Purification, characterization and crystallization of pyrroline-5-carboxylate reductase from the hyperthermophilic archeon Sulfolobus Solfataricus

Protein Expression and Purification 64 (2009) 125–130

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Purification, characterization and crystallization of pyrroline-5-carboxylate reductase from the hyperthermophilic archeon Sulfolobus Solfataricus Zhaohui Meng a,b,c,*, Zhe Liu b, Zhiyong Lou b, Xiaocui Gong c, Yi Cao c, Mark Bartlam d, Keqin Zhang c, Zihe Rao b,d a

Laboratory of Molecular Cardiology, Department of Cardiology, The First Affiliated Hospital of Kunming Medical College, 295 Xichang Road, Kunming, Yunnan 650032, China Laboratory of Structural Biology, Tsinghua University, Beijing 100084, China c Laboratory for Conservation and Utilization of Bio-resources, Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University, Kunming 650091, China d Tianjin State Laboratory of Protein Science, College of Life Sciences, Nankai University, Tianjin 300071, China b

a r t i c l e

i n f o

Article history: Received 1 July 2008 and in revised form 6 October 2008 Available online 7 November 2008 Keywords: P5CR Extremophile Crystallography Sulfolobus solfataricus

a b s t r a c t The gene SSO0495 (proC), which encodes pyrroline-5-carboxylate reductase (P5CR) from the thermoacidophilic archeon Sulfolobus solfataricus P2 (Ss-P5CR), was cloned and expressed. The purified recombinant enzyme catalyzes the thioproline dehydrogenase with concomitant oxidation of NAD(P)H to NAD(P)+. This archeal enzyme has an optimal alkaline pH in this reversible reaction and is thermostable with a half-life of approximately 30 min at 80 °C. At pH 9.0, the reverse activation rate is nearly 3-fold higher than at pH 7.0. The homopolymer was characterized by cross-linking and size exclusion gel filtration chromatography. Ss-P5CR was crystallized by the hanging-drop vapor-diffusion method at 37 °C. Diffraction data were obtained to a resolution of 3.5 Å and were suitable for X-ray structure determination. Ó 2008 Elsevier Inc. All rights reserved.

Pyrroline-5-carboxylate reductases (P5CRs, EC 1.5.1.2)1 are a growing superfamily including approximately 400 enzymes. They are currently found in every domain of life, including eukarya, bacteria and archea [1]. In almost all cases, they catalyze the final step in proline synthesis, converting D1-pyrroline-5-carboxylate (P5C) to proline via a NAD(P)H-dependent reaction [2]. Because P5CR plays more than just a housekeeping role in proline synthesis [3,4], this universal enzyme has received a great deal of attention of late. A number of studies have characterized the enzymes from Escherichia coli [5], rat tissues [6], human fibroblasts and erythrocytes [3,4] and spinach leaves [7]. Their physiological functions vary widely and include regulation of the intercellular redox potential [8,9], osmoregulation [10,11], enhancing nucleotide synthesis and energy production [3,4], and regulation of apoptosis [12–14]. More divergent roles have also been found in the metabolism of some proline analogs [1,5]. The P5CRs are relatively well-studied biochemically and the kinetic properties and regulation of P5CR are distinctly different, both in various cell types and in various growth phases in the same cell [7,15,16]. There is a general preference for NADH or * Corresponding author. Address: Laboratory of Molecular Cardiology, Department of Cardiology, The First Affiliated Hospital of Kunming Medical College, 295 Xichang Road, Kunming, Yunnan 650032, China. Fax: +86 871 5356221. E-mail address: [email protected] (Z. Meng). 1 Abbreviations used: P5CRs, pyrroline-5-carboxylate reductases; P5C, D1-pyrroline-5-carboxylate; AS, ammonium sulfate; EGS, ethylene glycolbis. 1046-5928/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2008.10.018

NADPH, as well as different inhibitor sensitivities to proline, NAD(P)+ and ATP from various sources. P5CRs are structurally fascinating because of their quaternary structural organization. The enzymes are reported to be a tetramer in yeast [17], an octamer in rat lens [18], a dimer in Neisseria meningitides [1], and a decamer in both Streptococcus pyogenes and humans [1,19]. Recently, three crystal structures of P5CRs have been reported [1,19]. Although they share low amino acid sequence identity (30%), the overall topology of these orthologs is similar. In particular, their monomer structure, dimer architecture and catalytic mechanism are in agreement with earlier biochemical studies. Interestingly, few of the residues in P5CR enzymes that are responsible for substrate recognition are structurally conserved, suggesting an additional catalytically competent structural arrangement might exist. To further investigate the evolutionary changes of this housekeeping protein from bacteria and mammals, P5CR from archeon Sulfolobus solfataricus (Ss-P5CR) was selected for crystallographic study to provide additional insights into the catalytic mechanism of P5CR enzymes. To date, no information is available for P5CR isolated from archea. Ss-P5CR consists of 277 amino acid residues, has a molecular weight of 30.5 kDa per subunit, and shares significant sequence identity (22–30%) with its counterparts from eukarya and bacteria. Here we report the purification, characterization and crystallization of wild-type SsP5CR.

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Materials and methods Materials Sulfolobus solfataricus genomic DNA was a gift from Dr. Huang Li (Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China). Ultrafree 10,000 NMWL filter units were purchased from Millipore (Bedford, MA). pET-28a(+) vector was obtained from Novagen (Madison, WI). RNaseA was purchased from Sigma–Aldrich (St. Louis, MO) and DNaseI was purchased from Takara (Dalian). Crystal screen kits (Crystal Screen I, II and PEG/ Ion kits) were obtained from Hampton Research (Riverside, CA). Protein expression and purification The proC gene (0.8 kb) encoding Ss-P5CR was amplified from S. solfataricus genomic DNA (a gift from Huang L., Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China) using the polymerase chain reaction (PCR) method. Two PCR primers, 50 GAAAGAATTCATGGAAGATTTAACTATTGGAATA-30 and 50 -GAAA CTCGAGTTATTTGCTATTATTCCTAATA-30 were designed. The PCR products were digested with EcoRI and XhoI, and ligated into EcoRI and XhoI restriction sites of the pET-28a(+) vector (Novagen Inc.) with a 6 His tag at the N-terminus. E. coli DH5a competent cells were transformed with the ligation mix and positive clones with an insert of the correct size were selected and confirmed by DNA sequencing. E. coli BL21 (DE3) cells were transformed by the recombinant plasmid and the transformants were selected on LB agar plates containing 100 lg/ml kanamycin. The cells were then cultured at 37 °C in LB medium containing 100 lg/ml kanamycin. When the culture density reached A600 = 0.5, the culture was induced with 1 mM IPTG and grown overnight before the cells were harvested. Cell lysis, heating and ammonium sulfate (AS) precipitation The bacterial cell pellet was resuspended in lysis buffer (Bicine 20 mM; NaCl 0.5 M; pH 9.0) and homogenized by sonication. The lysate was centrifuged at 15,000 rpm for 30 min to remove the cell debris. The protein pool was heat treated in a water bath at 70 °C for 20 min, and denatured proteins were removed by centrifugation. Next, 20% ammonium sulfate (AS) was added to the solution while stirring at 4 °C. The precipitate formed between 0% and 20% AS was collected. The protein was suspended in 1 ml lysis buffer, and incubated with 1 mg/ml RNaseA (Sigma) and 15 U/ml DNaseI (Takara) at room temperature overnight to remove nucleic acid. Chromatographic purification After further centrifugation at 15,000 rpm for 30 min to remove the precipitate, the sample was injected into a Superdex-200 10/ 300 column (Pharmacia) in running buffer (lysis buffer). The sample eluted isocratically at 10–13 ml running buffer volume was diluted to 100 mM NaCl in 20 mM Bicine, pH 9.0, and applied through a 10 ml superloop (Pharmacia) onto a Resource Q column (Pharmacia). The column was washed with 20 mM Bicine, pH 9.0, before being eluted in an increasing salt gradient from 0 to 1 M NaCl in 20 mM Bicine, pH 9.0. Ss-P5CR was concentrated with an Ultrafree 10,000 NMWL filter unit (Millipore) to 20 mg/ml and stored at 80 °C. Ss-P5CR enzymatic activity and thermostability assays According to the reported procedure [5], the thioproline dehydrogenase activity of Ss-P5CR were assessed spectrophotometri-

cally at 340 nm in 0.3 M Tris–Cl (pH 9.0, at which Ss-P5CR achieved the most steady activity), 0.1 lg/ll Ss-P5CR, 0.1–3 mM NAD(P)+, and 0.02–10 mM thioproline. Using the mM extinction coefficiency of NAD(P)H (6.22, 340 nm), the initial rates of product formation were calculated from the increase of absorbance per minute from the first 60 s of a 5 min recording period. For the thermal inactivation assay, the Ss-P5CR protein was first incubated in a water bath at a range of temperatures (20–95 °C) for 10 min, then the relative enzymatic activities of Ss-sP5CR were examined at 25 °C. Fine time-dependent thermal inactivation assays were performed at three temperatures (70, 80, 85 °C). The relative enzymatic activities of Ss-P5CR were measured at 25 °C. Reproducibility of all enzymatic assays was confirmed by taking each measurement at least two times and showed a fluctuation <10%. Samples with all components except Ss-P5CR served as negative controls. Cross-linking with EGS The Ss-P5CR protein was diluted to 6 mg/ml in 20 mM HEPES, pH 8.9, 100 mM NaCl. Ethylene glycolbis (EGS) (1 mM) in DMSO was added to 20 ll of protein sample with a final concentration of 100 and 400 lM. After the mixture was incubated at room temperature for 15 min, the reaction was quenched for 5 min by adding 1 M Tris–HCl (pH 7.5) to a final concentration of 100 mM. After loading the sample containing 4 ll SDS load buffer, the samples were run on SDS–polyacrylamide gels (8–12%). Crystallization and X-ray data collection The purified Ss-P5CR was concentrated to 10 mg/ml in 20 mM Bicine, pH 9.0, 0.5 M NaCl. The protein concentration was confirmed spectrophotometrically by the Bradford method. Crystallization was performed by the hanging-drop vapor-diffusion method at 37 °C. Hampton Research kits (Riverside, CA, USA) were used to supply sets of screening reagents for initial screening. To set up screening, 1 ll protein solution was mixed with 1 ll reservoir solution and the mixture was equilibrated against 400 ml reservoir solution at 37 °C. The best crystals were obtained in a 37 °C incubator by mixing 1 ll protein with 1 ll reservoir solution containing 0.6–1 M LiCl, 20–25% PEG3350. Initial cubic crystals appeared after 3–7 days. Crystals were immersed in cryoprotectant for 1 min, mounted into a nylon cryo loop and then flash-cooled in a stream of nitrogen gas cooled to 100 K. The cryoprotectant was prepared by adding 20% v/v glycerol to the mother-liquor reservoir. Preliminary diffraction data sets were collected on BL5A of the Photon Factory (Japan) with an ADSC Q315 CCD detector. All diffraction data were indexed, integrated and scaled using the HKL2000 package [20]. Results and discussion Ss-P5CR is expressed as a soluble protein Ss-P5CR was over-expressed in E. coli as a fusion protein with a 6 His and T7-tag at its N-terminus under the T7 promoter with IPTG induction. After being induced overnight, the expressed SsP5CR comprised 30% of the total protein and most of the expressed P5CR was soluble. High expression of Ss-P5CR does not affect cell growth. Soluble Ss-P5CR in the crude lysate was used for further purification, and Ss-P5CR was fractionated from the crude lysate by heating at 70 °C and 20% AS precipitation. The Ss-P5CR recovered was associated with small amounts of nucleic acid, and could be digested by incubation with RNaseA and DNaseI. Sequential systematic purification of P5CR using chromatographic methods, including size exclusion and anionic exchange, turned

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Z. Meng et al. / Protein Expression and Purification 64 (2009) 125–130 Table 1 Purification summary for Ss-P5CR. Purification step

Vol. (ml)

Protein (mg)

Total activity (U)a

Specific activity (U/mg)

Yield (%)

Soluble cell fraction Heated at 70 °C Ammonium sulfate Superdex-200 Resource Q

45 30 2.0 2.0 0.5

9 0.3 5.0 3.0 14

1,100 230 410 110 180

1.1 9.0 15 22 9.0

100 25 36 8.2 7.0

a

Units are defined as the amount of enzyme activity which will catalyze the transformation of 1 lM thioproline of the substrate per minute under standard conditions.

out to be efficient and convenient. Various purification steps are summarized in Table 1. Ss-P5CR forms a stable homopolymer and oligomer Affinity-purified protein showed a large peak at a very high molecular weight (>600 kDa) and a small peak at a high molecular weight (320 kDa, about ten times that of Ss-P5CR) from size exclusion chromatography (Fig. 1A). Both of them were determined to be target proteins by SDS–PAGE and Coomassie blue staining (Fig. 1B). Only the 320 kDa fraction protein was chosen for further purification with anionic exchange; the >600 kDa fraction had a low level of protein purity. Ss-P5CR eluted as a single peak with a high A280/A260 ratio of 1.8 at about 300 mM salt concentration and its purity was confirmed by SDS–PAGE. The 300 kDa Ss-P5CR protein was subsequently used for crystallization and enzyme activity assays. The cross-linking behavior of

Ss-P5CR supports the assembly mode of the supercomplex. At lower concentration of EGS (100 lM), the cross-linked dimer is the dominant product, while the homopolymer was also detected in cross-linking experiments at high EGS concentration (400 lM), (Fig. 1C). Taken together, these data demonstrate that under native conditions, Ss-P5CR self-associates to form large multimeric complexes. The most stable multimeric configuration appears to be a decamer, which can further self-associate to form higher order complexes. Homo-multimeric assembly is a feature of the housekeeping enzyme, as evidenced by X-ray crystallography structures [1,19] or gel filtration chromatography [17–19]. These interactions may be involved in rapid turnover of proteins in the cells [15]. Ss-P5CR implicated in dehydrogenation of thioproline The occurrence of a peak in the plot of initial velocities versus substrate concentrations (Fig. 2A) indicates that the kinetics of

Fig. 1. (A and B) Ss-P5CR (30.5 kDa) coded by proC gene and expressed in E. coli BL21 (DE3) appears to form a decamer and homopolymer, as determined by size exclusion chromatography and SDS–PAGE. Marker, molecular mass markers. Lane 1, before induction. Lane 2, after induction. Lane 3, supernatant. Lane 4, heating, Lane 5, ammonium sulfate precipitation. Lanes 6 and 7, peak 1 (>600 kDa) and peak 2 (320 kDa) from Superdex-200 after purification by Resource Q. (C) Cross-linking SDS–polyacrylamide gel of Ss-P5CR with EGS. The dimer (60 kDa) and homopolymers (600 kDa) were detected. Molecular weight markers are labeled at the side of each lane. EGS, ethylene glycolbis.

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Fig. 2. (A) Initial velocity (Vo) versus substrate concentration ([S]) plot for the thioproline dehydrogenase activities of Ss-P5CR. The activities were measured in reaction buffers described under Materials and methods with a fixed [NAD+] of 0.8 mM. (B and C) Thermal inactivation of Ss-P5CR. (B) After incubation at various temperatures (20– 90 °C) for 10 min, the relative activities of Ss-P5CR were measured at 25 °C. (C) The time dependent thermal inactivation of Ss-P5CR was assessed at 70, 80 and 85 °C. The relative activities were measured at 25 °C and plotted against time. (D) Ss-P5CR thioproline dehydrogenase activities were measured at various pH values (Tris–HCl 6.5–9.5) in 0.3 M Tris–HCl, 0.1 lg/ll Ss-P5CR, 2 mM NADP+, and 2 mM thioproline.

the thioproline dehydrogenase activity by Ss-P5CR deviate from classical Michaelis–Menten kinetics and are characterized by pronounced substrate inhibition. Substrate inhibition often occurs when two or more substrate molecules bind to different pockets of the active site at the same time and inhibit each other from catalysis [21]. Interestingly, this substrate inhibition phenomenon has not been observed in the kinetics of both human P5CR and S. pyogenes P5CR [1,19]. S. solfataricus is an aerobic crenarcheon that grows optimally at 80 °C [22]. To support growth at 80 °C, it is very important for Ss-P5CR to be thermostable. Fig. 2B and C show that the enzymatic activity of Ss-P5CR is not reduced after incubation

for 30 min at 70 °C. However, human P5CR is almost 95% inactivated after incubation for 15 min at 68 °C [19]. Thus, Ss-P5CR exhibits much greater thermostability than human P5CR. Another clear difference between human P5CR and Ss-P5CR is the strong preference of human P5CR (20-fold) for NAD+ (apparent KM = 0.151 ± 0.023) over NADP+ (apparent KM = 3.06 ± 0.042 mM) [19]. In contrast, Ss-P5CR has a 2-fold preference for NADP+ (apparent KM = 0.172 ± 0.010 mM) over NAD+ (apparent KM = 0.3896 ± 0.0355 mM). The biological significance and structural basis of these preferences are still largely unknown. We also found that Ss-P5CR works better at high pH in this reverse reaction (Fig. 2D). Actually, at pH 10, human P5CR can even use proline as a substrate (data not shown). The substrate we used (thioproline) does not exist in the natural habitat of S. solfataricus and, therefore, the results do not reflect the optimum pH for catalyzing the bona fide P5CR substrate, P5C. Crystallization and preliminary crystallographic characterization

Fig. 3. Crystals of Ss-P5CR. The larger crystal is about 500  20  20 lm in size.

To avoid auto-aggregation, crystallization experiments were established just after the incubation of Ss-P5CR and the reservoir solution at 37 °C for 15 min. Crystallization trials were conducted at 37 °C in 16-well plates using the hanging-drop, vapor-diffusion method. Initial crystals with poor diffraction quality were found in a solution containing 0.3 M LiCl, 20% PEG3350. Further, optimization was performed and better cubic crystals were obtained using 0.6–1 M LiCl, 25% PEG3350. Drops containing 1 ll protein solution and 1 ll reservoir solution were equilibrated against 400 ll reservoir solution (Fig. 3). Data from the native crystal were subsequently collected from this crystal (Fig. 4), which belongs to the I222 space group with unit-cell parameters a = 91.5 Å, b = 185.3 Å, c = 190.2 Å, a = b = c = 90°. Scaling and merging of the

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Fig. 4. A typical diffraction pattern collected from an Ss-P5CR crystal. The diffraction image was collected on a ADSC Q315 CCD detector. The detector edge corresponds to 3.5 Å resolution. The exposure time was 60 s, the crystal-to-detector distance was 120 mm and the oscillation range per frame was 1°.

Table 2 Data collection and refinement statistics.

References

Detector ADSC Q315 CCD 7606 native data set

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Data processing Space group Cell parameters (A, °) Wavelength (Å) Resolution range (Å) Total reflections Unique reflections Completeness (%) Rmerge (%)b I/r

I222 a = 91.5 Å, b = 185.3 Å, c = 190.2 Å, a = b = c = 90° 1.0000 50.0–3.5 (3.6–3.5)a 183,935 20,164 97.3 (80.9)a 14.8 (45.7)a 9.2 (3.3)a

a

Numbers in parentheses correspond to the highest resolution shell. P P P P Rmerge = h l|Iih|/ h I, where is the mean intensity of the observations Iih of reflection h. b

crystallographic data resulted in an overall Rmerge of 14.8% and an Rmerge in the highest resolution shell (3.6–3.5 Å) of 45.7%. We assume the presence of five molecules in one asymmetric unit, as defined by a self-rotation search, with an estimated solvent content of 65.6%. Data collection statistics from the crystals are summarized in Table 2. The selenomethionyl P5CR derivative protein and heavy-atom soaking derivations are currently underway to solve the structure. Acknowledgments The authors thank Dr. Huang Li, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China for kindly providing the proC gene. This work was funded by grants from Yunnan Province (2006C0071M, 2007C0001R), supported by the Seed Funding Scheme (2006bs08) from The First Affiliated Hospital of Kunming Medical College and the NSFC (grant No. 30860278).

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