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Logo of jsyncradthis articlesearchopen accesssubscribesubmitJournal of Synchrotron RadiationJournal of Synchrotron Radiation
J Synchrotron Radiat. 2011 January 1; 18(Pt 1): 88–91.
Published online 2010 November 5. doi:  10.1107/S0909049510037076
PMCID: PMC3004263

Improvement in the quality of hematopoietic prostaglandin D synthase crystals in a microgravity environment


Human hematopoietic prostaglandin synthase, one of the better therapeutic target enzymes for allergy and inflammation, was crystallized with 22 inhibitors and in three inhibitor-free conditions in microgravity. Most of the space-grown crystals showed better X-ray diffraction patterns than the terrestrially grown ones, indicating the advantage of a microgravity environment on protein crystallization, especially in the case of this protein.

Keywords: prostaglandin D synthase, hematopoietic, H-PGDS, crystal, microgravity, space experiment, anti-inflammation, anti-allergy, JAXA, counter-diffusion

1. Introduction

Hematopoietic prostaglandin (PG) D synthase (H-PGDS; EC is a clinically important protein that is widely distributed in hematopoietic systems, and produces PGD2 from cyclooxygenase-derived PGH2 (Kanaoka & Urade, 2003 [triangle]). PGD2 mediates allergic and inflammatory reactions through two distinct types of receptors: the D-type prostanoid receptor (DP) and the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). Although indomethacin and aspirin are known to suppress allergic and inflammatory reactions, they inhibit the production of all PGs including cytoprotective and anti-inflammatory PGs (Takeuchi et al., 2001 [triangle]; Halter et al., 2001 [triangle]). Therefore, selective inhibitors of H-PGDS are considered to be more useful candidates for anti-allergic and anti-inflammatory drugs as they suppress only the signals mediated by DP and CRTH2.

H-PGDS has been crystallized in a microgravity environment in Japan Aerospace Exploration Agency (JAXA)’s space experiments several times since 1997 as summarized in Table 1 [triangle]. Although it was not easy to obtain good crystals in earlier space experiments, high-quality crystals were obtained after JAXA improved crystallization technology and increased the accessibility of space experiments during a course of high-quality protein crystallization experiments on the International Space Station (ISS) starting in 2003 (Sato et al., 2006 [triangle]). Recently, Takahashi et al. (2010 [triangle]) reported that crystals of H-PGDS in complexes with novel inhibitors, which were grown in microgravity in 2007, diffracted up to a 1.1 Å resolution at 100 K using SPring-8 synchrotron radiation, one of the highest resolutions obtained to date for this protein. These novel inhibitors were derivatives of 4-benzhydryloxy-1-[3-(1H-tetrazol-5-yl)-propyl]-piperidine (HQL-79) which is an orally active anti-allergic drug and specifically inhibits the biological activity of H-PGDS (Aritake et al., 2006 [triangle]). Moreover, the novel inhibitors had more than 100 times lower inhibitory concentrations (IC50) than HQL-79, so they are expected to be candidates for novel drug design.

Table 1
H-PGDS crystallization experiments in space conducted by JAXA

In this report we show more crystallization results of H-PGDS in four recent space experiments performed from 2007 to 2009, and indicate that microgravity positively affects the solution environment improving the quality of the crystals of this protein.

2. Materials and methods

2.1. Microgravity experiments

H-PGDS was crystallized at 293 K inside the Russian TBU incubator on board the Russian Service Module on the ISS for 12, 11 and 10 weeks in JAXA-NGCF#1, #2 and #3, respectively, and inside the Protein Crystallization Research Facility on board the Japanese Experiment Module ‘Kibo’ on the ISS for 12 weeks in JAXA PCG#1 as shown in Table 1 [triangle].

2.2. Protein expression, purification and crystallization

Human H-PGDS was expressed and purified as previously described (Aritake et al., 2006 [triangle]; Takahashi et al., 2010 [triangle]). The crystallization conditions were as follows: a protein solution contained 4.0 mg ml−1 protein with or without 0.5 mM inhibitor in 150 mM sodium chloride, 15% PEG 6000, 5 mM dithiothreitol, 5 mM glutathione, 1% dioxane, 0.5 mM magnesium chloride and 20 mM Tris-HCl pH 8.0, and a precipitant solution contained 30% PEG 6000, 10 mM dithiothreitol, 10 mM glutathione, 1% dioxane and 1 mM magnesium chloride in 50 mM Tris-HCl pH 8.4. For other inhibitor-free conditions the divalent metal ion, magnesium chloride, was replaced by calcium chloride or a chelating agent EDTA.

For crystallization, JAXA adopted a gel-tube method for microgravity experiments, which modified the original counter-diffusion method introduced by García-Ruiz & Moreno (1994 [triangle]) (García-Ruiz, 2003 [triangle]; Ng et al., 2003 [triangle]; Tanaka et al., 2004a [triangle]; Gonzalez-Ramirez et al., 2008 [triangle]; Otálora et al., 2009 [triangle]). Assembly of a crystallization device and a micro-seeding technique were described previously (Takahashi et al., 2010 [triangle]). Briefly, 8 µl of the protein solution was put in each glass capillary (47 mm length, 0.5 mm diameter) to a length of 40 mm. A 6 mm piece of plastic tubing was attached to the end of the capillary, which contained 1% polymerized agarose gel pre-soaked in 15% PEG 6000, 10 mM dithiothreitol, 10 mM glutathione, 2% dioxane, 1 mM magnesium chloride (calcium chloride or EDTA) and 50 mM Tris-HCl pH 8.4. The top of the capillary was sealed with clay and epoxy adhesive. The agarose end of the capillary was placed in a tube which contained the precipitant solution. Each protein sample was loaded into three capillaries. A total of 22 kinds of inhibitors of H-PGDS were co-crystallized. The formulae of the inhibitors are proprietary information. Crystallization conditions were fixed to start crystallizing after the samples arrived in the microgravity environment. The same crystallization condition was applied to the terrestrial experiment as the control.

2.3. Data collection and analysis

Diffraction data were collected from a single crystal at 100 K using an X-ray wavelength of 0.85 Å on the BL41XU beamline at SPring-8, Harima, Japan, with an ADSC315 detector system, or using an X-ray wavelength of 1.0 Å on the X06SA beamline at the Paul Scherrer Institute (PSI), Villigen-PSI, Switzerland, with a MAR225 detector system. The methods of crystal extraction from capillaries and harvesting crystals were previously described (Tanaka et al., 2004a [triangle], 2007 [triangle]). Prior to data collection, a crystal was scooped with a nylon loop, briefly soaked in an artificial mother liquor supplemented with 15% glycerol as a cryoprotectant, and plunged into a nitrogen-gas stream at 100 K. The diffraction images collected at SPring-8 were integrated and scaled using the programs DENZO and SCALEPACK from HKL2000 (Otwinowski & Minor, 1997 [triangle]), and the images collected at PSI using XDS (Kabsch, 1993 [triangle]). X-ray diffraction data for each sample were obtained from two to three crystals with approximate dimensions of 0.03 × 0.1 × 0.05 mm. A summary of the best data is listed in Table 2 [triangle]. Data sets were collected up to the resolution range I/σ(I) > 2 and R merge < 50%.

Table 2
Summary of the X-ray diffraction experiments of H-PGDS crystals

3. Results and discussion

3.1. Crystallization and X-ray diffraction analysis

The sizes of the crystals grown in space were about the same or sometimes significantly larger than those grown on the ground and several times larger than those grown with the vapour-diffusion method. All the crystals chosen for diffraction data collection were grown in the upper part of the capillaries where crystals of better quality tend to grow (Lopez-Jaramillo et al., 2003 [triangle]), and those were of good quality as judged by a visual inspection.

As shown in Table 2 [triangle], H-PGDS was crystallized under 25 different conditions in total, both in space and on the ground: 22 with an inhibitor and three without an inhibitor but with a different divalent metal ion (Mg2+: sample ID S/G6; Ca2+: sample ID S/G7) or a chelating agent (EDTA: sample ID S/G8).

Comparisons of the quality of space-grown and ground-grown crystals are invalid if X-ray diffraction experiments are not performed or if the same beamline is not used for both space-grown and ground-grown crystals of the same sample. Therefore, we must exclude S/G7, S/G12, S/G13 and S/G15–19 because X-ray diffraction experiments were, unfortunately, not performed owing to the unavailability of beam time, and S/G2 because X-ray diffraction data were collected at SLS beamline X06SA for only the space-grown crystals for this condition.

In the case of S/G20–22, X-ray diffraction experiments were not performed both for space-grown and ground-grown crystals owing to the poor shape of the crystals. In the case of S/G11, no crystallization was observed both for space-grown and ground-grown crystals.

Therefore, there are 12 samples that were valid for comparison, as shown in bold face in Table 2 [triangle]. Among these samples, S/G24 and S/G25 showed improvement of crystallizability in space-grown crystals, which was indicated by the size and the shape of the crystals, respectively. The microgravity environment improved the maximum resolution and mosaicity in S/G1, S/G3, S/G4, S/G6, S/G8–10 and S/G14. In the case of S/G5, the microgravity environment improved the maximum resolution but not the mosaicity. For S/G23, the microgravity environment did not improve the maximum resolution but did improve mosaicity.

3.2. Microgravity effects

The microgravity environment definitely had a positive effect on the growth of high-quality H-PGDS crystals as shown by the better results of space-grown crystals of 12 comparable samples. When comparing two to three crystals of each sample, a superior quality improvement of space-grown crystals was mostly observed, although there were some deviations in the X-ray diffraction data quality of crystals grown both in space and on the ground. The absence of convective fluid motion in microgravity is known to decrease protein concentration around the growing crystal, forming a protein depletion zone (PDZ) (Otálora et al., 2001 [triangle]). The slower growth of crystals in the PDZ is thought to be preferable for a better-ordered intermolecular arrangement in a crystal, which may be one of the reasons for the improved quality of space-grown crystals. Impurity incorporation also affects the intermolecular order (Dold et al., 2006 [triangle]). An impurity depletion zone (IDZ) can also be expected to form around the growing crystal (Chernov, 1998 [triangle]), which may reduce the incorporation of impurity molecules into the crystals in space (Thomas & Chernov, 2001 [triangle]).

Tanaka et al. (2004b [triangle]) previously reported that D/β (diffusive coefficient of a protein molecule/kinetic constant for crystal growth), which represents the ratio between ‘the molecule transportation to a surface of a crystal’ versus ‘its uptake into a crystal’ (Chernov, 1998 [triangle]), can be used as an index of both PDZ and IDZ. In short, if D/β is less than the size of the growing crystal it means that depletion zones would be formed around the crystal in a diffusion-controlled environment. Since the measurement of D and β is not easy to calculate for most of the proteins, we applied a simplified method of estimating those values of H-PGDS, using the molecular weight of the protein and the precipitant, the concentration of the crystallization solution, the time to grow to half of the final crystal size, and the concentration and the solubility of the protein (Tanaka et al., 2004b [triangle]). We calculated that D/β of H-PGDS is 0.39 mm which is almost the comparative order to the crystal size. This may indicate that the H-PGDS crystals obtained in the space experiments were grown surrounded by a PDZ and IDZ, which may result in growing high-quality crystals in microgravity. Although the reason for the improvement of crystal quality in microgravity still remains a matter of speculation, H-PGDS is likely to be a better quality protein crystallized in microgravity. Using these high-quality X-ray diffraction data sets, a design for novel drug candidates is now underway.


This work is supported by the Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO). This study was funded in part through the High-Quality Protein Crystal Growth Experiment on JEM promoted by JAXA. We are grateful to the Federal Space Agency and RSC Energia for the use of the Russian Service Module and the Russian space crafts Progress and Soyuz for space transportation; to the European Space Agency (ESA) and the University of Granada for space crystallization technology; to the Japan Synchrotron Radiation Research Institute (JASRI) for access to and user support at the synchrotron facilities of BL41XU at SPring-8, Harima, Japan; and to the Paul Scherrer Institut (PSI) for MIX support for the synchrotron data collection at X065A of Swiss Light Source (SLS), Villigen, Switzerland.


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