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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2008 Jul 7;105(28):9552–9557. doi: 10.1073/pnas.0803787105

Crystal and cryoEM structural studies of a cell wall degrading enzyme in the bacteriophage φ29 tail

Ye Xiang †, Marc C Morais †,‡, Daniel N Cohen §, Valorie D Bowman †, Dwight L Anderson §, Michael G Rossmann †,
PMCID: PMC2474476  PMID: 18606992

Abstract

The small bacteriophage φ29 must penetrate the ≈250-Å thick external peptidoglycan cell wall and cell membrane of the Gram-positive Bacillus subtilis, before ejecting its dsDNA genome through its tail into the bacterial cytoplasm. The tail of bacteriophage φ29 is noncontractile and ≈380 Å long. A 1.8-Å resolution crystal structure of gene product 13 (gp13) shows that this tail protein has spatially well separated N- and C-terminal domains, whose structures resemble lysozyme-like enzymes and metallo-endopeptidases, respectively. CryoEM reconstructions of the WT bacteriophage and mutant bacteriophages missing some or most of gp13 shows that this enzyme is located at the distal end of the φ29 tail knob. This finding suggests that gp13 functions as a tail-associated, peptidoglycan-degrading enzyme able to cleave both the polysaccharide backbone and peptide cross-links of the peptidoglycan cell wall. Comparisons of the gp13− mutants with the φ29 mature and emptied phage structures suggest the sequence of events that occur during the penetration of the tail through the peptidoglycan layer.

Keywords: hydrolase, infection, structure, zinc ion

Bacterial cells, including Bacillus subtilis, are surrounded by a peptidoglycan-containing cell wall. The peptidoglycan consists of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) residues, cross-linked with oligopeptides at each NAM (1). This highly cross-linked peptidoglycan layer is not only essential for the integrity of bacteria in the face of the osmotic pressure created by the high concentration of molecules in the cell, but also serves as a physical barrier that restricts the passage of macromolecular complexes. In Gram-negative bacteria, the peptidoglycan layer is within the periplasmic space and is ≈25–75 Å thick (2), whereas in Gram-positive bacteria the peptiglycan layer is outside the cytoplasmic membrane and can be as much as 250 Å thick (3). Most bacteriophages have a tail that penetrates the host cell wall and membranes to deliver the genome. Bacteriophage tails are large macromolecular complexes that contain components for recognizing and puncturing the host cell and have a channel wide enough to provide passage for ejection of the viral genome (4–7). The peptidoglycan layer is a major barrier that must be spanned by a phage tail to infect a host cell. Unlike most eukaryotic viruses, infection of a host by tailed bacteriophages usually requires only one virion per bacterium, indicating that tailed bacteriophages have evolved mechanisms that easily surmount the peptidoglycan barrier. Virus-encoded peptidoglycan degrading enzymes, such as endopeptidases, N-acetylmuramyl-l-alanine amidases, N-acetylglucosaminidase, lysozymes, and lytic transglycosylases, have been found widespread in tailed bacteriophages (8, 9). These enzymes are known to play a significant role in facilitating tailed bacteriophage genome entry through localized peptidoglycan degradation or rearrangement (9, 10) and are frequently structural components of the virion. Their location within the virions is varied, but they are located mostly within the tail (8, 11, 12).

Bacteriophage φ29 infects the Gram-positive bacterium, B. subtilis, and is one of the smallest known tailed phages (13). The mature φ29 particle contains a prolate head and a short noncontractible tail. The head and part of the tail are filled with the 19-kbp DNA genome and the terminal protein gene product 3 (gp3) (7) that is covalently attached to both 5′ ends of the DNA genome (14). Previous studies show that the head consists of three proteins: the capsid protein (gp8), the head fibers (gp8.5), and the portal protein (connector) (gp10) (13, 15, 16). The tail had been found to consist of three proteins: gp11, gp12* (cleaved from the gp12 precursor), and gp9, that form the lower collar, appendages, and knob, respectively (Fig. 1) (7, 17). Biochemical data (8) have suggested that the peptidoglycan-degrading activity associated with φ29 lies in the DNA terminal protein gp3, although the crystal structure of gp3 does not support such a function (18). However, as gp9 is located at the distal end of the tail, it might be associated with the cell wall-degrading enzyme used by φ29.

Fig. 1.

Fig. 1.

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Diagram of bacteriophage φ29 showing its structural components. (Left) A surface-rendered presentation. (Right) A central section.

The gp13 of bacteriophage φ29 consists of 365 amino acid residues. Although gp13 had not been previously detected in the virion (19), it was known to be an essential morphogenetic factor that functions in the phage tail assembly stage and is indispensable for producing infectious particles (19, 20). In vitro complementation experiments showed that gp13 is required after gp11 has assembled onto the phage head. Furthermore, gp13 has been shown to interact with gp9 in vivo to yield infectious particles (19). Re-examination of gp13 using specific antisera in Western blots showed that it is a structural component of WT φ29, emptied φ29, free tails, and the mutant sus13(330) virus (20, 21). This mutant virus has a nonsense stop codon that results in production of gp13 that is missing the last 34 aa. On the other hand, gp13 was not detected by Western blotting in the tail for the sus13(342) mutant virus (21), which has a nonsense stop codon that results in the production of a vestigial gp13 consisting of only the 32 N-terminal residues. These results demonstrate that gp9, gp11, gp12, and gp13 are all components of φ29 tails (21).

Here, we report cryoEM image reconstructions that suggest gp13 to be located at the end of the tail knob. We also report that the crystal structure of gp13 has two domains that resemble lysozyme-like enzymes and metallo-endopeptidases, showing that it probably functions as a tail-associated peptidoglycan-degrading enzyme.

Results and Discussion

The Structure of gp13.

The full-length gp13 (residues 1–365) formed very thin needle crystals, too small to detect a diffraction pattern. Hence, the protein was degraded with trypsin and the major fragment (residues 1–347, gp13TD) was repurified and crystallized. The crystal structure of gp13TD [supporting information (SI) Text] showed that gp13TD had two spatially well separated domains, although the C-terminal domain was poorly defined. Therefore, three constructs, gp13NTD (residues 1–159, the N-terminal domain), gp13CTD (residues 166–365, the C-terminal domain), and gp13ΔC (residues 1–334, missing the end of the C-terminal domain), were made to study the domains independently. The gp13CTD construct failed to crystallize. Crystals of gp13ΔC were isomorphous with gp13TD, but produced a better electron density map. The resultant refined model of gp13ΔC (Fig. 2a) gave final Rworking and Rfree factors of 23.8% and 28.2%, respectively. Crystals of gp13NTD had a different crystal form and their structure was determined by molecular replacement. The rmsd between equivalent Cα atoms in the gp13ΔC and gp13NTD structures was 0.4 Å.

Fig. 2.

Fig. 2.

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The structure of gp13. (a) Ribbon stereo diagram of gp13ΔC with the N- and C-terminal domains shown in green and purple, respectively. Two flexible regions that were not clearly visible in the electron density maps are shown in coral. The active sites of the N- and C- terminal domains are indicated by arrows. (b) Stereo diagram showing surface charge distribution of the gp13 N-terminal domain. Sugar substrates are shown in a ball-and-stick representation with N, C, and O atoms colored blue, gray, and green, respectively. Sugar residues at sites A, B, C, and D (black bonds) are from the crystal structure of gp13NTD complexed with (NAG)4, whereas sugar residues at sites E and F (gray bonds) are based on homology model building. (c) Stereo diagram showing the interaction between sugar substrates and protein residues. Parts of the protein Cα backbone are shown in a ball-and-stick presentation with Cα atoms colored red. Sugar substrates and protein residues are shown in a ball-and-stick presentation and colored the same as in b.

The N-terminal domain of gp13ΔC had a structure closely similar to hen egg white lysozyme (Dali Z-score of 6.4) (22) and the C-terminal domain was similar to the active form of LytM (23) [Protein Data Bank (PDB) ID code 2B0P] a metallo-endopeptidase (Z score of 11.3) in the M23 family (24). The linker polypeptide between the two domains consists of six sequential glycine residues (160–165) defined by rather poor electron density in the gp13ΔC map, indicative of flexibility (Fig. 2a). Structure and sequence comparisons provided information about the putative substrate binding sites in the two gp13 domains (Figs. 2a and 3). A long groove (Fig. 2a) on one side of the C-terminal domain forms its putative substrate binding site.

Fig. 3.

Fig. 3.

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Structural-based sequence alignment of gp13, hen egg lysozyme [PDB ID code 3LZT (35)] and active form of lytM [PDB ID code 2B0P (23)]. The secondary structural elements are shown above the alignments. The glycine residues linking the two domains of gp13 are indicated by stars. Residues essential for catalysis are indicated by triangles. Completely conserved residues are shown in white on a gray background.

The N-Terminal Lysozyme-Like Domain of gp13.

Comparisons of φ29 gp13NTD with T4 lysozyme (PDB ID code 3LZM), bacteriophage lambda lysozyme (PDB ID code 1AM7), hen egg white lysozyme (PDB ID code 3LZT), and goose egg white lysozyme (PDB ID code 153L) showed that φ29 had the closest structural similarity to lambda lysozyme (Table 1). Although lambda lysozyme has a similar fold and substrate binding sites as the other lysozymes, unlike the other lysozymes it does not require a water molecule in its enzymatic action and hence should be referred to as a transglycosylase.

Table 1.

Structure and sequence comparison of gp13NTD and lysozyme-like proteins

Characteristics Hen egg white lysozyme (PDB ID code 3LZT) Goose egg white lysozyme (PDB ID code 153L) Phage lambda lysozyme (PDB ID code 1AM7) T4 lysozyme (PDB ID code 3LZM)
No. of residues aligned† 102 (129) 109 (185) 112 (154) 71 (164)
Identical residues among aligned residues, % 12 14 10 11
rmsd, Å 3.3 3.0 3.2 3.9
Secondary structure similarity‡ α1, α2, α3, α4, α5, α6, α7, β1, β2, β3 α1, α2, α3, α4, α5, α6, α7, β1, β2, β3 α1, α2, α3, α4, α5, α6, α7, β1, β2, β3 α1, α2, α3, α4, α5, α6, α7, β1, β2, β3
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†The complete amino acid sequence length of the corresponding lysozyme-like protein are given in parentheses.

‡α 1–7 and β 1–3 represent the secondary structural elements of gp13NTD. Equivalent secondary structures present in lysozyme-like proteins are shown in bold.

The substrate binding site of lysozymes is a groove that can accommodate a polysaccharide with up to six single sugar residues at sites A through F (25, 26) with the cleavage site occurring between sites D and E. Lysozymes cleave the β(1–4) glycosidic bonds between NAM at site D and NAG at site E (27). The catalytic center includes an invariable glutamic acid residue and a less conserved aspartic acid residue. The latter probably stabilizes the cleavage intermediate in some lysozyme-like proteins (28). A structural comparison of gp13NTD with other lysozymes shows that Glu-45 in gp13 corresponds to the catalytically essential glutamic acid residue. The aspartic acid residue in the active center, present in both hen egg white lysozyme (Asp-52) (25) and T4 lysozyme (Asp-20) (29), corresponds to Gly-90 in gp13. A glycine occurs at the corresponding position in bacteriophage lambda lysozyme (30) and goose egg white lysozyme (31). The structures of gp13NTD complexed with (NAG)x (where 3 ≤ x ≤ 6) showed that the side chain of Asn-54 is 4.3 Å from the C1 carbon of the sugar at substrate site D. Thus Asn-54 might play a role in stabilizing the substrate during catalysis.

Although the physiological substrate of lysozyme is a polysaccharide of alternating NAG and NAM residues, lysozymes can also bind and hydrolase chitin-like (NAG)n polysaccharides (27). Cocrystallizations of gp13NTD with (NAG)x (where 3 ≤ x ≤ 6) showed good density only at sites B, C, and D. These densities were interpreted as sugar rings in chair conformation (Fig. 2 b and c). This result indicated that sugar substrates longer than three residues were, in general, cleaved during the cocrystallization procedure, leaving only the cleaved product in the substrate-binding pocket. NAG hydrolysis was confirmed by electrospray ionization mass spectrometer analyses of sugar substrates before and after being incubated with gp13NTD (data not shown), showing that gp13NTD had lysozyme activity.

C-Terminal Domain.

The defining feature of the M23 family of metallo-peptidases is an antiparallel β-sheet and a conserved HXH motif that binds a metal ion (24) at the active center. There is a large electron density peak (8σ) in the gp13 C-terminal domain electron density, corresponding to a bound metal ion associated with the HXH motif. This metal cation is tetrahedrally coordinated by two histidine residues (H188 and H280), an aspartic acid residue (D195), and a water molecule (O59) (Fig. S1). Crystal structures of M23 metallo-endopeptidodases have a zinc ion bound at their metal binding center even when no exogenous zinc ions had been added. Thus, the ion in the crystal structure of gp13 was suspected to be zinc. This notion was confirmed by an x-ray fluorescence scan that showed the expected absorption maximum at a wavelength of 1.283 Å, near the K-edge of zinc.

LytM is synthesized as a preenzyme in vivo. The zinc ion is not solvent accessible in inactive LytM (23, 32). A proteolytic modification is required for activation of the enzyme that cleaves a loop blocking the active center. In the current gp13 structure, the substrate binding site of the C-terminal domain is accessible to solvent molecules, indicating that the gp13 C-terminal domain is in the active form.

LytM can cleave pentaglycine cross-links in peptidoglycan layers of staphylococci (32). The structure of active LytM (23) shows that a groove formed by four protruding loops from the central β-sheet is the substrate binding site. Comparison of the substrate binding groove of active LytM with that of the gp13 C-terminal domain shows differences in the lengths and conformations of the four loops forming the substrate binding groove, suggesting that the gp13 C-terminal domain may have different substrate binding specificity than that of LytM. This difference could have been anticipated as B. subtilis has not been shown to have pentaglycine cross-linkages (1).

Biological Functional Unit Formed by Neighboring Molecules in the Crystal Structure.

The largest buried surface area (764 Å2) between adjacent molecules in the gp13ΔC crystal is between the N- and C-terminal domains belonging to neighboring molecules related by a 21 screw axis along b (Fig. 4). A tetrasaccharide (NAG-NAM-NAG-NAM) built into the N-terminal domain of one molecule would place a pentapeptide (l-Ala-d-γ-Glu-l-Lys-d-Ala-d-Ala) associated with the NAM at site F (Fig. 2c), close to the peptide-binding groove in the C-terminal domain of the neighboring molecule. The docked peptidoglycan structure corresponds to the chemical composition of a bacterial cell wall (33). Thus, the docking suggests that the crystallographically observed association between neighboring molecules might be a biologically functional unit. The predicted cleavage sites derived from the modeling would be between the NAG and NAM residues at sites D and E and between the d-γ-Glu and l-Lys residues or between the l-Lys and d-Ala residues of the attached peptide. The predicted cleavage sites are consistent with the observations of the peptidoglycan cleaved product compositions (D.N.C., Yuk K. Sham, Greg D. Haugstad, Y.X., M.G.R., D.L.A., and David L. Popham, unpublished work) that occur when gp13 digests cell wall fragments.

Fig. 4.

Fig. 4.

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Schematic view of the four molecules in the unit cell and their contacts with symmetry-related neighboring molecules. Molecules A and B, related by a pseudo translation in c, are colored purple. Symmetry-related molecules to A and B are colored green. Molecules C and D, related by a pseudo translation in c, are colored blue. Symmetry related molecules to C and D are colored yellow. A potential biological functional unit is outlined in orange. The unit cell is outlined with a black line and the pseudo smaller orthorhombic unit cell is outlined with a dashed black line. The pseudo symmetry elements of the smaller unit cell are not shown. CTD, C-terminal domain; NTD, N-terminal domain.

Locating gp13 in the φ29 Tail.

The structure of the φ29 tail has a conical protrusion at its distal end in DNA-emptied WT particles (7). This protrusion appeared to be absent in WT full infectious particles, but re-examination has now shown that there is a weak density that might correspond to a somewhat disordered conical protrusion (Fig. 5ai). Asymmetric cryoEM reconstructions of the mutant sus13(330) (containing gp13 missing only the last 34 aa) and mutant sus13(342) (which should contain only the N-terminal 32 aa of gp13) particles both show the 5-fold symmetry of the head and the presence of 12 appendages, features present also in the WT mature and emptied particles. However, the sus13(342) particles were missing the protruding tail cone (Fig. 5aiv), whereas the sus13(330) particles (Fig. 5aiii) had a well formed, cone-shaped density at the distal end of the tail knob. This finding suggests that the tail cone is an assembly of gp13 molecules. A possible explanation is that the gp13 is somewhat disordered in the WT particle, but becomes more static after the genome has been ejected or when the carboxyl-terminal 34 residues of gp13 are missing. The external nature of gp13 is also made manifest by a difference map (Fig. 5biii) between the cryoEM density of the mutant sus13(330) particle (Fig. 5bi) and the mutant sus13(342) particle (Fig. 5bii), which shows only the cone-shaped density at the tip of the knob. Thus, there is no evidence for gp13 being within the phage tail. Whether gp13 is released from the virion in vivo during genome injection into the host remains unknown.

Fig. 5.

Fig. 5.

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The cone shaped gp13 density at the distal end of the phage tail. (a) Comparison of the cryoEM densities of WT DNA-filled (green; i), WT DNA-emptied (cyan; ii), mutant sus13(330) (blue; iii), and sus13(342) (purple; iv) tails. (b) Construction of the cryoEM density difference maps (iii and iv) [sus13(330) (blue; i)–sus13(342) (purple; ii)] showing the cone-shaped density at the distal end of the sus13(330) knob is the most significant difference in the tail. (c) Diagrammatic figure showing the WT, sus13(330), and sus13(342) gp13 peptides.

Variation in the hinge angle between the two domains observed crystallographically and the poor density of the hexa-glycine linker shows that the peptide link is extremely flexible. Thus, when fitting the gp13 molecular structure into the doughnut-shaped density representing the cone, it cannot be assumed that the two domains of gp13 have the same spatial relationship as they do in the crystal structure. The resolution of the cryoEM density was insufficient to be able to fit the rather spherical N and C domains independently. Instead, it was tentatively assumed that the close association of the N and C domains of neighboring molecules in the crystal structure of gp13ΔC might be the enzymatically active unit as suggested above. Two of these active units could be readily fitted into the 2-fold averaged cryoEM density (Fig. 6). This fit showed that two molecules of gp13 were able to account for the cone density, thus probably creating a symmetry mismatch with the knob containing about nine gp9 molecules.

Fig. 6.

Fig. 6.

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Stereo diagrams showing two gp13 monomers (blue and red) fitted into the 2-fold averaged cryoEM density of the emptied WT (green contours) and mutant full sus13(330) (black contours) cone densities. End-on (a) and side (b) views of a 40-Å-thick cross-section.

The fit of gp13 into the cone density placed the active sites of the two domains onto the outer distal ring of the cone, a location suitable for digesting the bacterial peptidoglycan cell wall. Furthermore, the gp13 fit into the tail cone places a lot of negative charge into the central channel, much as the gp10 dodecameric connector has a negatively charged inner channel (16). This feature may be to repel the negatively charged phosphates on the DNA, thereby focusing the DNA to the center of the channel, implying that the gp13 cone is present during DNA ejection from the virus.

Although the structural results are consistent within themselves, the location of gp13 in the cone at the distal end of the knob is in conflict with results on the aggregation of φ29 particles in the presence of anti-gp13 polyclonal antibodies (21). Finding a hypothesis that satisfies both the structural data presented here and the immunological data (21) is a challenge for future investigations.

Materials and Methods

Protein Expression, Purification, Crystallization, Data Collection, and Processing.

For details see SI Text.

Structure Determination and Refinement.

The structure of gp13TD was determined with a mercury derivative, using the single isomorphous replacement method enhanced by anomalous scattering data (SIRAS). The initial process was simplified by reindexing assuming a smaller unit cell (Fig. 4) so as to omit all reflections with l odd. The program SOLVE found four heavy atom sites in the asymmetric unit of the smaller pseudo unit cell. The structure had interpretable electron density for the N-terminal domain, but the C-terminal domain density was poorly defined. The partial structure was used as a molecular replacement search model for determining the orientation and position of each of the four independent molecules in the asymmetric unit of gp13ΔC (A, B, C, and D in Fig. 4) while using the l odd reflections. Further details are given in SI Text and Table S1.

CryoEM Reconstruction.

The production and purification of sus13(330) and sus13(342) particles has been described (21). Electron micrographs were recorded at a magnification of 33,000 with a CM300 FEG microscope under low-dose conditions. Both mutant virus reconstructions assumed no symmetry and were based on a procedure as described (4, 7, 34). The N- and C-terminal domains of gp13 were initially fitted manually into the cryoEM density map by using the program Chimera and then optimized with the program EMfit, assuming 2-fold symmetry. Further details are in SI Text and Table S2.

Supplementary Material

Supporting Information
supp_105_28_9552__index.html (642B, html)

Acknowledgments.

We thank Kay Choi and Petr Leiman for many helpful discussions; Sheryl Kelly and Cheryl Towell for the preparation of the manuscript; and Siyang Sun and the staff at the APS, BioCars, and GM/CA sectors for their help in x-ray diffraction data collection. Those facilities are supported by the U.S. Department of Energy and/or the National Institutes of Health. This work was supported by National Science Foundation Grant MCB-0443899 (to M.G.R.) and National Institutes of Health Grant DE03606 (to D.L.A. and M.G.R.).

Footnotes

The authors declare no conflict of interest.

Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 3CSQ, 3CSR, 3CSZ, 3CT0, 3CT1, and 3CT5). The cryoEM maps have been deposited in the Electron Microscopy Data Bank, www.ebi.ac.uk/msd-srv/docs/emdb (accession nos. EMD1506 and EMD5010).

This article contains supporting information online at www.pnas.org/cgi/content/full/0803787105/DCSupplemental.

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