Crystallization and preliminary X-ray analysis of the catalytic domains of Paenibacillus sp. strain FPU-7 cell-surface-expressed chitinase ChiW

The crystallization and preliminary X-ray diffraction analysis of the catalytic domains of cell-surface-expressed chitinase ChiW from Paenibacillus sp. strain FPU-7 are reported.

Abstract

The polysaccharide chitin is effectively hydrolyzed and utilized as a carbon and nitrogen source by the Gram-positive bacterium Paenibacillus sp. strain FPU-7. ChiW is a unique cell-surface-expressed chitinase among the Paenibacillus sp. strain FPU-7-secreted chitinases. An N-terminally truncated ChiW protein, primarily comprised of the two catalytic domains of the full-length protein, was successfully overexpressed in Escherichia coli, purified as a functional recombinant protein with a molecular mass of approximately 98 kDa and crystallized. Preliminary X-ray analysis showed that the crystal diffracted to 1.93 Å resolution and belonged to the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a = 112.1, b = 128.2, c = 162.6 Å, suggesting the presence of two molecules in an asymmetric unit.

1. Introduction  

The Gram-positive soil bacterium Paenibacillus sp. strain FPU-7 was reported to have a high degradation activity towards chitin (Itoh et al., 2013 ▶). It is noteworthy that ChiW was identified as a novel cell-surface-expressed multi-modular chitinase (Itoh et al., 2013 ▶). ChiW contains a signal peptide (amino acids 1–30) and three repeated surface-layer homology (SLH; amino acids 31–197) domains anchored to the bacterial surface layer (Mesnage et al., 2000 ▶; Schneewind & Missiakas, 2012 ▶; Fig. 1 ▶ a). The SLH domains are followed by two homologous catalytic domains (54% amino-acid sequence identity) belonging to the glycoside hydrolase family GH-18 chitinases (first GH-18, amino acids 557–927; second GH-18, amino acids 1045–1418; Itoh et al., 2014 ▶; http://www.cazy.org/; Henrissat & Bairoch, 1996 ▶; Fig. 1 ▶ a). The remaining regions, in particular amino acids 198–556, showed no sequence similarity to other known proteins. Although several chitinases with two catalytic domains in a single polypeptide chain have been identified in a wide variety of organisms (Tanaka et al., 1999 ▶; Howard et al., 2004 ▶; Hiramatsu et al., 1999 ▶), ChiW is the first chitinase that is expressed on the bacterial surface layer.

Figure 1.

Schematic representation of Paenibacillus sp. strain FPU-7 ChiW primary structure and SDS–PAGE analysis of purified ChiW-CD. (a) ChiW denotes full-length Paenibacillus sp. strain FPU-7 ChiW protein; ChiW-SLHd and ChiW-CD denote the truncated ChiW proteins. (b) SDS–PAGE protein profile of ChiW-CD. Lane M, molecular-weight standards (labelled in kDa); lane 1, purified ChiW-CD (1 µg).

A recombinant ChiW-SLHd construct (amino acids 198–1418; Fig. 1 ▶ a) lacking the SLH domains was purified from Escherichia coli cells and several of its enzymatic properties have been determined (Itoh et al., 2013 ▶, 2014 ▶). The detailed crystal structures of the catalytic domains (TIM-barrel) of GH-18 chitinases have been investigated (http://www.cazy.org/GH18_structure.html). However, to our knowledge, no information is currently available on the structures of the cell-surface-expressed multi-modular chitinases in the Protein Data Bank (PDB). Since crystallizations of full-length recombinant ChiW-SLHd protein have thus far been unsuccessful, we prepared the truncated mutant protein ChiW-CD (amino acids 557–1418; Fig. 1 ▶ a), which is primarily composed of two tandem catalytic GH-18 domains. Here, we report the crystallization and preliminary crystallographic analysis of ChiW-CD.

2. Materials and methods  

2.1. Macromolecule production  

The ChiW-SLHd gene (DDBJ/EMBL/GenBank database accession No. AB683965) was subcloned into the expression vector pColdIII (Takara, Otsu, Shiga, Japan) from Paenibacillus sp. strain FPU-7 genomic DNA as previously described (Itoh et al., 2013 ▶). Inverse PCR was used for constructing the truncated mutant ChiW-CD (Fig. 1 ▶ a) with a C-terminal 6×His tag using a KOD Plus mutagenesis kit (Toyobo, Osaka, Japan) and the expression vector for ChiW-SLHd as a template. Successful plasmid construction was confirmed by DNA sequencing with an ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems, Foster City, California, USA). The expression vector for ChiW-CD was transformed into competent E. coli BL21 cells (Novagen, Darmstadt, Germany; Table 1 ▶).

Table 1. ChiW-CD production information.

Source organism	Paenibacillus sp. strain FPU-7
DNA source	pColdIII_ChiW-SLHd expression vector with C-terminal 6×His tag
Forward primer	5′-CTCGAGGGTACCGAGCTCCATATGCAC-3′
Reverse primer	5′-GTGCAGGGCAAAATTGTTTCTTATATCCCGGCTTG-3′
Expression vector	pColdIII
Expression host	E. coli BL21

Recombinant ChiW-CD was purified from E. coli cells by the same procedure used for ChiW-SLHd with minor variations (Itoh et al., 2014 ▶). In brief, the cultured cells were chemically disrupted and the cell extract was subsequently purified by Ni-immobilized metal-affinity chromatography (HisTrap HP column; GE Healthcare, Little Chalfont, England) and anion-exchange chromatography (Mono Q HR 10/10 HR column; GE Healthcare). Enzyme-containing fractions were combined and dialyzed at 277 K overnight against 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer pH 7.0. The protein concentration was determined by UV spectrophotometry using the theoretical molar extinction coefficient ∊₂₈₀ = 193 900 M ⁻¹ cm⁻¹) according to the ExPASy ProtParam tool server (http://web.expasy.org/protparam/; Gasteiger et al., 2005 ▶). ChiW-CD was purified from the bacterial cells to homogeneity, as assessed by 12%(w/v) SDS–PAGE followed by Coomassie Brilliant Blue (CBB) R-250 staining.

2.2. Crystallization  

Initial screening for ChiW-CD crystallization was performed using the commercially available crystallization kits Crystal Screen and Crystal Screen 2 (Hampton Research, Aliso Viejo, California, USA) and Wizard Classic Crystallization Screens 1 and 2 (Emerald BioSystems, Bedford, Massachusetts, USA) by the sitting-drop vapour-diffusion method in 96-well plates at 293 K. Crystallization conditions were further refined using a 24-well plate. Final crystal preparation was performed by the sitting-drop vapour-diffusion method. The drop (4 µl) consisted of 2 µl enzyme solution (10 mg ml⁻¹) and 2 µl reservoir solution (0.5 ml) comprising 0.94 M ammonium phosphate, 0.1 M citrate–phosphate buffer (0.1 M citric acid and 0.2 M dibasic sodium phosphate solutions were mixed to adjust the pH to 5.5) (Table 2 ▶).

Table 2. Crystallization conditions.

Method	Sitting-drop vapour diffusion
Plate type	24-well plate
Temperature (K)	293
Protein concentration (mg ml−1)	10
Buffer composition of protein solution	20 mM HEPES pH 7.0
Composition of reservoir solution	0.94 M ammonium phosphate, 0.1 M citrate–phosphate pH 5.5
Volume and ratio of drop	4 µl, 1:1 enzyme solution:reservoir solution
Volume of reservoir (µl)	500

2.3. Data collection and processing  

Single crystals were soaked in a cryoprotectant solution consisting of 0.27 M ammonium phosphate, 0.029 M citrate–phosphate pH 5.5, 5 M sodium formate followed by cooling in a stream of cold nitrogen gas. The diffraction patterns were then examined on beamlines BL38B1 at SPring-8 and BL-17A at the Photon Factory. X-ray diffraction data for the ChiW-CD crystals were collected using Quantum 315r CCD detectors (Area Detector Systems Corporation, Poway, California, USA) and were indexed, integrated and scaled using HKL-2000 (Otwinowski & Minor, 1997 ▶). Molecular-replacement trials were performed using Phaser (McCoy et al., 2007 ▶) from the CCP4 software package (Winn et al., 2011 ▶) (Table 3 ▶).

Table 3. Data-collection statistics for ChiW-CD.

Values in parentheses are for the outer shell.

Diffraction source	BL-17A, Photon Factory
Wavelength (Å)	0.98
Temperature (K)	95
Detector	Quantum 315r CCD
Crystal-to-detector distance (mm)	256.1
Rotation range per image (°)	0.5
Total rotation range (°)	180
Exposure time per image (s)	5.0
Space group	P212121
Unit-cell parameters (Å)	a = 112.1, b = 128.2, c = 162.6
Mosaicity (°)	0.453
Resolution range (Å)	50.0–1.93 (1.96–1.93)
Total No. of reflections	1215285
No. of unique reflections	174215 (8716)
Completeness (%)	98.9 (100)
Multiplicity	7.0 (6.8)
〈I/σ(I)〉	29.7 (2.8)
R meas † (%)	8.6 (57.2)
Overall B factor from Wilson plot (Å2)	25.5

^† R _meas = , where I_i (hkl) is the ith observation of reflection hkl and 〈I(hkl)〉 is the weighted average intensity for all i observations of reflection hkl.

3. Results and discussion  

The overexpressed ChiW-CD in E. coli was successfully purified to homogeneity as observed by SDS–PAGE (Fig. 1 ▶ b). Other characteristics of ChiW-CD, such as enzymatic activity, N-terminal amino-acid residues and secondary-structural information evaluated from the far-UV circular-dichroism spectrum, are described in the Supporting Information1.

After incubation for 3 d, small crystals appeared in Wizard Classic 1 No. 9 condition consisting of 1.0 M ammonium phosphate, 0.1 M sodium acetate pH 4.5. Plate-like crystals were generated under the optimized condition of 0.94 M ammonium phosphate, 0.1 M citrate–phosphate buffer pH 5.5 (Table 2 ▶, Fig. 2 ▶). Several diffraction data sets were collected at the SPring-8 or Photon Factory synchrotron-radiation facilities. The highest quality X-ray diffraction data set was collected to 1.93 Å resolution on the BL-17A beamline of the Photon Factory (Fig. 3 ▶). A summary of the data-collection statistics is shown in Table 3 ▶. Based on a Matthews coefficient V _M of 2.99 ų Da⁻¹ (solvent content of 58.9%) we assume that two monomers exist in the asymmetric unit (Matthews, 1968 ▶). Initial molecular-replacement trials were performed with Phaser using the coordinates of Bacillus circulans WL-12 chitinase A1 catalytic domain (PDB entry 1itx; 42% sequence identity; Matsumoto et al., 1999 ▶) as the search model. Since ChiW-CD consists of two GH-18 catalytic domains (amino acids 557–927 and 1045–141; Fig. 1 ▶ a) and two molecules are proposed to exist in the asymmetric unit, four unambiguous solutions were generated (RFZ = 5.1, 4.2, 4.0 and 3.2; TFZ = 37.0; PAK = 40; LLG = 327). However, the entire polypeptide chain sequence could not be conclusively traced, except for the catalytic domains, because the electron densities of the remaining region (amino acids 928–1044; Fig. 1 ▶ a) were too low to identify the amino-acid structures.

Figure 2.

Crystals of ChiW-CD.

Figure 3.

X-ray diffraction image of ChiW-CD. The outer black circle corresponds to 1.8 Å resolution.