Role of bacteriophage T4 baseplate in regulating assembly and infection

Abstract

Bacteriophage T4 consists of a head for protecting its genome and a sheathed tail for inserting its genome into a host. The tail terminates with a multiprotein baseplate that changes its conformation from a "high-energy" dome-shaped to a "low-energy" star-shaped structure during infection. Although these two structures represent different minima in the total energy landscape of the baseplate assembly, as the dome-shaped structure readily changes to the star-shaped structure when the virus infects a host bacterium, the dome-shaped structure must have more energy than the star-shaped structure. Here we describe the electron microscopy structure of a 3.3-MDa in vitro-assembled star-shaped baseplate with a resolution of 3.8 Å. This structure, together with other genetic and structural data, shows why the high-energy baseplate is formed in the presence of the central hub and how the baseplate changes to the low-energy structure, via two steps during infection. Thus, the presence of the central hub is required to initiate the assembly of metastable, high-energy structures. If the high-energy structure is formed and stabilized faster than the low-energy structure, there will be insufficient components to assemble the low-energy structure.

Keywords: bacteriophage T4; baseplate assembly; conformational changes; cryo-EM reconstruction; near-atomic resolution.

Fig. 1.

Schematic diagram of bacteriophage T4. Bacteriophage T4 has a contractile tail and a complex baseplate. Six long-tail fibers are attached to the upper part of the baseplate and six short-tail fibers are folded under the baseplate before infection. Reproduced with permission from ref. , copyright American Society for Microbiology.

Fig. 2.

Cryo-EM 3D reconstruction of the in vitro-assembled hubless baseplate. (A) The cryo-EM density showing the various proteins colored according to the index at the bottom left. (B) Ribbon representation of the protein structures in a single wedge using the same color code. Domains II and III of gp10 are shown as cryo-EM density.

Fig. 3.

Ribbon diagrams of the newly determined protein structures gp7, gp10, gp53, and gp6. Each polypeptide chain is rainbow-colored from blue at the N terminus to red at the C terminus. Protein domains are indicated by roman numerals. Domains II and III of gp10 are shown as cryo-EM density. Helices of gp7 and gp6, involved in the formation of the three-helix coiled-coils located at the interface between wedges, are indicated by red arrows. Also shown diagrammatically is the ring of gp6 dimers that surround the gp27 hub in the dome-shaped baseplate. Each gp6 dimer has a different color. The amino end of each gp6 monomer makes a trimeric coiled-coil with a part of gp7. The wedge boundaries are indicated by a black outline. Each wedge contains one gp6 dimer formed by association of the gp6 C-terminal regions. Contact between the N-terminal regions of gp6 is outlined by a rectangle, whereas the contact between the C-terminal regions of gp6 is outlined by a circle.

Fig. 4.

Assembly of a baseplate based on present and earlier results. (A) Wedge assembly. Gp10, gp8, and gp6 bind sequentially to the gp7 backbone protein. The central hub of the baseplate is assembled independently. (B) Baseplate and tail assembly. Six wedges assemble around the central hub to form a baseplate. Gp53 binds adjacent wedges together. Subsequently, gp9 and the gp11–gp12 complex bind to the baseplate, further stabilizing the dome-shaped configuration. Then, gp48 and gp54 bind to the top of the central hub and initiate polymerization of the tail tube. Gp25 attaches to the gp48–gp54 complex, initiating polymerization of the tail sheath. For clarity, only three rings of the tail sheath are shown.

Fig. 5.

Position of proteins around the central hub. (A) Interactions of gp6 with the central hub protein gp27. The N-terminal domains of gp6 that interact with gp27 (olive) to associate the wedges around the central hub to assemble the dome-shaped baseplate are highlighted in blue. (B) Interactions of adjacent wedges in the baseplate. The helix-turn-helix motifs of gp7 (red) and the two gp6 chains (green and light brown) from adjacent wedges are held together by gp53 (light blue) in the baseplate. An enlargement of the interacting region is shown.

Fig. 6.

Conformational changes of the baseplate during infection. Domains II and III of gp10, gp48, gp54, and the proximal part of the tail tube are presented as electron density. The proximal end of LTFs and STFs are presented as rods. Three rings of the tail sheath are shown. The identity of each protein is indicated in the index. (A) The dome-shaped baseplate. (Right) The inner ring of gp6-gp53-gp25 and part of gp7. The ring is attached to the central hub. (B) The final star-shaped baseplate with three rings of the contracted tail sheath. (Right) The inner ring of gp6-gp53-gp25 and part of gp7. The ring is expanded and detached from the central hub. The intermediate star-shaped baseplate presumably would have star-shaped periphery (B, Center) but the inner ring of gp6-gp53-gp25 and part of gp7 remains attached to the central hub (A, Right).

Fig. S1.

Gold-standard FSC curve. Resolution corresponding to 0.143 FSC cutoff is 3.8 Å. The cryo-EM 3D reconstruction map for the in vitro-assembled, hubless baseplate-like structure reported in this study is estimated as 3.8 Å resolution.

Fig. S2.

Representative areas of the density map around (A) gp7, (B) gp6, and (C) gp53.