Crystallization and preliminary X-ray diffraction analysis of mouse prostaglandin F_2α synthase, AKR1B3

Aldo-keto reductase 1B3 (AKR1B3) produced in Escherichia coli has been crystallized in complex with NADPH by the sitting-drop vapour-diffusion method.

Abstract

Aldo-keto reductase 1B3 (AKR1B3) catalyzes the NADPH-dependent reduction of prostaglandin H₂ (PGH₂), which is a common intermediate of various prostanoids, to form PGF_2α. AKR1B3 also reduces PGH₂ to PGD₂ in the absence of NADPH. AKR1B3 produced in Escherichia coli was crystallized in complex with NADPH by the sitting-drop vapour-diffusion method. The crystal was tetragonal, belonging to space group P4₁2₁2 or P4₃2₁2, with unit-cell parameters a = b = 107.62, c = 120.76 Å. X-ray diffraction data were collected to 2.4 Å resolution at 100 K using a synchrotron-radiation source.

1. Introduction

Aldo-keto reductases (AKRs) are soluble monomeric proteins with a molecular mass of 37 kDa and NADPH-dependent oxidoreductase activity. AKR proteins are widely distributed in prokaryotes and eukaryotes, fall into 15 families (Penning & Drury, 2007 ▶) and metabolize a number of substrates, including aldehydes, monosaccharides, steroids, polycyclic hydrocarbons, isoflavonoids and prostaglandins (PGs), in the presence of NADPH (Jez et al., 1997 ▶). Aldose reductase (EC 1.1.1.21), named AKR1B1 in humans and AKR1B3 in mice, is considered to be the prototypical enzyme of the AKR superfamily. In addition to these canonical aldose reductases, a second group named aldose reductase-like proteins (ARLPs) have been characterized on the basis of sequence homology (at least 60–70% identity to aldose reductase). AKR1B7, initially characterized as a mouse vas deferens androgen-dependent protein, belongs to the ARLP group. X-ray crystallographic structures of members of the AKR superfamily have shown that these enzymes have a common three-dimensional fold known as the (α/β)₈-barrel fold, e.g. human AKR1B1 (Wilson et al., 1992 ▶), human AKR1B10 (Gallego et al., 2007 ▶), rat AKR1B14 (Sundaram et al., 2011 ▶), human AKR1C2 (Jin et al., 2001 ▶; Nahoum et al., 2001 ▶), human AKR1C3 (Lovering et al., 2004 ▶), rat AKR1C9 (Hoog et al., 1994 ▶; Bennett et al., 1996 ▶, 1997 ▶) and Trypanosoma brucei AKR5A2 (Kilunga et al., 2005 ▶). The nucleotide cofactor binds in an extended conformation at the top of the α/β-barrel, with the nicotin­amide ring projecting down towards the centre of the barrel and pyrophosphate straddling the barrel lip (Petrash, 2004 ▶). Kubiseski et al. (1992 ▶) have established that the enzyme follows a sequential ordered mechanism in which NADPH binds before the aldehyde substrate and NADP⁺ is released after the alcohol product is formed. When the first complete crystal structure of human AKR1B1 was solved in 1992, the conserved Tyr48 was shown to fulfil the role of a catalytic acid for NADPH-dependent reduction (Wilson et al., 1992 ▶).

Recently, we reported that human AKR1B1, mouse AKR1B3 and mouse AKR1B7 are associated with PGF_2α synthase (PGFS; EC 1.1.1.188) activity, which catalyzes the reduction of PGH₂, a common intermediate of various prostanoids, to PGF_2α (Kabututu et al., 2009 ▶). Moreover, we found that AKR1B1 and AKR1B3, but not AKR1B7 and AKR1C3, also catalyze the isomerization of PGH₂ to PGD₂ in the absence of NADPH. Although both AKR1B1 and AKR1B3 show similar pH–rate profiles for PGFS and PGD₂ synthase (PGDS) activities, the PGDS activities of AKR1B1 and AKR1B3 were found to be 89 and 24% when compared with their PGFS activities (Nagata et al., 2011 ▶). In order to further reveal the structure–function relationship of these isozymes, the three-dimensional structure of AKR1B3 and its comparison with that of AKR1B1 are required. Here, we report the crystallization and preliminary X-ray diffraction study of AKR1B3 in complex with NADPH.

2. Experimental

2.1. Protein expression and purification

Recombinant AKR1B3 was expressed in Escherichia coli BL21 Star (DE3) (Novagen) as described previously (Nagata et al., 2011 ▶). The E. coli cells were disrupted by sonication in buffer A (50 mM NaH₂PO₄ pH 8.0, 300 mM NaCl, 10 mM imidazole) containing a protease-inhibitor cocktail (Roche). The sample was applied onto Ni–NTA agarose resin (Qiagen) and eluted with 250 mM imidazole in buffer A. The sample was treated with thrombin (GE Healthcare) at 293 K for 14 h and then applied onto a HisTrap HP column (GE Healthcare) equilibrated with buffer A. The flowthrough fraction containing AKR1B3 without the His tag was pooled and applied onto a HiLoad 16/60 Superdex 75 column (GE Healthcare), which was developed with buffer B (20 mM Tris–HCl pH 8.0, 150 mM NaCl). The purified protein was concentrated to 30 mg ml⁻¹ using a Vivaspin (Sartorius) and stored at 193 K until the crystallization experiments. Up to 7.3 mg purified AKR1B3 was obtained from 1 l of E. coli cell culture.

2.2. Crystallization of AKR1B3

Initial crystallization screening of AKR1B3 in the presence of 1 mM NADPH was performed by the sitting-drop vapour-diffusion method on an Intelli-Plate 96 (Art Robbins Instruments). Needle-shaped crystals appeared in PEG/Ion Screen condition No. 48 [0.2 M ammonium citrate dibasic pH 5.1, 20% polyethylene glycol (PEG) 3350; Hampton Research] within 1 d. After optimizing the conditions in 24-well sitting-drop plates (Hampton Research), rod-shaped crystals with maximum dimensions of 0.25 × 0.25 × 1.25 mm appeared after one week at 293 K (Fig. 1 ▶). The drops consisted of 1 µl protein solution (15 mg ml⁻¹ AKR1B3, 2 mM NADPH, 20 mM Tris–HCl pH 8.0, 150 mM NaCl) and 1 µl reservoir solution (0.2 M ammonium citrate dibasic, 22.7% PEG 3350). 500 µl reservoir solution was added to the deep well of the 24-well sitting-drop plates.

Figure 1.

Crystals of AKR1B3. The crystals were obtained in a sitting drop.

3. Data collection and processing

X-ray diffraction data were measured on beamline BL44XU at SPring-8 (Harima, Japan). Prior to data collection, the crystal of AKR1B3 was soaked in cryoprotectant solution (0.2 M ammonium citrate dibasic, 25.5% PEG 3350, 1 mM NADPH, 10% 2-methyl-2,4-­pentanediol), fished out with a standard nylon loop and flash-cooled in a nitrogen-gas stream at 100 K. The diffraction patterns were recorded on a MX225HE CCD detector (Rayonix). The wavelength, crystal-to-detector distance, crystal oscillation angle per image and beam-exposure time were set to 0.9 Å, 300.0 mm, 0.5° and 5 s, respectively (Fig. 2 ▶). A complete data set was collected from 360 images covering 120° in total.

Figure 2.

X-ray diffraction image from an AKR1B3 crystal. The frame edge in the enlargement is 2.4 Å.

The data set was processed using the HKL-2000 program suite (Otwinowski & Minor, 1997 ▶). The crystal of AKR1B3 was tetragonal, belonging to space group P4₁2₁2 or P4₃2₁2, with unit-cell parameters a = b = 107.62, c = 120.76 Å. From the 1 214 259 accepted observations to 2.4 Å resolution, 28 017 unique reflections were obtained. Assuming the presence of two monomers of AKR1B3 in the asymmetric unit, the crystal volume per enzyme mass (V _M) and the solvent content were calculated to be 2.45 ų Da⁻¹ and 50%, respectively. A summary of the data statistics is presented in Table 1 ▶.

Table 1. Crystal parameters and X-ray diffraction data-collection statistics.

Values in parentheses are for the highest resolution shell.

Crystal system	Tetragonal
Space group	P41212 or P43212
Unit-cell parameters (Å)	a = b = 107.62, c = 120.76
Resolution range (Å)	50–2.4 (2.49–2.40)
No. of molecules in the asymmetric unit	2
VM (Å3 Da−1)	2.45
Vsolv (%)	50
No. of measured reflections	1214259
No. of unique reflections	28017
Rmerge† (%)	8.7 (22.7)
Completeness (%)	99.0 (95.7)
Average 〈I/σ(I)〉	24.5 (4.7)

^† R _merge = , where I _i(hkl) is the value of the ith measurement of the intensity of a reflection, 〈I(hkl)〉 is the mean value of the intensity of that reflection and the summation is over all measurements.

Molecular-replacement calculations using the program MOLREP (Vagin & Teplyakov, 2010 ▶) from the CCP4 program package (Winn et al., 2011 ▶) with human placenta aldose reductase (PDB entry 1ads; Wilson et al., 1992 ▶) as a search model and refinement of the structure are currently under way. The solved atomic structure should provide insights into the differences in PGDS activity between AKR1B1 and AKR1B3.