Crystallization and preliminary X-ray crystallographic analysis of the NmrA-like DDB_G0286605 protein from the social amoeba Dictyostelium discoideum

In order to investigate its structure and function, the NmrA-like domain-containing DDB_G0286605 protein from D. discoideum was expressed, purified and crystallized. X-ray diffraction analysis is reported to a resolution of 1.64 Å.

Abstract

The DDB_G0286605 gene product from Dictyostelium discoideum, an NmrA-like protein that belongs to the short-chain dehydrogenase/reductase family, has been crystallized by the hanging-drop vapour-diffusion method at 295 K. A 1.64 Å resolution data set was collected using synchrotron radiation. The DDB_G0286605 protein crystals belonged to space group P2₁, with unit-cell parameters a = 67.598, b = 54.935, c = 84.219 Å, β = 109.620°. Assuming the presence of two molecules in the asymmetric unit, the solvent content was estimated to be about 43.25% with 99% probability. Molecular-replacement trials were attempted with three NmrA-like proteins, NmrA, HSCARG and QOR2, as search models, but failed. This may be a consequence of the low sequence identity between the DDB_G0286605 protein and the search models (DDB_G0286605 has a primary-sequence identity of 28, 32 and 19% to NmrA, HCARG and QOR2, respectively).

1. Introduction

The DDB_G0286605 protein from Dictyostelium discoideum is a protein of unknown function that consists of 302 amino acids. Primary-sequence and secondary-structure analyses suggest that the DDB_G0286605 protein contains an NmrA-like domain and belongs to the short-chain dehydrogenase/reductase (SDR) superfamily. The members of the SDR superfamily have only low pairwise sequence identity (typically 20–30%), but have homologous three-dimensional structures with a single-domain nucleotide-binding Rossmann fold (Kallberg et al., 2010 ▶).

In the Protein Data Bank, the Aspergillus nidulans NmrA and human HSCARG proteins show highest sequence homology to the DDB_G0286605 protein (Fig. 1 ▶). NmrA is a negative transcriptional regulator that is involved in post-translational modulation of the GATA-type transcription factor AreA, forming part of a system that controls nitrogen-metabolite repression in various fungi (Stammers et al., 2001 ▶). The ability of NmrA to discriminate between oxidized and reduced forms of dinucleotides may be linked to a possible role in redox sensing (Lamb et al., 2003 ▶).

Figure 1.

Amino-acid sequence alignment of DDB_G0286605 protein with HSCARG, NmrA and QOR2. The predicted secondary structures (Cole et al., 2008 ▶) of the DDB_G0286605 protein are indicated by filled arrows (β-sheets) and empty boxes (α-helices). Asterisks indicate the consensus sequence among the four proteins.

Human HSCARG has structural similarity to NmrA-like SDR proteins and forms an asymmetric dimer with one subunit bound to an NADP(H) molecule and the other unoccupied; the two subunits have dramatically different conformations (Zheng et al., 2007 ▶). HSCARG has a much higher affinity for NADPH than for NADP⁺ and a decrease in the NADPH/NADP⁺ ratio induces the redistribution of HSCARG within cells. These results indicate that HSCARG may serve as an NADPH sensor (Zheng et al., 2007 ▶; Zhao et al., 2008 ▶; Lamb et al., 2008 ▶). Even though these proteins have high structural homology, they can discriminate between types of di­nucleotides and redox states of dinucleotides, and show various conformational changes in response to dinucleotides (Lamb et al., 2008 ▶).

In D. discoideum, several genes have been annotated as NmrA-like proteins and one of them, PadA (DDB_G0286385 protein), has been reported to be an essential gene for Dictyostelium development. padA ⁻ cells show many defects, most notably in the specification of the prestalk A cell population and ammonia sensitivity, and consequently show abnormal growth and development. The predicted three-dimensional structure of PadA is most similar to that of NmrA, but there is no other evidence that PadA acts as a transcription regulator or an NAD(P)(H)-sensing protein (Núñez-Corcuera et al., 2008 ▶). To investigate the structural and functional role of the NmrA-­like DDB_G0286605 protein from D. discoideum, we report its overexpression, crystallization and preliminary X-ray crystallo­graphic analysis as a first step towards structure determination.

2. Materials and methods

2.1. Expression and purification of DDB_G0286605 protein

The DDB_G0286605 gene was amplified by polymerase chain reaction (PCR) using D. discoideum cDNA, which was amplified by reverse transcriptase PCR with total RNA extract from the cells (Han & Kang, 1998 ▶). The PCR product was digested with NdeI and BamHI and inserted downstream of the T7 promoter of the expression plasmid pET-3a (Novagen). The resulting construct expresses residues 1–302 of the DDB_G0286605 protein without any additional residues. After verifying the DNA sequence, the plasmid DNA was transformed into Escherichia coli strain BL21 (DE3). The cells were grown to an OD₆₀₀ of approximately 0.6 in Luria–Bertani medium containing 0.1 mg ml⁻¹ ampicillin (Duchefa) at 310 K and expression was induced using 1 mM isopropyl β-d-1-thiogalactopyranoside (Duchefa). After 12 h induction at 295 K, the cells were harvested and resuspended in 50 mM potassium phosphate (Fluka) pH 7.5 containing 0.1 mM ethylenediaminetetraacetic acid (EDTA; Fluka). The cells were disrupted by sonication and the cell debris was discarded by centrifugation at 20 000g for 30 min. Ammonium sulfate (Fluka) was added to the supernatant to 55% saturation. After stirring the solution for 1 h, the precipitate was discarded by centrifugation at 20 000g for 30 min. The protein solution was loaded onto a Superdex 75 HR 16/60 column (GE Healthcare) pre-equilibrated with 25 mM Tris–HCl buffer pH 7.5 containing 150 mM NaCl. The fractions containing an overexpressed 35 kDa band on SDS–PAGE (Fig. 2 ▶) were pooled and concentrated. The proteins were loaded onto a Mono-­Q Sepharose column (Amersham Biosciences) and the DDB_G0286605 protein was eluted with washing buffer (25 mM Tris–HCl buffer pH 7.5). The purified proteins were dialyzed against 25 mM Tris–HCl buffer pH 7.5 containing 150 mM NaCl and then concentrated to approximately 30 mg ml⁻¹ for crystallization trials.

Figure 2.

SDS–PAGE analysis of purified DDB_G0286605 protein. Lane M, molecular-mass markers (kDa); lane P, 10 µg purified recombinant DDB_G0286605 protein.

2.2. Crystallization and X-ray data collection

Initial screening was conducted by the hanging-drop vapour-diffusion method using screening kits from Hampton Research and the Wizard I and II screening solutions from Emerald BioSystems. Droplets composed of 1.5 µl protein solution and an equal volume of crystallization screening solution were equilibrated against 350 µl reservoir solution at 295 K. Several bundles of rod-shaped crystals were produced using a condition consisting of 0.2 M sodium thiocyanate, 20% PEG 3350 in two weeks. The crystallization conditions were then optimized by the addition of 5 mM dithiothreitol (DTT) to the protein solution, which led to the growth of crystals that were large enough for data collection (Fig. 3 ▶). Since the crystals were not separated and formed chain-like bundles, we seperated one node of the crystal bundle using Micro-Tools from Hampton Research for data collection.

Figure 3.

Crystals of the DDB_G0286605 protein.

Crystals were maintained at ∼100 K during data collection in order to minimize radiation damage. Native data were collected at 100 K using an Area Detector Systems Corporation (ADSC) Quantum 210 charge-coupled device (CCD) area-detector system on BL-6B and BL-6C of the Pohang Light Source (PLS), South Korea (Fig. 4 ▶). The diffraction data were processed and scaled using the programs DENZO and SCALEPACK from the HKL-2000 program suite (Otwinowski & Minor, 1997 ▶).

Figure 4.

An X-ray diffraction pattern from a crystal of the DDB_G0286605 protein.

3. Results and discussion

Investigation of systematic absences in the reflections showed that the crystals of the DDB_G0286605 protein belonged to the monoclinic space group P2₁, with unit-cell parameters a = 67.598, b = 54.935, c = 84.219 Å, β = 109.620°. The crystal volume per unit molecular weight (V _M) was calculated to be 2.17 ų Da⁻¹, with a solvent content of 43.25% by volume (Matthews, 1968 ▶), when the asymmetric unit was assumed to contain two molecules (99% probability). In the self-rotation function, which was calculated with the GLRF program (Tong & Rossmann, 1997 ▶) using data in the resolution range 15–4 Å and an integration radius of 25 Å, no dominant features were found except in the κ = 180° section. The κ = 180° section revealed two peaks corresponding to twofold axes parallel to the crystallographic b axis (Fig. 5 ▶). Isothermal titration calorimetry analysis indicated that the DDB_G0286605 protein interacts with NADP(H) but not with NAD(H) (data not shown). Therefore, we attempted to obtain crystals of a cofactor-bound complex using the same conditions, but crystals did not grow. To solve the structure of the protein–cofactor complex, native crystals were soaked with NADP or NADPH for 5 min and diffraction data were then collected. The data-collection statistics are summarized in Table 1 ▶.

Figure 5.

The κ = 180° section of the self-rotation function from the data set of a native crystal. The self-rotation function was calculated using a 25 Å radius of integration and data in the resolution range 15–4 Å.

Table 1. Data-collection and processing statistics.

Values in parentheses are for the highest resolution shell.

	Native	NADP(H) soaking
Space group	P21	P21
Unit-cell parameters (Å, °)	a = 67.598, b = 54.935, c = 84.219, β = 109.62	a = 68.474, b = 54.274, c = 86.680, β = 112.77
Wavelength (Å)	0.97925	1.10000
Resolution (Å)	50–1.64 (1.64–1.62)	50–1.86 (1.91–1.86)
Completeness (>0σ) (%)	96.3 (90.7)	98.6 (92.1)
Rmerge† (%)	6.1 (31.1)	8.4 (21.6)
Average I/σ(I)	38.6 (3.2)	29.7 (3.6)
Unique reflections	68975 (3219)	48534 (2253)
Average multiplicity	5.5 (3.6)	5.4 (3.6)

^† R _merge = , where I _i(hkl) is the intensity of observed reflection hkl and 〈I(hkl)〉 is the mean intensity of symmetry-equivalent reflections.

Attempts were made to solve the crystal structure of the DDB_G0286605 protein by molecular replacement with MOLREP (Vagin & Teplyakov, 2010 ▶) and Phaser (McCoy, 2007 ▶) within the CCP4 software suite (Collaborative Computational Project, Number 4, 1994 ▶) using the structures of NmrA (PDB code 1k6i; Stammers et al., 2001 ▶), HSCARG (PDB code 2exx; Zheng et al., 2007 ▶) and QOR2 (PDB code 2zcu; Kim et al., 2008 ▶) as search models. However, all of the trials resulted in failure. Since the NmrA-like proteins of the SDR superfamily have well conserved N-terminal dinucleotide-binding Rossmann folds and relatively varied C-terminal substrate-binding domains (Jörnvall et al., 1995 ▶), the failure of the MR trials seems to be caused by the low primary-sequence identity and the different topology of the C-terminal domains.

The crystal structures of the NmrA-like DDB_G0286605 protein and of its complex with cofactor will help in understanding how the NmrA-like proteins of D. discoideum modulate biological functions in response to the redox state of the cell. Therefore, we are attempting to grow crystals of selenomethionine-substituted DDB_G0286605 protein in order to solve the crystal structure using the multiple-wavelength anomalous dispersion method.