Crystallization of the C-terminal domain of the bacteriophage T5 L-shaped fibre

The trimeric L-shaped fibre protein from bacteriophage T5 has been analysed by electron microscopy and a C-terminal domain has been crystallized. Diffraction data have been obtained for two native crystal forms and one selenomethionine derivative.

Abstract

Tails of bacteriophage T5 (a member of the Siphoviridae family) were studied by electron microscopy. For the distal parts of the L-shaped tail fibres, which are involved in host cell receptor binding, a low-resolution volume was calculated. Several C-terminal fragments of the fibre were expressed and purified. Crystals of two of them were obtained that belonged to space groups P6₃ and R32 and diffracted synchrotron radiation to 2.3 and 2.9 Å resolution, respectively. A single-wavelength anomalous dispersion data set to 2.5 Å resolution was also collected from a selenomethionine-derivatized crystal of one of the fragments, which belonged to space group C2.

1. Introduction  

Bacteriophage T5 belongs to the Siphoviridae family of the Caudovirales order of tailed bacteriophages, with a 90 nm wide icosahedral T = 13 capsid and a 200 nm long non-contractile flexible tail. In contrast to most tailed bacteriophages, T5 has a threefold rather than a sixfold symmetric tail (Effantin et al., 2006 ▶). Three L-shaped tail fibres (ltf) are connected to the bottom of the tail (Zivanovic et al., 2013 ▶) and each fibre is a homotrimer of protein pb1 (pb stands for ‘protein band’). Pb1 is expressed as a 1396 amino-acid protein, of which the C-terminal 132 or 133 amino acids function as an intramolecular chaperone and are removed by an autoproteolytic process after correct trimerization and folding (Heller & Krauel, 1986 ▶; Schwarzer et al., 2007 ▶; Xiang et al., 2009 ▶; Schulz et al., 2010 ▶).

The L-shaped tail fibres are involved in the first and reversible interaction of bacteriophage T5 with the host cell (Heller & Braun, 1979 ▶). They are not indispensable for infection, but increase the adsorption rate by a factor of 15. The ltf interact with the trimannose regions of the O-antigens of the O8 or O9 type of bacterial lipopoly­saccharides (LPS; Heller & Braun, 1982 ▶). After this first reversible interaction, the phage binds to the cell in an irreversible manner when the straight tail fibre (stf) engages FhuA, the Escherichia coli outer-membrane iron transporter (Heller, 1992 ▶), and DNA ejection into the bacteria is triggered (Boulanger et al., 1996 ▶). This interaction and DNA release have been characterized in vitro (Flayhan et al., 2012 ▶; Breyton et al., 2013 ▶). The stf is formed by the pb3, pb4 and pb5 proteins, of which pb5 is located at the end and is responsible for interacting with FhuA (Zivanovic et al., 2013 ▶).

Here, we report electron microscopy of bacteriophage T5 tails and analysis of the shape of the distal part of the L-shaped tail fibres, plus the expression, purification, crystallization and preliminary X-ray data collection of C-terminal fragments of the bacteriophage T5 L-shaped tail fibre, which is the part that presumably interacts with E. coli LPS. The intramolecular chaperone was autoproteolysed as expected after expression in E. coli. We also obtained diffraction data from selenomethionine-containing derivative crystals.

2. Methods  

2.1. Purification of bacteriophage T5 tails  

The E. coli strain F was used as a nonpermissive host for the bacteriophage T5 amber mutant T5amD2030d (Hendrickson & McCorquodale, 1971 ▶). T5amD2030d has an amber mutation in the gene coding for the major capsid protein pb8 (Zivanovic et al., 2013 ▶) and without it isolated tails accrue inside infected nonpermissive bacteria, while viable T5 phages can be obtained from permissive amber-suppressing strains. A 0.7 l exponentially growing culture was infected at a multiplicity of infection of 8. After cell lysis, DNase was added to 10 µg ml⁻¹ and 0.2 ml of chloroform per litre of culture was added. After 30 min incubation at 310 K, tails were prepared using a similar procedure to that reported by Huet et al. (2010 ▶) for the purification of bacteriophage T5 immature capsids. The procedure consisted of the precipitation of tails with polyethylene glycol 6000 at 8%(w/v) in the presence of 0.5 M sodium chloride, glycerol gradient centrifugation and ion-exchange chromatography on a HiTrap Q HP column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), which was eluted with a linear sodium chloride gradient of 0.075 to 1.0 M in 20 mM Tris–HCl pH 7.5, 1 mM calcium chloride, 1 mM magnesium chloride.

2.2. Electron microscopy  

Samples of purified bacteriophage T5 tails (2–5 µl at 0.1–0.5 mg ml⁻¹ in 20 mM Tris–HCl pH 7.5, 75 mM sodium chloride, 1 mM calcium chloride, 1 mM magnesium chloride) were applied to glow-discharged carbon-coated grids for 2 min. The grids were washed twice with water and negatively stained with 2%(w/v) aqueous uranyl acetate. Electron microscopy images were recorded on an FEI Eagle 4k CCD in a Tecnai G2 electron microscope (FEI, Hillsboro, Oregon, USA) operating at 200 kV and equipped with a field emission gun. Images were recorded at a detector magnification of 67 873× (sampling rate of 2.21 Å per pixel).

General image-processing operations were performed using XMIPP (Scheres et al., 2008 ▶) and BSOFT (Heymann & Belnap, 2007 ▶). Defocus was determined with CTFFIND3 (Mindell & Grigorieff, 2003 ▶). Choosing only the distal part of the fibre, 1129 images were selected using HELIXBOXER from the EMAN2 package (Tang et al., 2007 ▶). After classification employing maximum-likelihood routines (Scheres et al., 2005 ▶), 444 straight-rod images were selected to obtain a two-dimensional average image. The resulting image was rotationally averaged around the longitudinal fibre axis to render an averaged three-dimensional volume.

2.3. Protein expression and purification  

Secondary-structure prediction was performed with PSIPRED (Jones, 1999 ▶; Buchan et al., 2010 ▶). C-terminal fragments of the gene codifying amino acids 794–1396 and 970–1396 of pb1 were amplified from bacteriophage T5 DNA using forward primers containing BamHI restriction sites and a reverse primer containing a HindIII restriction site. The amplified fragments were cloned into the pET-28c(+) and pET-28a(+) vectors, respectively (Novagen, Merck, Darmstadt, Germany), and digested with the same restriction enzymes. The resulting proteins contain an N-terminal His and T7 tag. Both plasmid sequences were verified (Secugen SL, Madrid, Spain). The plasmids obtained, pET-28-pb1(794–1396) and pET-28-pb1(970–1396), were transformed into E. coli BL21(DE3). Cultures were grown overnight in LB medium (10 g l⁻¹ tryptone, 5 g l⁻¹ yeast extract, 10 g l⁻¹ sodium chloride) supplemented with 50 mg l⁻¹ kanamycin at 310 K. The following day, cultures were diluted 1:10 into the same medium and gene expression was induced with 1 mM IPTG when the optical density measured at 600 nm (OD) reached 0.6. After 3 h of further aerobic incubation, the cultures were harvested by centrifugation and the cells were resuspended in 50 mM Tris–HCl pH 8.5, 0.5 M sodium chloride, 10% glycerol. For expression of protein containing selenomethionine, pET-28-pb1(970–1396) was transformed into E. coli B834(DE3). The cultures were grown overnight at 310 K in selenomethionine medium (Molecular Dimensions, Newmarket, Suffolk, England) supplemented with 40 mg l⁻¹ l-methionine and 50 mg l⁻¹ kanamycin. The following day, cultures were diluted 1:10 into the same medium supplemented with 40 mg l⁻¹ selenomethionine and 50 mg l⁻¹ kanamycin and gene expression was induced with 1 mM IPTG when the OD reached 0.4. The bacteria were harvested in the same way as those grown in LB medium. Note, the plasmids are named after the expressed genes, i.e. pb1(794–1396) and pb1(970–1396), while the purified proteins are called pb1(794–1263) and pb1(970–1263) because the very C-terminal intramolecular chaperone domains consisting of amino acids 1264–1396 are not present in the final protein (they are autoproteolysed upon expression in E. coli and correct folding of the trimeric proteins).

Bacteria were lysed by performing three cycles of 30 s of sonication separated by 30 s on ice and cell debris was removed by 30 min centrifugation at 20 000g. The supernatant was incubated for 30 min with 0.5 ml Ni–NTA resin (Jena Biosciences, Jena, Germany) per litre of culture. The sample was loaded onto an empty column and was then washed with ten column volumes of 50 mM Tris–HCl pH 8.5, 0.3 M sodium chloride, 50 mM imidazole. The protein was eluted by a step gradient from 0.05 to 0.5 M imidazole. Fractions containing the protein [0.1–0.5 mM imidazole for pb1(794–1263) and 0.15–0.5 mM imidazole for pb1(970–1263)] were dialysed against 10 mM Tris–HCl pH 8.5 and loaded onto a Resource Q-6 column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) for anion-exchange chromatography. The protein was eluted with a linear gradient from 0 to 1 M sodium chloride in 10 mM Tris pH 8.5. The protein pb1(794–1263) eluted at around 0.28 M sodium chloride, while pb1(970–1263) eluted at around 0.30 M sodium chloride. Fractions containing the protein were concentrated using an Amicon Ultra 10K centrifugal filter device (Merck Millipore, Billerica, Massachusetts, USA) and washed with 10 mM Tris–HCl pH 8.5 three times to remove contaminants. The protein concentration was measured by UV spectroscopy (Nanodrop, Thermo Scientific, Wilmington, Delaware, USA) and samples were stored at 193 K until use.

2.4. Crystallization, data collection and processing  

Initial crystallization trials for pb1(794–1263) were performed by sitting-drop vapour diffusion in MRC crystallization plates (Molecular Dimensions, Newmarket, Suffolk, England) using a Genesis RSP 150 workstation nanodispenser robot (Tecan Trading AG, Männedorf, Switzerland). Crystals were obtained in drops in which the reservoir contained ammonium sulfate as a precipitant plus different buffers and additives. These crystals were reproduced and conditions were optimized using Compact Clover plates (Jena Bio­sciences, Jena, Germany) with 0.15 ml reservoirs and drops consisting of 2 µl protein solution mixed with 2 µl reservoir solution. Crystals of pb1(970–1263) were also obtained using these optimization screens. The final conditions for native crystals were 0.1 M MES pH 6.5, 1.5 M ammonium sulfate [at 2.5 mg ml⁻¹ protein concentration; pb1(794–1263)] and 0.1 M Tris–HCl pH 8.5, 1.5 M ammonium sulfate [at 3 mg ml⁻¹ protein concentration; pb1(970–1263)], while those for the selenomethionine-derivative crystals were 0.1 M Tris–HCl pH 8.5, 17.5%(w/v) PEG 4000 (at 3 mg ml⁻¹ protein concentration). To estimate protein concentrations, the absorbance was measured at 280 nm and the concentration was calculated using theoretical extinction coefficients of 1.04 and 1.59 ml mg⁻¹ cm⁻¹ for pb1(794–1263) and pb1(970–1263), respectively.

For data collection, crystals were soaked in reservoir solutions containing 15% glycerol, mounted using LithoLoops (Molecular Dimensions, Newmarket, Suffolk, England) or MicroMounts (Mitegen, Ithaca, New York, USA) and vitrified in liquid nitrogen for data collection at 100 K. Native crystallographic data collected at ALBA were integrated using iMosflm (Battye et al., 2011 ▶) and reduced using POINTLESS, SCALA and TRUNCATE (Evans, 2011 ▶), all integrated into CCP4 (Winn et al., 2011 ▶). Derivative data collected at Diamond Light Source were integrated using xia2 (Winter et al., 2013 ▶), which incorporates XDS (Kabsch, 2010 ▶) and AIMLESS (Evans & Murshudov, 2013 ▶).

3. Results and discussion  

Because no high-resolution structural information is available for any lateral tail fibres of bacteriophages belonging to the Siphoviridae family, we set about obtaining low-resolution information via transmission electron microscopy of negatively stained fibres of bacteriophage T5 and high-resolution information using X-ray crystallography of their likely receptor-binding C-terminal domains.

When performing negative-stain electron microscopy on bacteriophages, their capsids attract the heavy-atom stain strongly, obscuring their tail details. We therefore produced bacteriophage T5 tails alone, which were subsequently purified and analysed by electron microscopy as described in §2. As expected, the tails showed two types of fibres attached to their cone-shaped bases (Fig. 1 ▶ a): a single straight tail fibre and three L-shaped side or lateral tail fibres. The L-shaped tail fibres consisted of a thin proximal rod of approximately 30 × 3 nm connected by an apparently flexible hinge to a thicker longer distal rod of around 47 × 5 nm. Whereas the proximal part is rather smooth, the distal part has a bead-shaped appearance, suggesting different domains. To obtain a low-resolution volume of the distal rod, 444 straight-rod images were aligned and classified, and a three-dimensional model was generated applying rotational symmetry over the longitudinal axis.

Figure 1.

Electron microscopy of bacteriophage T5 tails. (a) Negative-stain electron microscopy image. Arrows indicate the central straight tail fibres (stf; made up of the pb5 protein), while asterisks indicate the lateral L-shaped tail fibres (ltf; made up of the pb1 protein). One of the beaded distal rods of the L-shaped tail fibre is boxed. The bar indicates 50 nm. (b) Three-dimensional model of the ltf distal rod domain. Domain numbers and their proposed border residues are indicated; D1 corresponds to the proximal thin rod domain (not shown).

In the protein sequence, the first 205 residues of the protein contain mainly small (Ala, Ser and Thr) and hydrophilic (Gln, Glu and Lys) amino acids, suggesting they might be folded in a triple-helical fashion (perhaps a coiled coil, as also supported by coiled-coil prediction software) and be responsible for forming the proximal thin rod. This would mean that residues 206–1263 (or 206–1264) would form the distal rod. The electron microscopy model clearly indicated a beaded structure for the distal rod (Fig. 1 ▶ b) containing seven domains, of which D2, D3, D6 and D8 are the widest (D1 has been assigned as the proximal rod domain). The total distal rod volume was calculated (1.16 × 10⁶ ų) and divided by the number of amino acids that it is proposed to contain (1058), and residues 206–1263 were then assigned to domains according to their volume, assuming a linear distribution of the amino acids along the fibre (N-terminal to C-terminal or hinge to distal end, with no backtracking of the protein chain).

Before the electron microscopy experiment was performed, several C-terminal fragments were designed based on the secondary-structure prediction alone (starting at amino acids 224, 413, 674, 693, 794 and 1196). Based on the volume estimates, additional C-terminal fragments were designed (starting at residues 728, 835 and 970) avoiding interruption of predicted secondary-structure elements. Corresponding DNA fragments were amplified by PCR and cloned into an expression vector as described in §2 (only the two that allowed the production of crystals, starting at amino acids 794 and 970, are discussed in detail). The N-terminal 6×His and T7 tag of the vector was retained and fused to the N-terminus of the target proteins to facilitate their purification by affinity chromatography. After transformation of expression vectors into E. coli BL21(DE3), soluble protein was obtained for both constructs. Proteins were purified by metal-affinity and ion-exchange chromatography (Fig. 2 ▶ a). For both constructs sufficient amounts of purified protein for crystallization trials could be obtained. Pb1(794–1263) showed a high degree of purity, but pb1(970–1263) contained impurities that were evident after denaturing gel electrophoresis (Fig. 2 ▶ a). A main impurity appeared to be the proteolysed C-terminal intramolecular chaperone. For pb1(970–1263), a small fraction of the protein remained trimeric unless heated to at least 368 K for a few minutes before gel electro­phoresis (Fig. 2 ▶ a); for pb1(794–1263) this behaviour was not observed. The C-terminal proteolysed intramolecular chaperone fragment present in the pb1(970–1263) preparation remained entirely trimeric unless heated (Fig. 2 ▶ a), suggesting that it formed more stable trimers than the C-terminal protein fragments whose folding it catalyses.

Figure 2.

Purification and crystallization of C-terminal fragments of the bacteriophage T5 L-shaped fibre protein pb1. (a) Denaturing gel electrophoresis of purified proteins. Lanes are marked M for molecular-weight markers (their sizes are indicated in kDa), 794 for purified pb1(794–1263), 970h for purified pb1(970–1263) protein heated to 368 K for 5 min and 970u for unheated purified pb1(970–1263) protein. Purified pb1(970–1263) protein contains a small amount of proteolysed intramolecular chaperone. The band corresponding to trimeric pb1(794–1263) is marked with a star, monomeric pb1(794–1263) is marked with an arrow and trimeric chaperone is marked with a diamond, while monomeric chaperone is marked with a circle. The figure is a composite of two regions of two different gels. (b) Native crystals of pb1(794–1263) shaped like hamburgers. (c) Native cubic shaped crystals of pb1(970–1263). (d) Bar-shaped crystals of selenomethionine-derivatized pb1(970–1263). The bars represent 0.5 mm.

Crystals of the pb1(794–1263) construct were obtained that presented rounded rather than the desired sharp edges; they had the shape of hamburgers (Fig. 2 ▶ b, used for data collection) or grew in the form of lenses. However, they turned out to diffract synchrotron radiation rather well. Crystallization solutions were supplemented with glycerol prior to cryocooling and data were collected from them using synchrotron radiation (for statistics, see Table 1 ▶). Both crystal types belonged to space group P6₃. The best crystal diffracted X-rays to around 2.3 Å resolution. The Matthews coefficient (Matthews, 1968 ▶) is 2.3 ų Da⁻¹ and the asymmetric unit is expected to contain one monomer, which would give a solvent content of 42%. We were not able to express selenomethionine-derivatized pb1(794–1263), even though it was attempted several times.

Table 1. Crystallographic data measured from native crystals.

Values in parentheses are for the highest resolution bin.

	Native 1	Native 2	Selenomethionine
Construct	pb1(794–1396)	pb1(970–1396)	pb1(970–1396)
Synchrotron beamline	BL13-XALOC, ALBA	BL13-XALOC, ALBA	I02, DLS
Detector	Pilatus 6M	Pilatus 6M	Pilatus 6M
Crystal-to-detector distance (mm)	478	434	487
Wavelength (Å)	1.0061	1.0061	0.9797
No. of images	180	180	2400
Oscillation range (°)	1.0	1.0	0.10
Space group	P63	R32	C2
Unit-cell parameters
a (Å)	52.2	80.4	227.4
b (Å)	52.2	80.4	58.2
c (Å)	299.1	80.4	69.9
α (°)	90.0	53.7	90.0
β (°)	90.0,	53.7	98.8
γ (°)	120.0	53.7	90.0
Resolution range (Å)	45.2–2.34 (2.47–2.34)	68.6–2.90 (3.06–2.90)	56.2–2.52 (2.59–2.52)
Unique reflections	17817 (2641)	4882 (681)	30585 (2237)
Multiplicity	2.8 (2.7)	8.1 (7.9)	6.3 (6.5)
Completeness (%)	92.0 (92.8)	100.0 (100.0)	96.8 (95.8)
〈I/σ(I)〉	5.3 (3.8)	8.4 (2.4)	11.4 (2.3)
R merge † (%)	17.0 (23.7)	19.2 (119.9)	10.0 (64.4)
CC Imean	0.926 (0.760)	0.991 (0.641)	0.996 (0.869)
CC anom	−0.167 (−0.001)	−0.137 (0.021)	0.508 (0.059)
Wilson B (Å2)	11.9	37.1	41.1

^† R _merge = , where I_i(hkl) is the intensity of the ith measurement of the same reflection and 〈I(hkl)〉 is the mean observed intensity for that reflection.

Crystals were also obtained for pb1(970–1263); these had a roughly cubic shape (Fig. 2 ▶ c). They belonged to space group R32 and diffracted to 2.9 Å resolution. The Matthews coefficient is 1.7 ų Da⁻¹ and the asymmetric unit is expected to contain one monomer, leading to a solvent content of only 28%. This low solvent content may suggest the possibility of partial proteolysis of this construct before crystallization. Preliminary attempts to introduce heavy atoms for phasing purposes were unsuccessful. Therefore, as the pb1(970–1263) construct contained five internal methionine residues, derivatization with selenomethionine was performed. The modified mutant protein could be purified using a very similar protocol to the native, and after crystallization elongated tombstone-shaped crystals were obtained (Fig. 2 ▶ d). The derivative crystals belonged to a different space group, C2, and had unit-cell parameters that were apparently unrelated to those of the native form (Table 1 ▶). They diffracted X-rays to 2.5 Å resolution. The Matthews coefficient is 2.4 ų Da⁻¹ and the asymmetric unit is expected to contain one trimer, with a solvent content of 47%.

4. Conclusions  

The bacteriophage T5 L-shaped tail fibre was analysed by electron microscopy and several C-terminal fragments were expressed, in part based on the electron microscopic analysis. Two of these proteins were crystallized, thus making T5 the first member of the Sipho­viridae family for which crystallization of fibre proteins has been reported. We also present crystals of the same protein modified with selenomethionine; the anomalous signal of the selenomethionine should allow structure solution. Together with the recently reported structure of the C-terminal domains of a myovirus fibre (bacterio­phage T4; Bartual et al., 2010 ▶) and a podovirus fibre (bacteriophage T7; Garcia-Doval & Raaij, 2012 ▶), this should lead to a deeper understanding of bacteriophage fibre structure and may lead to applications in bionanotechnology and novel tools employing bacteriophages, including those involving targeting of bacteriophage to specific pathogenic bacteria.