The Molecular Architecture of the Bacteriophage T4 Neck

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Abstract

A hexamer of the bacteriophage T4 tail terminator protein, gp15, attaches to the top of the phage tail stabilizing the contractile sheath and forming the interface for binding of the independently assembled head. Here we report the crystal structure of the gp15 hexamer, describe its interactions in T4 virions that have either an extended tail or a contracted tail, and discuss its structural relationship to other phage proteins. The neck of T4 virions is decorated by the “collar” and “whiskers”, made of fibritin molecules. Fibritin acts as a chaperone helping to attach the long tail fibers to the virus during the assembly process. The collar and whiskers are environment-sensing devices, regulating the retraction of the long tail fibers under unfavorable conditions, thus preventing infection. Cryo-electron microscopy analysis suggests that twelve fibritin molecules attach to the phage neck with six molecules forming the collar and six molecules forming the whiskers.

Graphical Abstract

Highlights

► The T4 gp15 protein and the phage tail tube proteins evolved from a common ancestor. ► Electrostatic interactions are important for T4 neck assembly and function. ► Phage T4 contains twelve fibritin molecules, with six of these molecules forming the phage collar and six forming the whiskers.

Introduction

Bacteriophage T4 has long served as a model system for molecular and structural biology. Its structure and assembly have been extensively studied using biochemical methods, cryo-electron microscopy (cryo-EM), and X-ray crystallography (for recent reviews, see Refs. [1], [2], [3]). T4 is a lytic phage that uses Escherichia coli as a host and belongs to the Myoviridae family, characterized by contractile tails. The T4 virion (Fig. 1) has a 1200-Å-long and 860-Å-wide head, and a 1000-Å-long tail containing a rigid central tube surrounded by a contractile sheath.[4], [5], [6] At the distal end of the tail, there is a multiprotein baseplate7 to which six long and six short tail fibers are attached. A different set of fibers attach to the neck region of T4 virions, near the head-to-tail interface, forming the phage collar and whiskers.[8], [9]

The T4 head and tail are assembled via independent pathways. Assembly of the T4 head is a complex process that includes a number of intermediate stages.[2], [10], [11] The head assembly is initiated by the dodecamer of the portal protein, gp20 (gp, gene product). First, a head precursor, called the prohead, is assembled, which is subsequently processed by a scaffold-associated protease. Then the phage genomic DNA is packaged into the capsid through the portal vertex by an ATP-driven motor composed of five gp17 molecules.12 Upon completion of the DNA packaging, the head assembly is finalized by attachment of several copies of the gp13 and gp14 proteins to the portal vertex.[9], [13] Monomers of gp13 and gp14 have a size of 309 and 256 amino acid (aa) residues, respectively. The gp13–gp14 complex seals the portal vertex and creates a site for attachment of the independently assembled tail. Mutant phages lacking these proteins produce heads that are unable to bind tails and lose their DNA.14

The T4 tail assembly begins with the baseplate formation and proceeds with polymerization of the tail tube and the contractile sheath. The tail tube is formed by gp19 molecules (163 aa residues) and has external and internal diameters of 90 Å and 40 Å, respectively. The length of the tube is controlled by a mechanism involving the “tape-measure protein”, gp29. The elongation of the tail tube is terminated by attachment of the hexamer of the 175-residue tail tube terminator protein, gp3, which binds to the last row of gp19 subunits (probably also to gp29) and stabilizes the tail tube.[15], [16] The tail tube assembly in the Myoviridae phages is similar to that of the Siphoviridae phages that have long non-contractile tails. The tail tube proteins of the Myoviridae phages have a fold that has similarity to the tail tube proteins of Siphoviridae phages and to the tube proteins of the bacterial secretion system VI.[17], [18], [19] The T4 tail tube is used as a scaffold for the polymerization of the contractile sheath. The gp18 sheath molecules (659 aa residues) assemble around the tube in the form of a six-start helix. The assembled sheath has 138 gp18 molecules arranged into 23 hexameric rings, stacked onto each other. Each ring is rotated by 17.2° and translated by 40.6 Å relative to the previous ring. The structure of the gp18M deletion mutant that consisted of three out of four protein domains was determined by X-ray crystallography.20 The deletion mutant did not contain the C-terminal domain that interacts with the tail tube. However, X-ray structures of two sheath proteins LIN1278 [Protein Data Bank (PDB) ID: 3LML] and DSY3957 (PDB ID: 3HXL) encoded in prophages of bacteria Listeria innocua and Desulfitobacterium hafniense were reported later.21 The structures are homologous to gp18 and include the C-terminal domains.

The T4 tail assembly is completed by the hexamer of the tail terminator protein, gp15 (the monomer is 272 aa residues long), which binds to the top† of the tail. The gp15 hexamer interacts with the gp3 hexamer and, presumably, with the C-terminal domains of gp18 molecules located in the last ring of the contractile sheath (Fig. 1c). Mutant tails lacking gp3 also lack gp15 and sheaths of the gp15-lacking tails are unstable.[22], [23]

Contraction of the tail during infection is associated with a substantial rearrangement of the gp18 subunits and results in shortening of the sheath to less than one-half of its original length. The contracted sheath is also a six-start helix with a pitch of 16.4 Å and a twist of 32.9°. After the tail contraction, the C-terminal domains of gp18 do not interact with the tail tube. However, the top gp18 ring of the sheath presumably remains in contact with the gp15 hexamer.

The gp15 protein also creates the binding site for attachment of the capsid. Mutant phage tails lacking gp15 are unable to join the heads. The tail binds to the head via interactions of gp15 with gp14 (or with both gp13 and gp14).

At a late stage of phage assembly, the collar and whiskers,9 made of fibritin molecules, attach to the phage neck via the gp13–gp14 complex (Fig. 1). Each 500-Å-long fibritin molecule is a trimer of the 486-residue product of the late gene wac (whisker antigen control).8 Sequence and structural analyses show that most of the fibritin structure consists of coiled-coil segments.24 Biochemical data suggest that the N-terminal domain of fibritin binds to the phage.25 Structures of the N- and C-terminal parts of fibritin, containing globular domains, were determined using X-ray crystallography.[26], [27], [28] The collar and whiskers serve as molecular chaperons facilitating attachment of the long tail fibers to the phage.29 Fibritin also serves as an environment-sensing device that controls the retraction of the long tail fibers under adverse conditions (e.g., low temperature, low ionic strength), thus preventing undesirable infection.30

Here we report the X-ray structure of the gp15 hexamer and describe its interactions with the T4 neck and tail proteins. We also present a cryo-EM reconstruction of the contracted T4 tail containing the collar and whiskers. The reconstruction suggests that twelve fibritin molecules bind to the phage neck via their N-terminal domains with six molecules forming the collar and six molecules forming the whiskers. The new interpretation of the structure of the collar–whisker complex is different from the previously proposed model6 that assumed six fibritin molecules per virion.

Section snippets

Structure of gp15 and its relationship to other phage proteins

The structure of the hexamer of the gp15 protein, which terminates the T4 tail assembly and stabilizes the contractile sheath, has been determined using X-ray crystallography (Fig. 2 and Table 1) to 2.7 Å resolution. The hexamer has a shape resembling a hexagonal nut with a 40-Å pore through which the genomic DNA exits during the infection. The wall of the pore is formed by six 4-stranded antiparallel β-sheets and six α-helices coming from different gp15 monomers. The central pore and the side

Conclusion

Bacteriophages probably represent the most abundant biological entities on Earth with the estimated number of phage particles to be in the order of 1032 (Refs. [42], [43]). The number of phages outnumber about 10-fold the number of bacteria.44 As phages are a major cause of bacterial mortality, they have a considerable impact on the Earth's ecology. Phages also play an important role in evolution as they participate in the horizontal transfer of genes. Furthermore, phages have potential for

Expression and purification of the full-length gp15 protein and its C-terminal truncation mutant containing residues 1–261

The vector for the expression of the full-length gp15 and the protein over-expression in E. coli cells were described previously.16 Ammonium sulfate was added to the supernatant of gp15 over-expressed cell lysate at a concentration of 25% saturation and gp15 was fractionated by centrifugation. The gp15 precipitates were resuspended and solubilized in 25 mM Tris–HCl (pH 8.0) and subsequently applied to HiTrap Q HP (5 ml; GE Healthcare) anion-exchange column. Proteins were eluted with a 0- to 1-M

Author Contributions

A.F., Z.Z., S.K., A.A.A., F.A., V.B.R., and M.G.R. designed research. A.F., Z.Z., S.K., V.D.B., and A.A.A. performed research. A.F., V.B.R., and M.G.R. wrote the paper.

Conflict of Interest Statement

The authors declare that they have no conflict of interest.

Acknowledgements

We thank Sheryl Kelly for help in the preparation of the manuscript. Use of the Advanced Photon Source (sectors 23 and 14) was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. Support for this project was provided to M.G.R. by the National Institutes of Health (AI081726) and, for early work, by the National Science Foundation (MCB-0443899). Figures were prepared using the program Chimera63 and PyMOL.64

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    Present address: A. A. Aksyuk, Laboratory of Structural Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

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