ATP and Mg²⁺ Promote the Reversible Oligomerization and Aggregation of Chloroplast 2-Cys Peroxiredoxin^*

Materials

Biochemicals were purchased from Sigma-Aldrich.

Construction of 2-Cys Prx Mutants

The pET-22b(+) vector (Novagen, Madison, WI) was used to express the variants of 2-Cys Prx as C-terminal hexahistidine-tagged fusion proteins in JM109 (DE3) Escherichia coli cells. Site-directed mutagenesis was carried out by the megaprimer method with primers described in supplemental Table S1.

Protein Purification

Recombinant chloroplast rapeseed 2-Cys Prx was prepared as described previously (18). Briefly, JM109 (DE3) E. coli cells harboring 2-Cys Prx expression plasmids were grown at 37 °C in Luria-Bertani medium supplemented with 100 μg/ml ampicillin. After induction with 0.6 mm isopropyl β-d-thiogalactoside, bacteria were harvested by centrifugation (3,000 × g, 10 min), washed with 20 mm Tris-HCl buffer (pH 8.0), resuspended in the same buffer containing 0.5 m NaCl, and subjected to sonication. After centrifugation (20,000 × g, 30 min), the supernatant fraction was loaded on a Ni²⁺-iminodiacetate-Sepharose column that was washed with 20 mm Tris-HCl buffer (pH 8.0) containing successively 60 and 600 mm imidazole. The protein fraction released in the latter elution was dialyzed against and stored in 20 mm Tris-HCl buffer (pH 8.0). The purified chloroplast 2-Cys Prx exhibited a single band of 46 and 23 kDa in non-reducing and reducing SDS-PAGE, respectively (18). A molar extinction coefficient of 23,555 m⁻¹ cm⁻¹ at 280 nm was used to quantitate the homogeneous preparation of the recombinant protein. Recombinant chloroplast rapeseed Trx-m was prepared as reported previously (18).

Assay of Peroxidase and Chaperone Activities

The peroxidase activity was assayed at 25 °C as described previously (7). The chaperone activity of 2-Cys Prx was measured using bovine pancreas insulin as substrate. To this end, 2-Cys Prx (8–18 μm) was incubated at 25 °C in 20 mm Tris-HCl buffer (pH 8) containing 0.5 mg/ml insulin. After 5 min, dithiothreitol was added (20 mm), and the aggregation of insulin β-chain was followed at 25 °C by monitoring optical density at 600 nm.

8-Anilinonaphthalene-1-sulfonic Acid (ANS) Fluorescence

Fluorescence studies with ANS were performed at 25 °C in solutions of 20 mm Tris-HCl buffer (pH 8) containing 10 μm 2-Cys Prx, 75 μm ANS (ϵ_{350 nm} = 5,000 m⁻¹ cm⁻¹), 2 mm MgCl₂, and appropriate concentrations of ATP (0–5 mm). After 10 min, the samples were excited at 370 nm, and emission spectra were scanned from 400 to 600 nm (bandwidth 5 nm). Reported spectra were averaged over four scans taken at 90° relative to the direction of the incident beam. All measurements were corrected for both buffer and non-bound ANS. The spectral center of mass, CM, was calculated using the following equation,

where mi is the emission intensity at the yi wavelength and N is the spectrum range (23).

Dynamic Light Scattering (DLS)

Solutions of 20 mm Tris-HCl buffer (pH 8) containing 5 μm 2-Cys Prx were centrifuged at 14,000 rpm for 30 min. Insoluble debris was removed, and MgCl₂ (3 mm) containing appropriate concentrations of ATP (0–5 mm) were added to the supernatant fraction. DLS measurements were performed at 25 ± 0.1 °C using non-invasive back scatter equipment (Malvern Nano S, Malvern, UK) with a constant 173° scattering angle (sample volume 0.5 ml). A total of 25 scans (10 repetitions each) with an individual duration of ∼2.4 min were obtained for each sample. Scattering intensities were analyzed using the instrument software for the calculation of hydrodynamic diameter (D_H), size, and volume distribution.

Static Light Scattering (SLS)

The measurement of the molecular mass of 2-Cys Prx was carried out at 25 °C on a Precision Detector PD2010 light-scattering instrument connected in tandem to a Sephadex G-50 column and an LKB 2142 differential refractometer. 2-Cys Prx (43 μm) was incubated for 5 min in 50 mm Tris-HCl buffer (pH 7.8) in the presence and in the absence of 3 mm ATP and 3 mm MgCl₂. The protein was loaded on the Sephadex G-50 column equilibrated with the incubation solution and eluted with the same solution. The 90° light scattering and refractive index signals of the eluting material were analyzed with the Discovery32 software supplied by the manufacturer. Molecular masses were determined from the values of light scattering and refractive index using the model of Rayleigh-Debye-Gans for dilute polymer solutions.

Aggregation of 2-Cys Prx

2-Cys Prx (1–15 μm) was incubated at 25 °C for 10 min in 0.4 ml of 20 mm Tris-HCl buffer (pH 8) containing appropriate perturbants. The aggregation of the protein was started by the addition of both ATP and MgCl₂ and followed by monitoring the optical density at 600 nm in a Hitachi 220A dual beam spectrophotometer or by analysis of the supernatant and precipitate fractions on non-reducing SDS-PAGE.

Analysis of 2-Cys Prx Aggregation with Surface Plasmon Resonance (SPR)

The aggregation of 2-Cys Prx mediated by ATP/Mg was carried out on an IAsys instrument (NeoSensors Ltd., Durham, UK), using standard carboxymethyl-dextran chips where neither 2-Cys Prx nor ATP was covalently attached to the surface of the cuvette. For base-line stabilization, the cuvette was washed several times with 20 mm Tris-HCl buffer (pH 8) until the resonant angle response was constant. The instrument parameters were set at 100% for stirring and 0.3 s for the sampling interval, and all measurements were performed at 25 °C. To estimate the association parameters, 4.2 μm 2-Cys Prx, 3 mm MgCl₂, and appropriate concentrations of ATP were sequentially added. After every analysis, the cuvette surface was regenerated by washing three times with 4 m KOH (100 μl each) followed by several times with 20 mm Tris-HCl buffer (pH 8). The apparent association (k_a), dissociation (k_d), and equilibrium (K_D = k_d/k_a) constants were obtained from the linear regression plot (k_on = k_a × [ATP] − k_d) using the FAST plot program. Alternatively, k_d was estimated by adding 10 mm EDTA to the cuvette after the association signal remained constant.

Transmission Electron Microscopy (TEM)

2-Cys Prx (0.1 mg/ml) was incubated at 25 °C in 20 mm Tris-HCl buffer (pH 8) and 3 mm MgCl₂, in the presence or in the absence of 3 mm ATP. After 5 min, an aliquot (20 μl) was disseminated onto a carbon-coated copper grid (mesh 300, Ted Pella, Redding, CA), the excess of solution was gently removed using filter paper, and the grid was air-dried for 1 min. The protein was negatively stained with 0.5% uranyl acetate (pH 4.5) for 1 min. The grid was air-dried, and samples were examined under a transmission electron microscope (Philips EM 301). Particle dimensions were estimated using ImageJ. At least three independent experiments were performed for each treatment of 2-Cys Prx.

Photochemical Cross-link of ATP with 2-Cys Prx

UV-mediated binding of ATP to 2-Cys Prx was carried out as described previously (24). Briefly, the protein (10 μm) in 20 mm Tris-HCl buffer (pH 8) containing 5 mm MgC1₂ and appropriate concentrations of ATP was irradiated at 25 °C with a 15-watt germicidal lamp (total volume 0.2 ml). After 35 min, the solution was dialyzed overnight at 4 °C against 20 mm sodium phosphate buffer (pH 8).

Circular Dichroism (CD)

CD spectra of 2-Cys Prx (2 μm) in 20 mm sodium phosphate buffer (pH 8) were measured at 20 °C with a quartz microcuvette (light path 0.2 cm) placed in a J-810 spectropolarimeter equipped with a thermoelectric temperature controller (Jasco, Easton, MD). Spectra were collected at 0.5 nm resolution and a scan rate of 200 nm/min, using at least two different preparations of freshly prepared protein solutions. Reported spectra were averaged over four scans. Ellipticities of CD spectra were analyzed with the software package CDPro, and the outputs of SELCON3, CONTINLL, and CDSSTR programs were averaged to quantitate the percentage of secondary structure of each spectrum.

Binding of ANS to 2-Cys Prx

ANS is a fluorescent probe that interacts with solvent-accessible non-polar regions of partially folded proteins, which would be relatively rare in the native state (25, 26). ANS binds to chloroplast 2-Cys Prx, causing a marked blue shift in the emission maximum wavelength (512 nm → 497 nm) and concurrently increasing the intensity of the fluorophore (7). Fig. 1A illustrates that the emission spectra of ANS exhibited modifications of the center of mass and intensity upon the incorporation of ATP in the presence of 2 mm Mg²⁺. As illustrated in Fig. 1B, the response of the center of mass to increasing concentrations of ATP showed three well defined stages: (i) a significant blue shift (497 nm → 492 nm) at concentrations lower than 0.5 mm, (ii) a stabilization at 492 nm from 0.5 to 2 mm, and (iii) a second blue shift (492 nm → 489 nm) between 2 and 5 mm. Notably, these transitions of fluorescence emulated the ATP/Mg-mediated inhibition of the peroxidase activity reported previously (cf. Fig. 1 in Ref. 7). Taken together, the ATP-induced transitions appear to be related to changes in the tertiary structure and/or the self-association of 2-Cys Prx, which, in turn, drag the protein to populate states with more exposed hydrophobic regions. The interaction with ATP/Mg did not imply changes in the chemical state of the nucleotide because we found that neither ADP nor phosphate is incorporated to the protein or released to the surrounding milieu.

ATP/Mg Drive the Quaternary Structure of 2-Cys Prx to Larger Assemblies

The weight average molar mass of native 2-Cys Prx was 230 kDa in SLS experiments but the major species corresponded to a weight average molar mass of 46 kDa when estimated by gel filtration or non-reducing SDS-PAGE. In line with previous studies, these results confirmed that covalently linked homodimers of 2-Cys Prx have a tendency to associate non-covalently into decamers (27–29). Moreover, SLS experiments showed that the weight average molar mass of 2-Cys Prx increased from 230 kDa to 2.2 MDa upon the addition of 3 mm ATP/Mg, but it returned immediately to the former value when ATP was removed by gel filtration (not shown). Given the fast interconversion between 2-Cys Prx decamers and large oligomers, we turned to DLS for studying the self-polymerization in real time. In the presence of 3 mm Mg²⁺, the particle size distribution of 2-Cys Prx showed a homogeneous population centered at a D_H of 13 nm that remained invariable during the assay (Fig. 2A). This size was in line with the values obtained from structures of 2-Cys Prx decamers from different organisms (15, 28). When 4 mm ATP was added, the peak maximum shifted slowly to different species that approached a maximum of 40 nm in 30 min (Fig. 2B). This irregular time-dependent size distribution revealed that ATP/Mg induced the emergence of new oligomeric species from the monodisperse population of decamers. Two complementary experiments further indicated that the ATP/Mg-mediated oligomerization was an important feature of typical 2-Cys Prx. First, the E. coli homolog AhpC exhibited similar variations in the profile of D_H upon the addition of 4 mm ATP/Mg (not shown). Second, ATP/Mg did not modify the D_H of chloroplast Trx-m (4.2 nm), a monomeric protein that, like typical 2-Cys Prx, exhibits the Trx fold (supplemental Fig. S1) (30, 31). At this stage, we asked whether ATP/Mg drive 2-Cys Prx to the formation of irreversible or reversible oligomers in solution. The former process is often associated with modifications that denature proteins, whereas the latter is ascribed to self-associations that generally contribute to regulatory functions. The almost instantaneous dissociation of the large oligomers to initial decamers by the addition of EDTA demonstrated the reversibility of the process (Fig. 2C). Although we were able to trap oligomers that accumulate in solution when 2-Cys Prx was incubated with low concentrations of ATP, large supramolecular assemblies appeared beyond 4 mm (Fig. 2D). These novel species originated from the decameric form were far larger than most previous reports of soluble 2-Cys Prx oligomers (Fig. 2E is the enlarged boxed section of Fig. 2D).

Morphologic Analysis of 2-Cys Prx Assemblies

DLS experiments do not convey information on the shapes of particles. Therefore, TEM analyses were performed to assess the dependence of protein morphology on the binding of ATP/Mg. In the absence of ligands, proteins appear as ring-shaped structures with an average external diameter of 13 nm and an internal diameter of 6 nm (Fig. 3A), consistent with the decameric toroids observed in counterparts from other species (9, 32, 33). In contrast to these regular morphologies, treatment with 3 mm ATP/Mg (Fig. 3B) produced large ball-like particles with an average diameter of ∼30 nm. Most of the spheres exhibited irregular surfaces that contained small discernible substructures, suggesting a residual heterogeneity within each class. Overall, TEM corroborated that the binding of ATP/Mg triggered the formation of oligomers whose quaternary structures were markedly different from the ring-shaped forms observed in the absence of ATP.

Secondary Structure of 2-Cys Prx·ATP/Mg Complexes

To further unravel the structural changes that promote the modification of the quaternary structure, we relied on far-UV CD spectroscopy to study the action of ligands on the elements of secondary structure. Although the approach was conceptually straightforward, the high extinction coefficient of ATP in the far-UV region precluded the direct use of CD spectroscopy due to a high noise/signal ratio. If the non-covalent binding of ATP to 2-Cys Prx has an effect upon the structure, the immobilization of the nucleotide through covalent binding should mimic the conformational change. We therefore used the method of photoaffinity labeling, which enabled the covalent binding of the nucleotide to regions of the protein in close contact with the adenine ring (34). The exposure of 2-Cys Prx to UV light in the presence of both [8-¹⁴C]ATP and Mg²⁺ and subsequent analysis of the products by SDS-PAGE provided direct evidence for the formation of the photoadduct (supplemental Fig. S2, A and B). Next, we examined whether far-UV CD spectra of 2-Cys Prx were affected by the concentration of ATP during photolabeling. To this end, 2-Cys Prx was (i) incubated with 5 mm MgCl₂ and varying concentrations of ATP, (ii) exposed to UV light, and (iii) separated from the unbound nucleotide by dialysis (Fig. 4A). Fig. 4B shows that increasing concentrations of ATP during the photochemical reaction brought about two important changes in CD spectra: (i) a considerable decrease in the large positive ellipticity at 192 nm and (ii) a pronounced shift of negative minima at 222 and 208 nm to a unique minimum at 219 nm with a concurrent increase of ellipticity. The lack of an absolute convergence of the spectra onto an isodichroic point suggested that a simple two-state process did not account for changes in the secondary structure of 2-Cys Prx (35, 36). Analysis of the data showed that the proportion of α-helical structures fell from 33.2 to 10.0% and β-strands increased from 17.6 to 38.8%, whereas unordered and β-turn structures remained practically unchanged (Fig. 4B, inset). Taken together, data presented here are the first evidence of a physiological modulator, ATP, that interacts with 2-Cys Prx, inducing α-helix → β-sheet transitions, which, in turn, may lead to the self-polymerization of the protein.

Higher Concentrations of ATP/Mg Cause the Reversible Aggregation of 2-Cys Prx

Investigating the relationship between the quaternary structure of 2-Cys Prx and ATP/Mg binding, we unexpectedly found that 2-Cys Prx no longer remained soluble at concentrations of ATP over 5 mm. The presence of 5 mm ATP/Mg caused the immediate increase of light scattering to values proportional to 2-Cys Prx concentrations from 1 to 15 μm (Fig. 5A). Interestingly, no significant lag time preceded the formation of insoluble 2-Cys Prx that was completed in about 100 s. When the ligands were added separately, the insolubilization of 2-Cys Prx started upon the addition of the second ligand, indicating that the aggregation process was dependent on the simultaneous presence of both ATP and Mg²⁺ (supplemental Fig. S3A). Although ADP partially replaced ATP in this capacity, neither other nucleotides nor phosphate were functional (supplemental Fig. S3B). On the contrary, Mn²⁺, Ca²⁺, and Zn²⁺ were as efficient as Mg²⁺ in assisting ATP for the insolubilization of 2-Cys Prx (not shown).

We confirmed by SDS-PAGE the appearance of insoluble aggregates when the protein was incubated in solutions containing 5 mm ATP/Mg (Fig. 5B). Strikingly, these precipitates completely redissolved by adding EDTA (5 mm) or by dilution with 25 mm Tris-HCl buffer (pH 8) containing 5 mm MgCl₂. In line with a specific link between ATP/Mg and the structure of 2-Cys Prx, other chloroplast proteins (Trx-m and fructose-1,6-bisphosphatase) did not aggregate in the presence of ATP/Mg (supplemental Fig. S4) (18). It is evident from these results that the transition of 2-Cys Prx from soluble oligomers to insoluble aggregates is specifically induced by high concentrations of ATP/Mg (≥5 mm) and completely reversed by removal of either ligand.

The ATP/Mg-mediated Aggregation of 2-Cys Prx Is Prevented by the Induction of Non-native Helical Conformations

If the ATP/Mg-mediated insolubilization of 2-Cys Prx was dependent on the transition α-helix → β-sheet, the enhancement of more stable α-helix forms should favor the solubility of the protein. We thus explored initially the secondary structure of 2-Cys Prx by far-UV CD upon the addition of 2,2,2-trifluoroethanol (TFE), a cosolvent commonly used to induce α-helical conformations (37, 38). Both the small positive maximum at 192 nm and the negative minima at 208 and 222 nm indicated the presence of α-helices in the native protein (Fig. 6A). Increasing concentrations of TFE, however, enhanced the dichroic signals with a dramatic increment of the α-helical content (12.1% → 37.1%) at the expense of β-sheet structures (35.2% → 17.2%) beyond 5% (v/v) TFE (Fig. 6A, inset). Subsequently, we investigated the aggregation of 2-Cys Prx by light scattering when ATP/Mg were added into solutions containing different concentrations of TFE. As predicted, concentrations of TFE beyond 5% (v/v) inhibited the aggregation of 2-Cys Prx (Fig. 6B). These data were in line with the view that ATP/Mg promoted the formation of β-sheet structures and, in so doing, predisposed 2-Cys Prx to the relatively fast and reversible process of oligomerization and subsequent aggregation.

SPR Analysis of ATP/Mg-mediated Aggregation of 2-Cys Prx

We next implemented a new application of SPR to assess the kinetics of 2-Cys Prx aggregation. Monitoring the association of interacting molecules through SPR relies upon variations of the refractive index close to the sensor surface when soluble ligands bind to the partner molecule immobilized on SPR chips (39, 40). Because the resonant condition is extremely sensitive to the refractive index of the medium adjacent to the metal film, adsorption of large polypeptides or conformational changes of the attached proteins can be accurately detected (41, 42). On this basis, we reasoned that the formation of large aggregates on the surface of unmodified chips should also provide an optical measurement of ligand-mediated protein precipitations, both reactants being soluble notwithstanding. As predicted, the technique of SPR was effective for estimating the reversible aggregation of 2-Cys Prx caused by ATP/Mg (Fig. 7A). The sensorgram exhibited slight increases upon the addition of 5 mm Mg²⁺ but rose significantly after the incorporation of 6 mm ATP, resulting in a nearly 7-fold increase of SPR angle shift. The removal of Mg²⁺ by the addition of 10 mm EDTA diminished the SPR response. The dynamic data collected by the SPR sensor were further employed for the estimation of kinetic binding parameters. Using the linear regression of the pseudo-first-order rate constant (k_on = k_a. [ATP] − k_d) versus the respective concentration of ATP, we derived the apparent association (k_a = 0.94 ± 0.05 m⁻¹ s⁻¹) and dissociation (k_d = (2.3 ± 0.4) × 10⁻³ s⁻¹) rate constants for the reversible aggregation of 2-Cys Prx (Fig. 7B). The apparent equilibrium dissociation constant (K_D) was calculated as K_D = k_d/k_a. Notably, this K_D (2.4 ± 0.1 mm) approached the previous second constant of ATP-mediated inhibition of the peroxidase activity (I_0.5 = 1.4 mm) (7). In summary, the dynamic monitoring of 2-Cys Prx aggregation by the novel use of SPR sensors not only facilitated the estimation of association and dissociation rates but also avoided interference frequently associated with spectrophotometry or fluorescence techniques, such as absorbance and quenching. However, our approach was not suitable to envision conformational changes, as previously suggested for dihydrofolate reductase (41) and transglutaminase (42), because 2-Cys Prx was not bound to the sensor surface.

Site-directed Mutants Reveal the Structural Independence of 2-Cys Prx Functions

The results described here provided the first evidence that two physiological ligands devoid of redox activity, ATP and Mg²⁺, promote the self-polymerization of 2-Cys Prx, an additional feature to the specific regulation of peroxidase and chaperone activities (7). Earlier studies have revealed the necessary participation of two conserved arginines, Arg¹²⁹ and Arg¹⁵², in the peroxidase activity, but detailed information on the chaperone activity and the ATP/Mg-dependent oligomerization/aggregation was missing (43–47). Therefore, we set out to find whether mutants whose positively charged residues were replaced by the neutral alanine were functional in the latter functions. As expected, the substitution of Arg¹²⁹ and Arg¹⁵² by alanine abrogated the ability of the active site to reduce H₂O₂ (Fig. 8). We next assayed the capacity of 2-Cys Prx variants to prevent the insolubilization of the β-chain upon the reductive cleavage of insulin. Surprisingly, this activity was greatly enhanced and almost abolished when the alanine residue replaced Arg¹²⁹ and Arg¹⁵², respectively. These contrasting effects unexpectedly revealed that the chaperone activity of 2-Cys Prx was not only independent of a functional peroxidatic site but also highly dependent on the position of amino acid residues crucial for the redox activity. When we next compared the responses of the wild type and mutant forms to ATP/Mg, the positively charged residues also appeared as important for the stabilization of the aggregates. However, at variance with the peroxidase and chaperone activities, the substitution of Arg¹²⁹ reduced the propensity of 2-Cys Prx to aggregate in the presence of ATP/Mg, whereas the variant at Arg¹⁵² was mostly equivalent to the wild type form. Interestingly, neither the mutants nor the wild type counterpart exhibited an appreciable lag phase upon the addition of ATP/Mg. Overall, the site-directed mutagenesis of Arg¹²⁹ and Arg¹⁵² confirmed earlier work concerning the peroxidase activity but revealed that the manifold functions of 2-Cys Prx are not strictly linked and, thus, can be uncoupled.