Crystallization and preliminary crystallographic analysis of the central domain of Drosophila Dribble, a protein that is essential for ribosome biogenesis

A soluble domain spanning amino-acid residues 16–197 of the Drosophila Dribble protein was overexpressed in E. coli, purified and crystallized. X-ray diffraction data were collected to beyond 2 Å resolution.

Abstract

Dribble (DBE) is a Drosophila protein that is essential for ribosome biogenesis. Bioinformatics analysis revealed a folded central domain of DBE which is flanked by structural disorder in the N- and C-terminal regions. The protein fragment spanning amino-acid residues 16–197 (DBE_16–197) was produced for structural determination. In this report, the crystallization and preliminary X-­ray diffraction data analysis of the DBE_16–197 protein domain are described. Crystals of DBE_16–197 were grown by the sitting-drop vapour-diffusion method at 289 K using ammonium phosphate as a precipitant. The crystals belonged to space group P2₁2₁2₁. Data were collected that extended to beyond 2 Å resolution.

1. Introduction

Dribble (DBE; Gene ID 33269) is a Drosophila protein that represents a class of evolutionarily conserved proteins in higher eukaryotes (Chan et al., 2001 ▶; Xin et al., 2005 ▶; Gromadka et al., 2004 ▶; Sasaki et al., 2000 ▶). This class of proteins is essential for early ribosomal RNA (rRNA) processing (Chan et al., 2001 ▶) and ribosome assembly (Sasaki et al., 2000 ▶). It has previously been shown that the yeast orthologue of DBE, Krr1p, is a member of the small-subunit (SSU) processome (Dragon et al., 2002 ▶; Bernstein et al., 2004 ▶). The SSU processome is not only an early ribosome intermediate but is also actively involved in pre-rRNA processing. Having no endonuclease activity, this class of proteins is believed to play a scaffolding role in linking both the specific regions of rRNA and protein factors together for pre-rRNA cleavage. The proposed scaffolding role of this protein family is supported by the fact that DBE possesses RNA-binding affinity (Yiu et al., 2006 ▶) and Krr1p interacts with a range of ribosomal proteins as well as other factors that are essential for ribosome biogenesis (Grandi et al., 2002 ▶). Biophysical studies on DBE demonstrated that a protein domain exists in the central part of the protein that is flanked by structurally disordered N- and C-terminal regions (Yiu et al., 2006 ▶). DisProt VL3H disorder-prediction analysis (Peng et al., 2005 ▶) suggested that approximately amino-acid residues 20–200 are likely to be ordered. Furthermore, both Pfam (Finn et al., 2008 ▶) and SUPERFAMILY (Wilson et al., 2009 ▶) database searches demonstrated that DBE contains a KH-like domain that approximately spans amino-acid residues 120–200 (Yiu et al., 2006 ▶). However, the structural identity of the DBE_20–119 fragment is currently unknown. A DBE protein fragment spanning amino-acid residues 16–197 was generated with the aim of studying the protein architecture of this family of proteins and, more importantly, shedding light on their molecular functions (Chan et al., 2001 ▶; Xin et al., 2005 ▶; Gromadka et al., 2004 ▶; Sasaki et al., 2000 ▶).

2. Experimental procedures

2.1. Expression and purification

A bacterial expression construct, pET-HS-DBE_16–197, encoding amino-acid residues 16–197 (20.9 kDa) of DBE was generated. The primers used were 5′-AAC TCG ACC GGT GGA GTG GAC AAT GCG TGG TCC-3′ and 5′-AAC TCG CTC GAG TTA TCA GGG GTG CAC ATT GTT CAT-3′. pET-HS is a homemade bacterial expression vector derived from pET3d (Novagen). The DBE_16–197 fragment carries an N-terminal His/SUMO (HS) tag that can be removed by a specific protease SENP1C derived from the SUMO-specific protease SENP1 (Xu et al., 2006 ▶). The sequence of the HS tag was MRGSHHHHHHHMSDQEAKPSTEDLGDKKEGEYIL­KKVIGQDSSEIHFKVKMTTHLKKLKESYCQRQGVPMNSLRF­LFEGQRIADNHTPKELGMEEEDVIEVYQEQTGG. No extra residues were left on DBE_16–197 after proteolytic cleavage of the HS tag. Escherichia coli BL21 (DE3) pLysS cells transformed with the pET-HS-DBE_16–197 construct were grown in LB medium at 310 K. Recombinant protein expression was induced by 0.1 mM isopropyl β-­d-1-thiogalactopyranose at 295 K with shaking for 16–20 h. The cells were then harvested by centrifugation and lysed by sonication in 50 mM sodium phosphate pH 7.4, 300 mM sodium chloride, 50 mM imidazole and 1 mM phenylmethylsulfonyl fluoride. The total lysate was centrifuged at 20 000g for 30 min at 277 K. The supernatant was collected and loaded onto an Ni²⁺-charged HiTrap IMAC column (GE Biosciences) equilibrated with 50 mM sodium phosphate pH 7.4, 300 mM sodium chloride, 50 mM imidazole and 1 mM phenylmethylsulfonyl fluoride. Using an ascending linear gradient of imidazole from 50 to 300 mM over 100 ml, the HS-DBE_16–197 protein eluted from ∼220 mM imidazole onwards. The specific protease SENP1C was used at an enzyme:substrate molar ratio of 1:500 to cleave the HS tag from the DBE_16–197 protein fragment. The reaction was carried out at room temperature for 30 min. The DBE_16–197 fragment was released after SENP1C cleavage; this fragment was then separated from the cleaved HS tag by loading onto an Ni²⁺-charged HiTrap IMAC column equilibrated with 50 mM sodium phosphate pH 7.4, 300 mM sodium chloride, 50 mM imidazole and 1 mM phenylmethylsulfonyl fluoride and the DBE_16–197 fragment was collected in the flowthrough. The protein was then concentrated and loaded onto a HiLoad 26/60 Superdex 75 column (GE Biosciences) equilibrated with 50 mM sodium phosphate pH 7.4, 400 mM sodium chloride. The purified DBE_16–197 fragment was subsequently con­centrated to 10 mg ml⁻¹ under the same buffering conditions as described for the Superdex 75 column purification step and subjected to protein crystallization.

2.2. Crystallization

Crystallization-condition screening was carried out using the Crystal Screen 1 and 2 and Index kits from Hampton Research. Crystallization of the DBE_16–197 protein fragment was performed at 289 K using sitting drops consisting of 1 µl protein mixed with 1 µl reservoir solution in Greiner CrystalQuick plates. The initial crystallization condition was 0.4 M ammonium phosphate monobasic from Crystal Screen 1, which was then optimized by extensive grid screening. The best condition for crystal growth was found to consist of using a sitting drop comprising 2 µl protein solution and 4 µl precipitant solution equilibrated against 500 µl well solution con­sisting of 0.6 M ammonium phosphate pH 4.8 at 289 K. Crystals often grew as stacked plates; occasional single plates of dimensions ∼0.3 × 0.4 × 0.02 mm were obtained after 4 d incubation (Fig. 1 ▶).

Figure 1.

Protein crystals of DBE_16–197. Single thin crystals grew to dimensions of ∼0.3 × 0.4 × 0.02 mm after 4 d incubation.

2.3. Preliminary X-ray diffraction data analysis

Crystals were cryoprotected by soaking them in crystallization solution containing 30%(v/v) ethylene glycol for 1 min. X-ray diffraction data were collected on beamline I04 at the Diamond Light Source (UK) at 100 K using an ADSC Q315 detector. 720 images were collected using 0.5° oscillations at a wavelength of 0.9704 Å. Data processing was performed using MOSFLM (Leslie, 2006 ▶) and SCALA (Evans, 2006 ▶) via CCP4i (Potterton et al., 2003 ▶). Pseudo-precession images revealed mmm Laue symmetry and reflection conditions h = 2n, k = 2n, l = 2n, suggesting that the space group was P2₁2₁2₁. The unit-cell parameters were a = 68.99, b = 78.76, c = 79.57 Å. Matthews coefficient (V _M; Matthews, 1968 ▶) analysis suggested that each asymmetric unit contained two protein molecules, giving a V _M value of 2.58 ų Da⁻¹ and a solvent content of 52.4%. Diffraction data were integrated and scaled to a maximum resolution of 1.98 Å. Statistics of data processing are summarized in Table 1 ▶. Attempts at phasing by multiple anomalous dispersion (MAD) using selenomethionine-substituted protein are currently under way.

Table 1. Data-collection and processing statistics.

Values in parentheses are for the highest resolution shell.

No. of crystals	1
Beamline	I04
Wavelength (Å)	0.9704
Detector	ADSC Q315
Crystal-to-detector distance (mm)	274.6
Rotation range per image (°)	0.5
Total rotation range (°)	280
Exposure time per image (s)	1.5
Resolution range (Å)	52.13–1.98 (2.09–1.98)
Space group	P212121
Unit-cell parameters (Å, °)	a  =  68.99, b = 78.76, c = 79.57
Mosaicity (°)	0.73
Total No. of measured intensities	322992
Unique reflections	30870
Multiplicity	10.5 (10.8)
Mean I/σ(I)	11.0 (3.9)
Completeness (%)	99.9 (100)
Rmerge† (%)	9.6 (39.2)
Rmeas or Rr.i.m. (%)	10.1 (41.1)
Overall B factor from Wilson plot (Å2)	25.5

^† R _merge = , where I_i(hkl) and 〈I(hkl)〉 are the observed intensity and the mean intensity of symmetry-related reflections, respectively.