 mM NaCl, 0.02% DDM and 50% glycerol with cobalt concentrations in the 1–1.5 mM range. Two independent replicates of cobalt-loaded, WT- and C327A-sCoaT were prepared. Samples were loaded into Lucite cells wrapped with Kapton tape, frozen in liquid nitrogen and stored at −80 °C until data collection. XAS data were collected at the Stanford Synchrotron Radiation Laboratory (SSRL) on beamline 7-3 and at the National Synchrotron Light Source (NSLS) on beamline X3-b. The SSRL beamline was equipped with a Si[220] double crystal monochromator and the NSLS beamline was equipped with a Si[111] monochromator; both beamlines were equipped with a harmonic rejection mirrors. During data collection, samples were maintained at 10 K using an Oxford Instruments continuous-flow liquid helium cryostat at SSRL and at 24 K using a He Displex Cryostat at NSLS. Protein fluorescence excitation spectra were collected using 30-element Ge solid-state detectors at both beamlines. At SSRL, iron filters, 0.3 μm in width, were placed between the cryostat and detector to filter background fluorescence scattering not associated with Co signals. XAS spectra at both facilities were measured in 5 eV increments in the pre-edge regions (7542–7702 eV), 0.25 eV increments in the edge regions (7702–7780 eV), and 0.05 Å⁻¹ increments in the extended X-ray absorption fine structure (EXAFS) region (to k = 13.0 Å⁻¹), integrating from 1s to 25s in a k³ weighted manner for a total scan length of approximately 40 min. X-ray energies were individually calibrated by collecting Co foil absorption spectra simultaneously with protein data. The first inflection point of the Co foil spectrum was assigned to 7709.5 eV. Each fluorescence channel of each scan was examined for spectral anomalies prior to averaging and spectra were closely monitored for photoreduction. SSRL data represent the average of 6 to 7 scans, and NSLS data represent the average of 9 to 10 scans.

XAS data were processed using the Macintosh OS X version of the EXAFSPAK program suite (50) integrated with the Feff v8 software (51) for theoretical model generation. Data reduction utilized a Gaussian function in the pre-edge region and a three-region cubic spline throughout the EXAFS region. Data were converted to k-space using a cobalt E₀ value of 7745 eV. The k cubed weighted EXAFS was truncated at 1.0 and 12.5 Å⁻¹ for filtering purposes. This k range corresponds to a spectral resolution of ca. 0.14 Å for all cobalt-ligand interactions; therefore only independent scattering environments outside 0.14 Å were considered resolvable in the EXAFS fitting analysis (52). EXAFS fitting analysis was performed on raw/unfiltered data. EXAFS data were fit using both single and multiple scattering amplitude and phase functions calculated with the program Feff v8. Single scattering theoretical models were calculated for carbon, oxygen, sulfur and cobalt coordination to simulate copper-nearest neighbor ligand environments. A multiple scattering Co-imidazole theoretical model was calculated to simulate the numerous scattering interactions observed from the linear ring. A scale factor (Sc) of 0.98% and an E₀ value of −3 eV for a Co-O/N bond, utilized during the protein simulations, were determined by fitting a crystallographically characterized hexaquacobalt (II) nitrate (53). Criteria for judging the best-fit simulation utilized both the lowest mean square deviation between data and fit (F′), corrected for the number of degrees of freedom (54), and a reasonable Debye-Waller factor (σ² < 0.006 Ų).

RESULTS AND DISCUSSION

Homology Modeling

On the basis of sequence alignments, seven conserved residues were proposed to participate in metal binding by P_1B-4-ATPases: serine, proline, and cysteine in TM4, and histidine, glutamine, glycine, and threonine in TM6 (24) (Figure 2). Additional insight into specific ligands can be obtained by comparison of sCoaT to LpCopA, the only structurally characterized P_1B-ATPase to date (14). Although there are no bound metal ions in the structure, LpCopA is predicted to have two metal binding sites, one composed of Asn 689, Met 717, and Ser 721 (site I), and the second involving the transmembrane cysteines Cys 382 and Cys 384 as well as Tyr 688 (site II). Homology modeling reveals that four of the putative LpCopA metal binding residues align with corresponding conserved residues in sCoaT (Figure 2, right). The residues in the SPC motif, Ser 325 and Cys 327, align with LpCopA Cys 382 and Cys 384, and residues His 657 and Thr 661 align with LpCopA proposed ligands Met 717 and Ser 721. The other two LpCopA binding residues, Asn 689 and Tyr 688, align with nonconserved valine residues. Conserved sCoaT residue Glu 658 corresponds to an alanine in LpCopA, but would be close enough to His 657 and Thr 661 to participate in cobalt binding.

Expression and Purification of WT-sCoaT and Variants

Recombinant expression of WT-sCoaT yielded approximately 2 mg of purified protein/L of cultured media. Overnight induction of cells at 18 °C increased the yield of purified WT-sCoaT compared to a 3 h induction period at 37 °C. DDM-solubilized WT-sCoaT was purified in a single step by Ni²⁺-affinity chromatography (Figure 3), and cleavage of the hexahistidine tag using TEV protease yielded ~70% recovery of untagged WT-sCoaT. The removal of the hexahistidine tag was verified by Western blot using an anti-hexahistidine antibody (Figure 3) and by N-terminal sequence analysis, which indicated complete cleavage. Variant proteins in which Ser 325, Cys 327, His 657, Glu 658 and Thr 661 were mutated to alanine were also prepared to assess the role of these conserved residues in metal binding and activity. These mutants did not express as well as WT-sCoaT, yielding approximately 0.3–0.5 mg of pure protein/L, but utilization of the same WT-sCoaT purification protocol resulted in >90% pure proteins (Figure S1). The low yields precluded TEV protease cleavage of the sCoaT variants, however.

Figure 3.

SDS-PAGE and Western analysis of purified WT-sCoaT. Lane 1, SDS-PAGE of purified tagged WT-sCoaT; lane 2, SDS-PAGE of purified TEV-cleaved WT-sCoaT; lane 3, anti-6xHis tag Western blot of purified tagged WT-sCoaT; lane 4, anti-6xHis tag Western blot of purified TEV-cleaved WT-sCoaT.

ATPase Activity

The ATPase activity of WT-sCoaT was specifically stimulated by Co²⁺, and was also stimulated by Ni²⁺, but to a much lesser extent than Co²⁺ (Figure 4A). None of the other metals tested (Zn²⁺, Cd²⁺, Cu²⁺, Cu⁺, Pb²⁺) were able to activate the enzyme. The Co²⁺-dependent activity of WT-sCoaT has a K_M value of 20.05 ± 3.6 μM and a V_max value of 0.71 ± 0.03 μmol mg⁻¹min⁻¹ (Figure 4B). The V_max value is similar to those reported for the Pb²⁺-stimulated activity of ZntA (17) and the Ag⁺-stimulated activity of CopA (19). The activity of the variants was negligible, with the notable exception of the C327A variant (C327A-sCoaT), which has approximately 50% of the WT-sCoaT activity (Figure 5). This result is unexpected and differs from previous studies of P_1B-1-, P_1B-2- and P_1B-3-ATPases. For A. fulgidus CopA, mutation of either or both cysteines in the CPC motif to alanine abolishes ATPase activity (26), and in the case of E. coli CopA, mutation of these cysteines results in loss of copper resistance, transport, and phosphoenzyme formation (25). Replacement of either cysteine with alanine in the E. coli ZntA CPC motif also results in inactive enzyme, but ZntA variants in which the cysteines are replaced with histidine retain some activity (27). For the E. hirae CopB Cu²⁺-ATPase, alteration of the CPH transmembrane motif to SPH abolished the ability to restore copper resistance in a CopB knockout mutant in vivo, suggesting impaired Cu²⁺ transport (55).

Figure 4.

Activity of WT-sCoaT. (A) Co²⁺ stimulates the highest ATP hydrolysis activity. Results are the average of at least three independent replicates. Maximal activity for 100 μM Co²⁺ is 0.57 μmoles ATP mg⁻¹ min⁻¹. (B) Co²⁺ stimulated ATPase activity of WT-sCoaT. Fits were performed using Kaleidagraph software with the equation y = (V_max*x)/(K_M+x).

Figure 5.

Comparison of ATP hydrolysis activity of WT-sCoaT, C327A-sCoaT, and variants in which His 657, Ser 325, Glu 658, and Thr 661 are replaced with alanine.

Metal-Binding Stoichiometry

Tagged WT-sCoaT binds 1.55 ± 0.07 Co²⁺ ions per protein monomer, and cleaved WT-sCoaT binds 1.38 ± 0.21 Co²⁺ ions per protein monomer, indicating that the hexahistidyl tag does not bind a significant amount of Co²⁺ during the loading procedure. WT-sCoaT is thus proposed to contain one metal binding site for Co²⁺. ZntA from E. coli also is reported to contain a single metal binding side (35) whereas A. fulgidus CopA binds two Cu⁺ ions with high affinity (56). Interestingly, C327A-sCoaT also appears to have a single Co²⁺ binding site, with a stoichiometry of 1.49 ± 0.53 Co²⁺ ions per monomer. This result suggests that the cysteine in the TM metal binding SPC motif is not essential for Co²⁺ binding and is consistent with the observation of ATPase activity for C327A-sCoaT (Figure 5). None of the other mutants were able to bind Co²⁺, further indication that the hexahistidyl tag does not bind Co²⁺. This finding is consistent with the lack of ATPase activity of these variants and indicates that Ser 325, His 657A, Glu 658 and Thr 661 are essential for Co²⁺ binding and activity, and may be ligands as predicted by the homology modeling based on the LpCopA structure.

The optical spectra of Co²⁺-loaded WT-sCoaT and C327A-sCoaT both exhibit a broad shoulder extending from the 280 nm protein absorbance to ~400 nm (Figure 6). Although this region of the spectrum is typically associated with Co²⁺-thiolate ligand-to-metal charge transfer (LMCT) bands, the lack of distinct peaks observed for these intense transitions (57–59) and the similarity between the spectra of WT-sCoaT and the cysteine-lacking C327A-sCoaT suggest that this absorbance does not arise from interactions with sulfur ligands. This shoulder is more likely attributable to histidine-to-Co²⁺ LMCT, and resembles features observed in the spectrum of Co²⁺-substituted copper,zinc superoxide dismutase (60). The geometry of Co²⁺ sites can be correlated with the position and intensity of d-d transitions in the visible region (59, 61). The broad features in the 500–600 nm region of the sCoaT spectra (ε ~ 300 M⁻¹ cm⁻¹) are most consistent with regular tetrahedral geometry (61, 62). The shift of the peak in C237A-sCoaT towards ~525 nm may indicate some distortion of the Co²⁺ binding site, perhaps due to perturbation of the secondary coordination environment.

Figure 6.

Difference spectra of the Co²⁺-bound forms of WT- and C327A-sCoaT, using the respective apo forms as reference spectra.

EPR and ENDOR

WT-, tagged WT-, and C327A-sCoaT all exhibit an S = 3/2 Co²⁺ EPR signal with a broad feature at g_⊥ = 3.9 and g_|| ~ 2, consistent with tetrahedral symmetry (63). Tagged and untagged WT-sCoaT show identical EPR signals, indicating that Co²⁺ does not bind to the hexahistidyl tag (Figure 7a). The presence of the same feature in the C327A-sCoaT spectrum indicates that the Co²⁺ coordination sphere is not altered by deletion of the conserved cysteine residue.

Figure 7.

35 GHz EPR and ENDOR spectra. (A) Absorption-display CW Q-band EPR spectra of tagged WT-sCoaT, C327A-sCoaT and WT-sCoaT at 2K. Each sample exhibits a S = 3/2 Co²⁺ signal as characterized from the broad feature at g_⊥ = 3.9. Adventitious Cu²⁺ in variable amounts is observed at higher fields, but no copper is detectable in the samples by ICP-OES. (B) ¹⁴N Davies ENDOR spectra taken near g_⊥. Goalpost centered at A′/2, split by 2 × ν(¹⁴N). (C) ¹H CW ENDOR spectra of WT-CoaT in ¹H₂O (solid line) and ²H₂O buffer (dashed line) taken near g_⊥. Shading highlights intensity from exchangeable proton. Goalpost centered at ν(¹H), split by A′. CW EPR and ENDOR conditions: microwave frequency, 34.9 GHz; modulation amplitude, 2 G; time constant, 32 ms; EPR scan time, 4 min; ENDOR scan rate, 2 MHz/sec. Pulse ENDOR conditions: microwave frequency, 34.6 GHz; π, 80 ns; τ, 600 ns; T_RF, 30 μs; repetition rate, 20 ms.

ENDOR spectra collected at g_⊥ = 3.9 from WT-, C327A- and tagged WT-sCoaT (Figure 7b), reveal identical signals from a single, strongly-coupled ¹⁴N. The signal is a doublet of broad peaks that is centered at half the hyperfine coupling of A′ = 13.6 MHz (45), with a separation of twice the ¹⁴N nuclear Larmor frequency. This coupling treats the Co²⁺ as having an effective spin, S′ = 1/2; in an S = 3/2 representation the hyperfine coupling is A = 21.0 MHz, comparable to histidine bound to Cu²⁺, S = 1/2. The observed hyperfine coupling A′, taken at a given g-value is modified from the intrinsic A value. A′ = gA/ge. The higher-frequency (ν₊) partner shows poorly resolved structure that indicates the Co²⁺ site is structurally heterogeneous, with several contributing conformers; an additional contribution from the nuclear quadrupole interaction in at least one conformer may also be present. An attempt to measure this interaction with ESEEM spectroscopy failed because rapid spin dephasing quenched the spin-echo modulation. The strongly-coupled nitrogen can be assigned to a bound histidine residue, based on the near identity of its signal to that of a histidine bound to Co²⁺ of the Zn-finger in transcription factor IIIA (TFIIIA) from Xenopus laevis (64). This residue is very likely to be His 657, which is found in transmembrane helix 6 of sCoaT (Figure 2), is conserved in the P_1B-4 subgroup, and has been proposed to be a metal binding ligand (24). The equivalence of the ¹⁴N signals from untagged and tagged WT-sCoaT shows that there is no coordination to Co²⁺ by a residue from the hexahistidine tag. The equivalence of the ¹⁴N signals from WT-sCoaT and C327A-sCoaT, like the invariance of the EPR signal, indicates that the C327A mutation does not alter the Co²⁺ coordination sphere. Lastly, ¹H ENDOR spectra taken near g_⊥ exhibit a ¹H doublet, hyperfine coupling A′ = 13 MHz, that is lost when the sample is prepared with ²H₂O buffer. As this coupling is of the magnitude expected from the proton(s) of an H_xO coordinated along the g_|| direction, these observations indicate that Co²⁺ binds a single aqua species.

XAS Spectroscopy

The XANES spectra of WT- and C327A-sCoaT resemble that of Co(NO₃)₂•6H₂O (Figure 8), indicating cobalt is stable in the divalent redox state and most likely dominated by coordination of oxygen/nitrogen based ligands. The Fourier transforms of the EXAFS spectrum for Co²⁺ bound to WT- and to C327A-sCoaT suggest complex patterns of Co-nearest neighbor ligand environments at R < 3 Å, with additional scattering patterns of ligands at R > 3 Å (Figure 9). Best-fit simulations of the WT- and C327A-sCoaT Co EXAFS indicate a nearest neighbor environment constructed completely of oxygen and/or nitrogen based ligands. Nearest neighbor metrical parameters for include a short Co-O/N distance at 1.95 and 1.96 Å, and a longer distance set of Co-O/N ligands at 2.14 and 2.12 Å for WT- and C327A-sCoaT, respectively. The shorter ~1.95 Å distances are highly reminiscent of the values observed for four-coordinate Co-O small molecule compounds in the Cambridge structural database, whereas the longer ~2.13 Å distance would be consistent with Co-imidazole nitrogen distances (65). Long-range carbon scattering environments at ca. 3.1, 3.5, and 4.1 Å are also consistent with imidazole multiple scattering interactions expected for histidines coordinated to Co (66) (Table 1). There is no evidence for a Cu-S interaction in the EXAFS, consistent with the activity assays, metal binding studies, UV-Vis, and EPR data, indicating Cys 327 of the SPC motif is not involved in Co²⁺ binding. Moreover, the presence of only oxygen/nitrogen ligands is compatible with the predicted metal binding residues that are conserved in the P_1B-4 subclass, including the serine in TM4, and the histidine, glutamate, and threonine residues in TM6 (Figure 2). The observed imidazole multiple scattering interactions support the EPR data and are consistent with His 657 in TM4 being a ligand.

Figure 8.

Normalized XANES spectra of Co bound WT- (solid) and C327A-sCoaT (dashed line), displayed with the spectrum of solid Co(NO₃)₂ (gray triangles).

Figure 9.

EXAFS and Fourier transform for WT- and C327A-sCoaT. Raw unfiltered EXAFS data (black) and simulations (gray) for cobalt bound to WT- (A) and C327A-sCoaT (C). Fourier transforms of the raw EXAFS (black) and best fit simulations (gray) for cobalt bound to WT- (B) and C327A-sCoaT (D).

Table 1.

Summary of best fit EXAFS simulations for cobalt bound to WT- and C327A-sCoaT. Data fit over a k range of 1 to 12.5 Å⁻¹.

Sample	Nearest Neighbor Ligand Environmenta				Long Range Ligand Environmentb				F′g
	Atomc	R (Å)d	CNe	σ2f	Atomc	R (Å)d	CNe	σ2f
WT	O/N	1.95	2	3.62	C	3.04	1	5.21	0.49
	O/N	2.14	2	3.42	C	3.33	1	3.62
					C	4.09	1	3.31
C327A	O/N	1.96	1	1.97	C	3.03	1	4.85	0.49
	O/N	2.12	3	5.69	C	3.39	0.5	3.15
					C	4.08	2	5.06

^aIndependent metal-ligand scattering environment at R < 3.0 Å

^bIndependent metal-ligand scattering environment at R > 3.0 Å

^cScattering atoms: O (oxygen), N (nitrogen), C (carbon)

^dAverage metal-ligand bond length for multiple independent samples

^eAverage metal-ligand coordination number for multiple independent samples

^fAverage Debye-Waller factor in Ų × 10³ for multiple independent samples

^gNumber of degrees of freedom weighted mean square deviation between data and fit

Implications for the sCoaT metal binding site

The combined data on WT-sCoaT and the sCoaT variants indicate the presence of a single Co²⁺ binding site that is coordinated by nitrogen and oxygen ligands, including a histidine and a water molecule, in a tetrahedral geometry. Surprisingly, the data indicate that the cysteine residue in the conserved SPC transmembrane metal binding motif is not directly involved in Co²⁺ binding in this state of the enzyme. In several P_1B-ATPases that contain cysteines in this motif (vide supra), such as E. coli CopA, A. fulgidus CopA, E. hirae CopB, and E. coli ZntA, the transmembrane cysteines appear to play essential and equivalent roles in the catalytic cycle (25–27, 55). However, in other P_1B-ATPases, the cysteine residues in this motif are believed to be nonequivalent and function at different stages of the transport mechanism. For example, mutation of both cysteines in S. cerevisiae Ccc2 yields no in vivo or ATPase activity, but phosphorylation by ATP is still possible for a CPC to SPC variant, although not for a CPC to CPS variant. These findings indicate that the second cysteine is necessary for metal binding at the transport site whereas the first cysteine plays a role in metal dissociation and/or enzyme dephosphorylation (67). Similarly, mutation of either cysteine in the CPC motif of CadA, a Zn²⁺/Cd²⁺/Pb²⁺-ATPase from Listeria monocytogenes, inhibits in vivo Cd²⁺ transport, but only the CPC to CPA variant retains ATP hydrolysis activity. In addition, the CPA variant can be phosphorylated by ATP, suggesting that the first cysteine is more directly involved in Cd²⁺ binding and the second cysteine may participate in occlusion and release (29). The data for C327A-sCoaT are consistent with this type of scenario in which Cys 327 does not play a role in initial Co²⁺ binding and catalytic steps involving the E1 conformation (Figure 1), but may play a role in the E2 conformation, perhaps participating in metal release or dephosphorylation. Taken together, our data indicate that Co²⁺ can bind in the absence of the TM4 cysteine and that histidine, probably His 657, and water are coordinated. Besides His 657, Ser 325, Glu 658, and Thr 661 are necessary for Co²⁺ binding and transport and thus likely comprise the additional nitrogen and oxygen ligands. Further studies to elucidate the molecular details of Co²⁺ coordination are underway.

Abbreviations

sCoaT

Sulfitobacter sp. NAS-14.1 P_1B-4-ATPase

WT-sCoaT

wild type sCoaT

C327A-sCoaT

variant sCoaT in which Cys 327 is replaced with alanine

LpCopA

Legionella pneumophila Cu⁺ P_1B-1-ATPase

ATPBD

ATP binding domain

N-domain

nucleotide binding domain

P-domain

phosphorylation domain

A-domain

actuator domain