Crystal structure of a heat and protease-stable part of the bacteriophage T4 short tail fibre.

Adsorption of T4 bacteriophage to the Escherichia coli host cell is mediated by six long and six short tail fibres. After at least three long tail fibres have bound, short tail fibres extend and bind irreversibly to the core region of the host cell lipopolysaccharide (LPS), serving as inextensible stays during penetration of the cell envelope by the tail tube. The short tail fibres consist of a parallel, in-register, trimer of gene product 12 (gp12). The 1.9 A crystal structure of a heat and protease-stable fragment of gp12 reveals three new folds: a central right-handed triple beta-helix, a globular C-terminal domain containing a beta-sandwich and an N-terminal beta-structure reminiscent of but different from the adenovirus triple beta-spiral. The centre of the C-terminal domain shows weak homology to gp11, a trimeric protein connecting the short fibre to the base-plate, suggesting that the trimerisation motifs of gp11 and gp12 are similar. Repeating sequence motifs suggest that the N-terminal beta-structure extends further towards the N terminus and is conserved in the long tail fibre proteins gp34 and gp37.     

Structure and location of gene product 8 in the bacteriophage T4 baseplate

Many bacteriophages, such as T4, T7, RB49, and phi29, have complex, sometimes multilayered, tails that facilitate an almost 100% success rate for the viral particles to infect host cells. In bacteriophage T4, there is a baseplate, which is a multiprotein assembly, at the distal end of the contractile tail. The baseplate communicates to the tail that the phage fibers have attached to the host cell, thereby initiating the infection process. Gene product 8 (gp8), whose amino acid sequence consists of 334 residues, is one of at least 16 different structural proteins that constitute the T4 baseplate and is the sixth baseplate protein whose structure has been determined. A 2.0A resolution X-ray structure of gp8 shows that the two-domain protein forms a dimer, in which each monomer consists of a three-layered beta-sandwich with two loops, each containing an alpha-helix at the opposite sides of the sandwich. The crystals of gp8 were produced in the presence of concentrated chloride and bromide ions, resulting in at least 11 halide-binding sites per monomer. Five halide sites, situated at the N termini of alpha-helices, have a protein environment observed in other halide-containing protein crystal structures. The computer programs EMfit and SITUS were used to determine the positions of six gp8 dimers within the 12A resolution cryo-electron microscopy image reconstruction of the baseplate-tail tube complex. The gp8 dimers were found to be located in the upper part of the baseplate outer rim. About 20% of the gp8 surface is involved in contacts with other baseplate proteins, presumed to be gp6, gp7, and gp10. With the structure determination of gp8, a total of 53% of the volume of the baseplate has now been interpreted in terms of its atomic structure.     

The tail lysozyme complex of bacteriophage T4

The tail baseplate of bacteriophage T4 contains a structurally essential, three-domain protein encoded by gene 5 in which the middle domain possesses lysozyme activity. The gene 5 product (gp5) undergoes post-translational cleavage, allowing the resultant N-terminal domain (gp5*) to assemble into the baseplate as a trimer. The lysozyme activity of the undissociated cleaved gp5 is inhibited until infection has been initiated, when the C-terminal portion of the molecule is detached and the rest of the molecule dissociates into monomers. The 3D structure of the undissociated cleaved gp5, complexed with gp27 (another component of the baseplate), shows that it is a cell-puncturing device that functions to penetrate the outer cell membrane and to locally dissolve the periplasmic cell wall.     

gpwac of the T4-type bacteriophages: structure, function, and evolution of a segmented coiled-coil protein that controls viral infectivity

The wac gene product (gpwac) or fibritin of bacteriophage T4 forms the six fibers that radiate from the phage neck. During phage morphogenesis these whiskers bind the long tail fibers (LTFs) and facilitate their attachment to the phage baseplate. After the cell lysis, the gpwac fibers function as part of an environmental sensing device that retains the LTFs in a retracted configuration and thus prevents phage adsorption in unfavorable conditions. A comparative analysis of the sequences of 5 wac gene orthologs from various T4-type phages reveals that the approximately 50-amino-acid N-terminal domain is the only highly conserved segment of the protein. This sequence conservation is probably a direct consequence of the domain's strong and specific interactions with the neck proteins. The sequence of the central fibrous region of gpwac is highly plastic, with only the heptad periodicity of the coiled-coil structure being conserved. In the various gpwac sequences, the small C-terminal domain essential for initiation of the folding of T4 gpwac is replaced by unrelated sequences of unknown origin. When a distant T4-type phage has a novel C-terminal gpwac sequence, the phage's gp36 sequence that is located at the knee joint of the LTF invariably has a novel domain in its C terminus as well. The covariance of these two sequences is compatible with genetic data suggesting that the C termini of gpwac and gp36 engage in a protein-protein interaction that controls phage infectivity. These results add to the limited evidence for domain swapping in the evolution of phage structural proteins.     

Structure, stability, and biological activity of bacteriophage T4 gene product 9 probed with mutagenesis and monoclonal antibodies

Gene product (gp) 9 connects the long tail fibers and triggers the structural transition of T4 phage baseplate at the beginning of infection process. Gp9 is a parallel homotrimer with 288 amino acid residues per chain that forms three domains. To investigate the role of the gp9 amino terminus, we have engineered a set of mutants with deletions and random substitutions in this part. The structure of the mutants was probed using monoclonal antibodies that bind to either N-terminal, middle, or C-terminal domains. Deletions of up to 12 N-terminal residues as well as random substitutions of the second, third and fourth residues yielded trimers that failed to incorporate in vitro into the T4 9(-)-particles and were not able to convert them into infectious virions. As detected using monoclonal antibodies, these mutants undergo structural changes in both N-terminal and middle domains. Furthermore, deletion of the first twenty residues caused profound structural changes in all three gp9 domains. In addition, N-terminally truncated proteins and randomized mutants formed SDS-resistant "conformers" due to unwinding of the N-terminal region. Co-expression of the full-length gp9 and the mutant lacking first 20 residues clearly shows the assembly of heterotrimers, suggesting that the gp9 trimerization in vivo occurs post-translationally. Collectively, our data indicate that the aminoterminal sequence of gp9 is important to maintain a competent structure capable of incorporating into the baseplate, and may be also required at intermediate stages of gp9 folding and assembly.     

Domain organization and polarity of tail needle GP26 in the portal vertex structure of bacteriophage P22

The attachment of tailed bacteriophages to the host cell wall as well as the penetration and injection of the viral genome into the host is mediated by the virion tail complex. In phage P22, a member of the Podoviridae family that infects Salmonella enterica, the tail contains an approximately 220 A elongated protein needle, previously identified as tail accessory factor gp26. Together with tail factors gp4 and gp10, gp26 is critical to close the portal protein channel and retain the viral DNA inside the capsid. By virtue of its topology and position in the virion, the tail needle gp26 is thought to function as a penetrating device to perforate the Salmonella cell wall. Here, we define the domain organization of gp26, characterize the structural determinants for its stability, and define the polarity of the gp26 assembly into the phage portal vertex structure. We have found that the N-terminal 27 residues of gp26 form a functional domain that, although not required for gp26 trimerization and overall stability, is essential for the correct attachment to gp10, which is thought to plug the portal vertex structure. The region downstream of domain I, domain II, folds into helical core, which exhibits four trimerization octad repeats with consensus Ile-xx-Leu-xxx-Val/Tyr. We demonstrate that in vitro, domain II represents the main self-assembling, highly stable trimerization core of gp26, which retains a folded conformation both in an anhydrous environment and in the presence of 10% SDS. The C terminus of gp26, immediately downstream of domain II, contains a beta-sheet-rich region, domain III, and a short coiled coil, domain IV, which, although not required for gp26 trimerization, enhance its thermodynamic stability. We propose that domains III and IV of the tail needle form the tip utilized by the phage to penetrate the host cell wall.     

Structure and folding of bacteriophage T4 gene product 9 triggering infection. I. Production and properties of recombinant protein

Gene product 9 (gp9, 288 amino acid residues per monomer, molecular weight 30.7 kD) of bacteriophage T4 triggers the baseplate reorganization and the sheath contraction after interaction of the long tail fibers with the receptors of the bacterial cell. In this work we have produced the recombinant protein and determined that gp9 is a stable homotrimer and active in in vitro complementation assay completing the defective phage particles which lack gp9. According to CD-spectroscopy data, the gp9 polypeptide chain contains 65-73% beta-structure and 11-16% alpha-helical segments, this being in good agreement with secondary structure prediction results. Additionally, we have constructed a set of plasmid vectors for expression of gp9 deletion mutants. The fragments with consecutive truncations of the N-terminus of the molecule, as well as the full-length protein, are trimers resistant to SDS treatment and decrease infective phage particle formation in in vitro complementation assay with native gp9. The deletion of the molecule C-terminal region results in failure of trimerization and decreases the stability of the protein.     

Evolution of bacteriophage tails: Structure of T4 gene product 10

The success of tailed bacteriophages to infect cells far exceeds that of most other viruses on account of their specialized tail and associated baseplate structures. The baseplate protein gene product (gp) 10 of bacteriophage T4, whose structure was determined to 1.2 A resolution, was fitted into the cryo-electron microscopy structures of the pre and post-infection conformations of the virus. gp10 functions as a molecular lever that rotates and extends the hinged short tail fibers to facilitate cell attachment. The central folding motif of the gp10 trimer is similar to that of the baseplate protein gp11 and to the receptor-binding domain of the short tail fiber, gp12. The three proteins comprise the periphery of the baseplate and interact with each other. The structural and functional similarities of gp10, gp11, and gp12 and their sequential order in the T4 genome suggest that they evolved separately, subsequent to gene triplication from a common ancestor. Such events are usual in the evolution of complex organelles from a common primordial molecule.     

Structure of the central hub of bacteriophage Mu baseplate determined by X-ray crystallography of gp44

Bacteriophage Mu is a double-stranded DNA phage that consists of an icosahedral head, a contractile tail with baseplate and six tail fibers, similar to the well-studied T-even phages. The baseplate of bacteriophage Mu, which recognizes and attaches to a host cell during infection, consists of at least eight different proteins. The baseplate protein, gp44, is essential for bacteriophage Mu assembly and the generation of viable phages. To investigate the role of gp44 in baseplate assembly and infection, the crystal structure of gp44 was determined at 2.1A resolution by the multiple isomorphous replacement method. The overall structure of the gp44 trimer is similar to that of the T4 phage gp27 trimer, which forms the central hub of the T4 baseplate, although these proteins share very little primary sequence homology. Based on these data, we confirm that gp44 exists as a trimer exhibiting a hub-like structure with an inner diameter of 25A through which DNA can presumably pass during infection. The molecular surface of the gp44 trimer that abuts the host cell membrane is positively charged, and it is likely that Mu phage interacts with the membrane through electrostatic interactions mediated by gp44.     

Identification and crystallisation of a heat- and protease-stable fragment of the bacteriophage T4 short tail fibre.

Irreversible binding of T-even bacteriophages to Escherichia coli is mediated by the short tail fibres, which serve as inextensible stays during DNA injection. Short tail fibres are exceptionally stable elongated trimers of gene product 12 (gp12), a 56 kDa protein. The N-terminal region of gp12 is important for phage attachment, the central region forms a long shaft, while a C-terminal globular region is implicated in binding to the bacterial lipopolysaccharide core. When gp12 was treated with stoichiometric amounts of trypsin or chymotrypsin at 37 degrees C, an N-terminally shortened fragment of 52 kDa resulted. If the protein was incubated at 56 degrees C before trypsin treatment at 37 degrees C, we obtained a stable trimeric fragment of 3 x 33 kDa lacking residues from both the N- and C-termini. Apparently, the protein unfolds partially at 56 degrees C, thereby exposing protease-sensitive sites in the C-terminal region and extra sites in the N-terminal region. Well-diffracting crystals of this fragment could be grown. Our results indicate that gp12 carries a stable central region, consisting of the C-terminal part of the shaft and the attached N-terminal half of the globular region. Implications for structure determination of the gp12 protein and its folding are discussed.     

Structural changes in proteins of the bacteriophage T4 basal plate resulting from the attachment of long fibrils

The effect of the attachment of long tail fibers on the structure of proteins of the bacteriophage T4 baseplate was studied by digital processing of electron microscopic images. The attachment of the long fibers was found to result in dramatical changes of the proteins of the baseplate plag, while the wedges, to which the long fibers are attached, undergo only slight changes. We studied the baseplates with one to six attached fibers and found that the attachment of one fiber resulted in the change of the entire baseplate, although the wedge located in the vicinity of the fiber attachment changed to a greater extent. Only after the attachment of three and more fibers the changes of the same kind occurred through the entire baseplate.     

A shifty stop for a hairy tail

The tail apparatus of the bacteriophage SPP1 is an extraordinary approximately 1600-A-long molecular machine. The tail mediates attachment of the virus to the host surface receptor, as well-as ejection of the viral genome into the host. The distal tip of the tail binds the extracellular ectodomain of the Bacillus subtilis receptor YueB, while the tail tube provides a conduit to funnel the viral genome into the host. This process, which culminates with the ejection of the approximately 44 kb of viral DNA across the thick, cell envelope of the Gram-positive bacterial cell, takes place in a time scale of seconds to minutes and represents a remarkable example of biotransformation. In this issue of Molecular Microbiology, Auzat et al. provide compelling evidence that the two major structural proteins of the SPP1 tail, gp17.1 (approximately 19.1 kDa) and gp17.1* (approximately 28 kDa), share a common N-terminal sequence, and that gp17.1* is generated by a translational frameshift in the gene 17.1. The extra domain fused to gp17.1* is synthesized by a +1 programmed translational frameshift at the end of gene 17.1, which leads to the synthesis of approximately one gp17.1* for every three equivalents of gp17.1. This finding extends our current knowledge of translational frameshifts and provides a framework to understand how Siphoviridae phages like SPP1 have developed long-tail machines using only two major structural proteins.     

Design and crystal structure of bacteriophage T4 mini-fibritin NCCF

Fibritin is a fibrous protein that forms "whiskers" attached to the neck of bacteriophage T4. Whiskers interact with the long tail fibers regulating the assembly and infectivity of the virus. The fibritin trimer includes the N-terminal domain responsible for attachment to the phage particle and for the collar formation, the central domain forming a 500 A long segmented coiled-coil structure, and the C-terminal "foldon" domain. We have designed a "mini" fibritin with most of the coiled-coil domain deleted, and solved its crystal structure. The non-helical N-terminal part represents a new protein fold that tightly interacts with the coiled-coil segment forming a single domain, as revealed by calorimetry. The analysis of the crystal structure and earlier electron microscopy data on the collar-whisker complex suggests the necessity of other proteins to participate in the collar formation. Crystal structure determination of the N-terminal domain of fibritin is the first step towards elucidating the detailed structure and assembly mechanism of the collar-whisker complex.     

Biogenesis of a Bacteriophage Long Non-Contractile Tail

Siphoviruses are main killers of bacteria. They use a long non-contractile tail to recognize the host cell and to deliver the genome from the viral capsid to the bacterial cytoplasm. Here, we define the molecular organization of the Bacillus subtilis bacteriophage SPP1 ~ 6.8 MDa tail and uncover its biogenesis mechanisms. A complex between gp21 and the tail distal protein (Dit) gp19.1 is assembled first to build the tail cap (gp19.1-gp21Nter) connected by a flexible hinge to the tail fiber (gp21Cter). The tip of the gp21Cter fiber is loosely associated to gp22. The cap provides a platform where tail tube proteins (TTPs) initiate polymerization around the tape measure protein gp18 (TMP), a reaction dependent on the non-structural tail assembly chaperones gp17.5 and gp17.5* (TACs). Gp17.5 is essential for stability of gp18 in the cell. Helical polymerization stops at a precise tube length followed by binding of proteins gp16.1 (TCP) and gp17 (THJP) to build the tail interface for attachment to the capsid portal system. This finding uncovers the function of the extensively conserved gp16.1-homologs in assembly of long tails. All SPP1 tail components, apart from gp22, share homology to conserved proteins whose coding genes’ synteny is broadly maintained in siphoviruses. They conceivably represent the minimal essential protein set necessary to build functional long tails. Proteins homologous to SPP1 tail building blocks feature a variety of add-on modules that diversify extensively the tail core structure, expanding its capability to bind host cells and to deliver the viral genome to the bacterial cytoplasm.     

The C-terminal fragment of the precursor tail lysozyme of bacteriophage T4 stays as a structural component of the baseplate after cleavage

Tail-associated lysozyme of bacteriophage T4 (tail lysozyme), the product of gene 5 (gp 5), is an essential structural component of the hub of the phage baseplate. It is synthesized as a 63-kDa precursor, which later cleaves to form mature gp 5 with a molecular weight of 43,000. To elucidate the role of the C-terminal region of the precursor protein, gene 5 was cloned and overexpressed and the product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, analytical ultracentrifugation, and circular dichroism. It was shown that the precursor protein tends to be cleaved into two fragments during expression and that the cleavage site is close to or perhaps identical to the cleavage site in the infected cell. The two fragments, however, remained associated. The lysozyme activity of the precursor or the nicked protein is about 10% of that of mature gp 5. Both the N-terminal mature tail lysozyme and the C-terminal fragment were then isolated and characterized by far-UV circular dichroism and analytical ultracentrifugation. The latter remained trimeric after dissociation from the N-terminal fragment and is rich in beta-structure as predicted by an empirical method. To trace the fate of the C-terminal fragment, antiserum was raised against a synthesized peptide of the last 12 C-terminal residues. Surprisingly, the C-terminal fragment was found in the tail and the phage particle by immunoblotting. The significance of this finding is discussed in relation to the molecular assembly and infection process.     

Functional Role of the N-Terminal Domain of Bacteriophage T4-Gene Product 11

Bacteriophage T4 late gene product 11 (gp11), the three-dimensional structure of which has been solved by us to 2.0 A resolution, is a part of the virus' baseplate. The gp11 polypeptide chain consists of 219 amino acid residues and the functionally active protein is a three-domain homotrimer. In this work, we have studied the role of gp11 N-terminal domain in the formation of a functionally active trimer. Deletion variants of gp11 and monoclonal antibodies recognizing the native conformation of gp11 trimer have been selected. Long deletions up to a complete removal of the N-terminal domain, containing 64 residues, do not affect the gp11 trimerization, but considerably change the protein structure and lead to the loss of its ability to incorporate into the baseplate. However, the deletion of the first 17 N-terminal residues results in functionally active protein that can complete the 11(-)-defective phage particles in in vitro complementation assay. This region of the polypeptide chain is probably essential for gp11-gp10 stable complex formation at the early stages of phage baseplate assembly in vivo. A study of the gp10 deletion variants suggests that the central domain of gp10 trimer is responsible for the interaction with gp11.     

Crystal Structure of pb9, the Distal Tail Protein of Bacteriophage T5: a Conserved Structural Motif among All Siphophages

The tail of Caudovirales bacteriophages serves as an adsorption device, a host cell wall-perforating machine, and a genome delivery pathway. In Siphoviridae, the assembly of the long and flexible tail is a highly cooperative and regulated process that is initiated from the proteins forming the distal tail tip complex. In Gram-positive-bacterium-infecting siphophages, the distal tail (Dit) protein has been structurally characterized and is proposed to represent a baseplate hub docking structure. It is organized as a hexameric ring that connects the tail tube and the adsorption device. In this study, we report the characterization of pb9, a tail tip protein of Escherichia coli bacteriophage T5. By immunolocalization, we show that pb9 is located in the upper part of the cone of the T5 tail tip, at the end of the tail tube. The crystal structure of pb9 reveals a two-domain protein. Domain A exhibits remarkable structural similarity with the N-terminal domain of known Dit proteins, while domain B adopts an oligosaccharide/oligonucleotide-binding fold (OB-fold) that is not shared by these proteins. We thus propose that pb9 is the Dit protein of T5, making it the first Dit protein described for a Gram-negative-bacterium-infecting siphophage. Multiple sequence alignments suggest that pb9 is a paradigm for a large family of Dit proteins of siphophages infecting mostly Gram-negative hosts. The modular structure of the Dit protein maintains the basic building block that would be conserved among all siphophages, combining it with a more divergent domain that might serve specific host adhesion properties.     

Three-dimensional structure of bacteriophage T4 baseplate

The baseplate of bacteriophage T4 is a multiprotein molecular machine that controls host cell recognition, attachment, tail sheath contraction and viral DNA ejection. We report here the three-dimensional structure of the baseplate-tail tube complex determined to a resolution of 12 A by cryoelectron microscopy. The baseplate has a six-fold symmetric, dome-like structure approximately 520 A in diameter and approximately 270 A long, assembled around a central hub. A 940 A-long and 96 A-diameter tail tube, coaxial with the hub, is connected to the top of the baseplate. At the center of the dome is a needle-like structure that was previously identified as a cell puncturing device. We have identified the locations of six proteins with known atomic structures, and established the position and shape of several other baseplate proteins. The baseplate structure suggests a mechanism of baseplate triggering and structural transition during the initial stages of T4 infection.     

Mapping of functional sites on the primary structure of the contractile tail sheath protein of bacteriophage T4 by mutation analysis

In order to determine the functional roles of amino acid residues in gp18 (gp: gene product), the contractile tail sheath protein of bacteriophage T4, the mutation sites and amino acid replacements of available and newly created missense mutants with distinct phenotypes were determined. Amber mutants were also utilized for amino acid insertion by host amber suppressor cell strains. It was found that mutants that gave rise to a particular phenotype were mapped in a particular region along the polypeptide chain. Namely, all amino acid replacements in the cold-sensitive mutants (cs, which grows at 37 degrees C, but not at 25 degrees C) and the heat-sensitive mutant (hs, lose viability by incubation at 55 degrees C for 30 min) except for one hs mutant were mapped in a limited region in the C-terminal domain. On the other hand, all the temperature-sensitive mutants (ts, grow at 30 degrees C, but not at 42 degrees C) and carbowax mutants (CBW, can adsorb to the host bacterium in the presence of high concentrations of polyethylene glycol, where wild-type phage cannot) were mapped in the N-terminal protease-resistant domain, except for one ts mutant. The results suggested that the C-terminal region of gp18 is important for contraction and assembly, whereas the N-terminal protease-resistant domain constitutes the protruding part of the tail sheath.     

The opening of the SPP1 bacteriophage tail, a prevalent mechanism in Gram-positive-infecting siphophages

The SPP1 siphophage uses its long non-contractile tail and tail tip to recognize and infect the Gram-positive bacterium Bacillus subtilis. The tail-end cap and its attached tip are the critical components for host recognition and opening of the tail tube for genome exit. In the present work, we determined the cryo-electron microscopic (cryo-EM) structure of a complex formed by the cap protein gp19.1 (Dit) and the N terminus of the downstream protein of gp19.1 in the SPP1 genome, gp21(1-552) (Tal). This complex assembles two back-to-back stacked gp19.1 ring hexamers, interacting loosely, and two gp21(1-552) trimers interacting with gp19.1 at both ends of the stack. Remarkably, one gp21(1-552) trimer displays a "closed" conformation, whereas the second is "open" delineating a central channel. The two conformational states dock nicely into the EM map of the SPP1 cap domain, respectively, before and after DNA release. Moreover, the open/closed conformations of gp19.1-gp21(1-552) are consistent with the structures of the corresponding proteins in the siphophage p2 baseplate, where the Tal protein (ORF16) attached to the ring of Dit (ORF15) was also found to adopt these two conformations. Therefore, the present contribution allowed us to revisit the SPP1 tail distal-end architectural organization. Considering the sequence conservation among Dit and the N-terminal region of Tal-like proteins in Gram-positive-infecting Siphoviridae, it also reveals the Tal opening mechanism as a hallmark of siphophages probably involved in the generation of the firing signal initiating the cascade of events that lead to phage DNA release in vivo.