Structure of bacteriophage ϕ29 head fibers has a supercoiled triple repeating helix-turn-helix motif

Abstract

The tailed bacteriophage ϕ29 capsid is decorated with 55 fibers attached to quasi-3-fold symmetry positions. Each fiber is a homotrimer of gene product 8.5 (gp8.5) and consists of two major structural parts, a pseudohexagonal base and a protruding fibrous portion that is about 110 Å in length. The crystal structure of the C-terminal fibrous portion (residues 112–280) has been determined to a resolution of 1.6 Å. The structure is about 150 Å long and shows three distinct structural domains designated as head, neck, and stem. The stem region is a unique three-stranded helix-turn-helix supercoil that has not previously been described. When fitted into a cryoelectron microscope reconstruction of the virus, the head structure corresponded to a disconnected density at the distal end of the fiber and the neck structure was located in weak density connecting it to the fiber. Thin section studies of Bacillus subtilis cells infected with fibered or fiberless ϕ29 suggest that the fibers might enhance the attachment of the virions onto the host cell wall.

Bacteriophages are viruses that infect bacteria and are probably the most abundant organisms on Earth (1). More than 96% of bacteriophages are tailed bacteriophages that consist of either an isometric or a prolate head with an attached tail (2). The head capsids of bacteriophages are tough shells with complex elastic properties (3). The major capsid protein is the primary unit from which the head capsid is assembled and in most tail phages adopts the “HK97” fold, named for the major capsid protein of phage Hong Kong 97 (4–6). Other than the major capsid protein, phage capsids are often decorated with different types of minor proteins. One type of minor protein that is frequently found in many phages is an immunoglobulin-like protein (7). Although many minor capsid proteins are not essential for infection in laboratory conditions (8–11), they may have provided favorable properties for the survival of phage in hostile environments. For instance, the minor capsid protein Soc of phage T4 provides enhanced capsid stability (9).

Bacteriophage ϕ29 infects the Gram-positive bacteria Bacillus subtilis using a short, 380 Å-long, noncontractile tail (12, 13). The B. subtilis cell wall teichoic acid is the primary receptor recognized by the ϕ29 tail appendages for initiating infection (14). After recognition of the host, the tail is brought closer to the peptidoglycan by digesting the cell wall teichoic acids. With the aid of gene product 13 (gp13) (15, 16), the phage tail can penetrate the cell wall peptidoglycan layer and cell membrane to release the genomic DNA that had been packaged in the prolate capsid. The mature ϕ29 capsid consists of two proteins: the major capsid protein gp8 and the head fiber protein gene product 8.5 (gp8.5), assembling into a T = 3, Q = 5 icosahedral shell elongated along a 5-fold axis of symmetry (Fig. 1) (5, 17). The major capsid protein gp8 has two domains that resemble the HK97 capsid protein and the group 2 bacterial immunoglobulin-like (BIG2) protein. The HK97-like domains of ϕ29 gp8 pack tightly against one another in the capsid assembly. The BIG2 domains protrude from the surface of the capsid and provide the attachment sites for the head fibers at quasi-3-fold symmetry positions. Cryoelectron microscopy (cryoEM) maps indicate that the head fibers possess two distinct structural domains: a pseudohexagonal base that attaches to the capsid and a protruding stem that is about 110 Å long. Additional unconnected weak densities at the distal ends of the fibers may be part of the fibers or noise (5, 17). The fibers are not essential for phage infection in laboratory conditions (10, 11).

Fig. 1.

The fibered structure of phage ϕ29. (Left) A 25 Å resolution cryoelectron microscope reconstruction of ϕ29. The fibers are colored in gold. Note the lower density connecting the distal ends of the fibers. (Right) Diagrammatic representation of the icosahedral and quasi-symmetry elements (T = 3, Q = 5) of the phage head. The fibers attach only to quasi-3-fold axes (green dots). The icosahedral 5-fold and 3-fold axes are represented by black and red dots, respectively. The 6-fold symmetry axes in the cylindrical part of the prolate head are represented by orange dots.

Here we report the crystal structure of the gp8.5 C-terminal fibrous portion, which has a unique type of supersecondary structure, helix-turn-helix supercoil. The fibers might enhance the attachment of phage ϕ29 to the host cells during infection.

Results and Discussion

The gp8.5 Structure.

A construct containing the full length gp8.5 and a C-terminal His₆ tag was expressed in Escherichia coli cells. The expressed gp8.5 was cleaved in vitro with chymotrypsin. A stable fragment was obtained and purified. Western blotting showed that the fragment has the His₆ tag and, therefore, was identified as being a C-terminal fragment.

The crystal structure of the gp8.5 C-terminal fragment was determined using single isomorphous replacement with anomalous scattering (SIRAS) employing a sodium iodine derivative. In the final 1.6 Å resolution electron density map (Fig. S1), residues 118–280 could be identified. Leu110, which is the closest upstream substrate appropriate for chymotrypsin cleavage, is probably the N terminus.

The structure of the gp8.5 C-terminal fragment is an elongated homotrimer with its 3-fold axis coinciding with a crystallographic 3-fold axis. The molecule has a rod-like shape and is approximately 150 Å long. The molecule can be divided into three distinct domains, which form the head, neck, and stem (Fig. 2A). The stem domain is about 90 Å in length and is about 24 Å in diameter. Each monomer of the stem region consists of the N-terminal 110 residues (118–227) that fold mainly into 9 short α-helices. The neck region is about 20 Å in length and is about 14 Å in diameter and has 10 residues in each monomer. The head is a three-stranded α-helical bundle, flanked by three extended peptide chains each about 40 Å long that are antiparallel to the helices. The head domain has a higher temperature factor (for these main chain atoms the mean temperature factor is 35 Å²) compared to the rest of the structure (for these the equivalent value is 18 Å²), indicating that it is flexible. Fitting of the crystal structure into the cryoEM map of the virus showed that the stem domain can be fitted into the extended density of the head fiber (Fig. S2), placing the head into the disconnected densities at the distal end and the neck into the weak connecting densities. Thus, the disconnected densities in the cryoEM reconstruction were identified as being a part of the gp8.5 structure. The cryoEM densities corresponding to the head and neck parts of gp8.5 are weak, probably as a result of averaging the flexible head and neck structures. Dali searches (http://ekhidna.biocenter.helsinki.fi/dali_server/) did not find any significant similar structure to gp8.5.

Fig. 2.

Structure of the fibrous portion of gp8.5. (A) Ribbon diagram of the trimeric fiber. Amino acid numbers and helix nomenclature are indicated for the red molecule. (B) Diagrammatic representation of the red monomer showing the repeating double helical motif (Table 1). (C) Hydrophobic core within the fiber shown for helices α4 to α9. (D) Sequence analysis for the three repeating motifs along the fiber. The consensus sequence is represented by the characters H for hydrophobic, P for polar and X for any kind of residue.

A Unique Super Helix-Turn-Helix Coiled Coil.

The stem domain of the structure contains short α-helices, each with two to three turns. In each gp8.5 monomer the helices are connected with “β-turns.” Although, as in all other defined β-turn structures, the β-turns in the stem domain change the direction of the main chain by approximately 180°, they do not correspond to any previously defined β-turn groups (18) (Table S1). Each β-turn raises the following α-helix by about 10 Å. Of the nine helices in each monomer, three helices at the proximal end of the fiber are almost antiparallel to each other. The remaining six helices make a right-handed super helix. The basic repeat unit in the super helix is a helix-turn-helix (Fig. 2B and Table 1). Each repeat unit corresponds to a 45° turn and a translation of 19.5 Å along the super helical axis. There would be eight helix-turn-helix repeats per turn with a pitch of 156 Å and an external radius of 24 Å if the helix were extended.

Table 1.

Super secondary structure helical parameters

	U2*	U3
U1	trans: 19.6 Å	trans: 38.8 Å
α4–α5	rot: 48°	rot: 90°
Residues 149–174	Cα rmsd: 0.28 Å	Cα rmsd: 0.63 Å
	sequence identity: 40%	sequence identity: 38%
U2		trans: 19.4 Å
α6–α7		rot: 43°
Residues 175–200		Cα rmsd: 0.60 Å
		sequence identity: 28%
U3
α8–α9
Residues 201–226

^*See Fig. 2B for the definitions of U1, U2, and U3.

The three super helices in the trimer are coiled around the central vertical 3-fold axis to form a super helical coiled coil (Fig. 2A). The core of the coiled coil is made of hydrophobic side chains from horizontal helices that belong to different molecules (Fig. 2C). The vertical interactions in the coiled coil are mainly hydrogen bonds between side chain atoms of the antiparallel helices within the same monomer. Sequence analysis shows that the super coiled coil contains the repeat motif HPPXPHXXHHPXPXPPPPPXXHPPHH, where H represents a hydrophobic residue, P represents a polar residue and X represents any residue (Fig. 2D). A search for the presence of this motif in sequence databases showed that similar sequences exist in many other proteins. However, none of these proteins contains sequential repeats of this motif except for the phage ϕ29 fiber-like proteins.

The α-helix is a basic secondary structural element of proteins. Helix-helix interactions are ubiquitous in the structure of proteins and protein assemblies. The most common supersecondary structure built on helix–helix interactions is the coiled coil. Coiled coils have a hydrophobic core and are characterized with a heptad sequence repeat. The helix-turn-helix motif is another type of helical supersecondary structure that has been frequently found in DNA-binding proteins (19). The gp8.5 super helix coiled coil is a combination of the coiled coil and the helix-turn-helix supersecondary structures (Fig. 3). Although the exact function of this supersecondary structure is not clear, it could provide information for protein structure prediction and protein design as well as the folding mechanism of fibrous proteins.

Fig. 3.

Model of a trimeric super helix-turn-helix coiled coil. (Top) The monomers are colored red, green, and blue with black dots on the red monomer showing the positions of the helix-turn-helix repeating motifs. (Middle) One repeat motif. (Bottom) Diagrammatic representation of the coiled coil structure showing individual helices.

Structural Comparisons with Other Fiber Structures in Viruses.

Many viruses encode proteins that often form homotrimeric fibrous structures of various folds. A few of these proteins, such as the T4 fibritin (20) and the P22 tail needle (21), fold into α-helical fibrous structures that each has an α-helix coiled coil fold (Fig. 4A). However, most of these viral fiber molecules consist entirely, or nearly entirely, of β-structures and usually function as host-cell recognition molecules (22). Among the viral fibrous β-structures, phage tail spike proteins contain a “β-helix” fold and are probably the most ubiquitous viral fibers (Fig. 4A) (14). The bacteriophage T4 tail needle fiber (23), the T4 long tail fiber (24), and the adenovirus fiber (25) structures each represent a different β-structured fold (Fig. 4A). Each monomer of the adenovirus triple β-spiral structure contains eight short antiparallel β-strands of which two adjacent β-strands are a repeating unit that makes up a left-handed spiral. Although the secondary structural components of gp8.5 are quite different from that of the adenovirus fiber, the overall organization of the gp8.5 α-helices is surprisingly similar to that of the adenovirus fiber antiparallel β-strands (Fig. 4B). Gp8.5 uses a pair of α-helices whereas the adenovirus fiber uses a pair of β-strands as a repeating unit to make a super helix. However, these super helices have opposite hands and slightly different helical parameters. The adenovirus triple β-spiral structure repeats by translating a β-hairpin motif along the 3-fold axis by about 13 Å and rotating it clockwise by about 50°. In contrast, the α-helical gp8.5 fibrous structure has a displacement of about 20 Å between two neighboring α-helical repeats and an anticlockwise rotation of about 45° (Fig. 2).

Fig. 4.

Trimeric virus fiber structures. (A) A comparison of different virus fiber structure folds. (B) Stereographic detail structure comparison of a fibrous β-structure (Left) and a fibrous α-structure (Right).

Possible Role of the Fibers During Phage Infection.

The minor surface decorating protein Soc of bacteriophage T4 enhances the stability of the capsid. Thus, possibly, gp8.5 might have a similar function for bacteriophage ϕ29. This possibility was investigated by treating fibered and fiberless ϕ29 particles each with buffers at pH 3, pH 10.0, or in 3 M guanidinium chloride. The treated particles were then inspected using cryoEM. The results showed that almost all the capsids were still intact. However, most of them had lost their packaged DNA (Fig. S3). Furthermore, the treated particles produced fewer plaques compared to untreated particles in a standardized plaque assay (Fig. S3). Both types of particles were affected about equally by the above conditions. Thus, gp8.5 does not appear to be required for stabilizing a ϕ29 capsid.

Crystallization of the fiber proteins showed that phosphate was essential for obtaining well-diffracting crystals, suggesting that the protein may have weak interactions with the phosphate buffer. The major component of the Bacillus subtilis cell wall teichoic acid is the glycosylated polyglycerol phosphate. Thus the head fibers may also have weak interactions with the cell teichoic acids and function as an enhancer for the attachment of the phage to the cell wall. To further investigate the role of the fibers during infection, fibered or fiberless ϕ29 infected cells were frozen under high pressure 30 min after infection. The frozen samples were then slowly substituted with resin. The fixed cells were sliced and examined with an electron microscope. The results showed that most of the fibered particles were well aligned on the cell wall, whereas the fiberless particles attached to the cell wall in a roughly random fashion (Fig. 5 and Fig. S4). Although the infectivity of the fiberless particles had apparently not been affected, more of the particles with fibers recognize the cell successfully or can reach the next stage of infection more efficiently. The difference between the fibered and fiberless particles may have a great impact on the evolution of the virus when the concentration of the virus is low.

Fig. 5.

Transmission electron micrographs of thin sections showing a B. subtilis bacterial cell surrounded by ϕ29 phages. (Left) Fibered phages that have arranged themselves in ordered arrays on the bacterial surface. (Right) Fiberless phage that associate with the bacterial surface in a random manner.

Materials and Methods

Protein Expression and Purification.

Gene 8.5 was amplified by PCR from the genome DNA of bacteriophage ϕ29 using the following primers, 5′-GGTGGTCATATGATGGTTTCATTTACT-3′ and 5′-GGTGGTCTCGAGTGATATGATTCCTGCGTT-3′. Preparation of the genome DNA has been reported previously (26). Purified PCR products were cloned into pET30b (Novagen) using the NcoI-XhoI sites that introduce a C-terminal His₆ tag into the recombinant protein. The recombinant 8.5 gene was expressed in E. coli BL21(DE3) CodonPlus-RIL cells at 20 °C. The produced recombinant protein was affinity-purified using cobalt charged BD TALON™ resins and was eluted from the cobalt beads by using an elution buffer containing 50 mM sodium phosphate at pH8.0, 300 mM sodium chloride and 200 mM imidazole. The eluted protein was concentrated to approximately 3 mg/mL and then digested overnight at 37 °C with chymotrypsin (final concentration 0.1 mg/mL). The digested gp8.5 was further purified with a Superdex 200 column (GE) and was eluted with a buffer containing 50 mM Tris at pH 8.0 and 100 mM sodium chloride. Size exclusion chromatography purification of the gp8.5 indicated that it exists mainly as a multimer, most probably a trimer, in solution.

Phage Production and Thin Section Sample Preparation.

Fibered and fiberless ϕ29 particles were produced in Bacillus subtilis su44⁺ or SpoOA12(sup^-1) cells infected with the mutants sus16(300)–sus14(1241) or sus14(1241)–sus8.5(900), respectively, and purified by centrifugation in an isopycnic 60% (w/v) CsCl gradient. The effect of pH or guanidinium chloride on ϕ29 infection was tested by using standard plaque assays. Prior to phage infection, the phage particles were variously incubated for approximately 20 min in buffers at pH 3.0, pH 8.0, pH 10.0, or in 3 M guanidinium chloride solution. The treated phage particles were also examined by using a CM300 transmission electron microscope.

To prepare thin sectioned samples of infected cells, Bacillus subtilis su 44+ cells were grown in 5 mL LB media to an OD600 value of approximately 1.2. The cells were centrifuged at 200  × g for 3 min and then were resuspended in 1 mL buffer containing 50 mM Tris HCl pH 8.0, 100 mM sodium chloride, and 10 mM magnesium chloride. The resuspended cells were split into two equal portions infected with fibered or fiberless ϕ29 particles, respectively, at a multiplicity of infection of 100. About 30 min after the infection, the infected cells were flash-frozen at a high pressure and were fixed at -90 °C in acetone containing 2% (w/w) O_sO₄ and 1% (w/w) uranyl acetate. The fixed samples were slowly substituted over 80 h with resins and then sliced into approximately 100 nm thin sections.

Crystallization.

All crystals were obtained by hanging-drop vapor diffusion at 20 °C, using 3 μL protein (15 mg/mL) mixed with an equal volume of well solution. Crystals of the gp8.5 C-terminal fragment were grown in 100 mM sodium phosphate at pH approximately 6.8 and 8% w/v PEG 3000. It took about one week for crystals to appear. Crystals were soaked for 30 s in the well solution containing a final concentration of 15% w/v PEG 3000 and 15% v/v MPD (2-methyl-2,4-pentanediol) to flash-freeze in a N₂ stream at 100 K. The iodine derivative of the gp8.5 C-terminal fragment was prepared by soaking the crystals in the cryowell solution containing 1 M sodium iodine for 1 min.

X-ray Data Collection, Processing, Structure Determination, Refinement, and Analysis.

X-ray diffraction data were collected using either synchrotron radiation or home source Cu K_α radiation (Table S2). Crystals of the gp8.5 C-terminal fragment diffracted to 1.6 Å and belong to space group R32 with one molecule in the asymmetric unit and cell parameters of a = b = 47 Å and c = 425 Å. All data were integrated and scaled with the HKL2000 suite (Table S2) (27).

The structure of the gp8.5 C-terminal fragment was determined by SIRAS using the anomalous signals of iodine atoms measured at the Cu K_α wavelength. Heavy atom sites were located by using the program SHELX (28). The heavy atom parameters were refined and initial phases were calculated using reflections in the resolution region of 50 Å to 2.2 Å with the program SHARP (29, 30) The calculated phases were gradually improved and extended to higher resolution by using solvent density flattening with the program DM (31). The resultant electron density maps are of good quality in which most of the residues can be clearly recognized. See SI Text for further model building procedures.