Role of bacteriophage T4 baseplate in regulating assembly and infection

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.Most bacteriophages have a tail. At the distal end of the tail there is usually a baseplate that is decorated by some fibers (1). The baseplate initiates infection when the tail fibers bind to a host cell. Signals are transmitted from the tail fibers via the baseplate to the tail that then trigger the ejection of the phage genome from the head into the host cell through the tail tube. Two evolutionary related structures, of pyocin (2, 3) and of the type VI secretion system (4, 5), are found in bacteria as defense systems to kill competing bacteria. These structures are remarkably similar to the tail baseplate structure of bacteriophages, suggesting that tail baseplate-like structures are effective organelles for infecting bacteria (6, 7).T4 is a member of the Myoviridae family of bacteriophages. These phages have a sheath around the tail tube that contracts during infection (Fig. 1) (8). T4 has a complex baseplate that is essential for assuring a highly efficient infection mechanism (8). After recognition of an Escherichia coli host cell by some of the six long-tail fibers (LTF), the short-tail fibers (STF) that are a part of the baseplate, bind irreversibly to the cell. This process is accompanied by a large conformational change in the baseplate from a “high-energy” dome- to a “low-energy” star-shaped structure (9, 10), although each of these structures represent an energy minimum in the energy landscape of the baseplate assembly. This change triggers contraction of the tail sheath, driving the tail tube into the outer host cell membrane and further across the periplasmic space to the inner membrane. The genomic DNA is then ejected into the host’s cytoplasm. Hence, the baseplate serves as the nerve center for transmitting signals from the tail fibers to the head for the release of DNA into the host.Open in a separate windowFig. 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. 50, copyright American Society for Microbiology.The hexagonal dome-shaped T4 baseplate assembles from six wedges surrounding a central hub (8). A total of 134 protein subunits from 15 different proteins form the ∼6.5-MDa baseplate (8, 11). The structure of the T4 baseplate has been studied extensively by cryoelectron microscopy (cryo-EM) of the whole virus and X-ray crystallography of individual proteins (12). Cryo-EM maps of the baseplate when in the dome- (9) and star-shaped (10) conformations were previously reported to 12 Å and 16 Å resolution, respectively. The dome-shaped baseplate was found to be ∼520 Å in diameter and ∼270 Å high, whereas the diameter and height of the star-shaped baseplate were ∼610 Å and ∼120 Å, respectively.

Table S1.

List of the baseplate proteins, their position and interacting protein partners in the tail and their putative role

Protein	No. of residue	Oligomeric state in solution	Position and interacting protein partner(s)	Putative role
gp5	575	Trimer	Hub	Lysis of host cell membrane
			gp5.4, gp27
gp5.4	97	Monomer	Distal end of the tail	Puncturing of host cell membrane
			gp5
gp6	660	Dimer	Wedge	Inter wedge interaction and wedge to hub interaction
			gp7, gp8, gp25, gp27, gp53
gp7	1032	Monomer	Wedge	Connecting the wedge proteins
			gp6, gp8, gp9, gp10, gp53
gp8	334	Dimer	Wedge	Raising the baseplate periphery
			gp6, gp7, gp10
gp9	288	Trimer	Wedge	Attachment of LTF
			gp7, LTF
gp10	602	Trimer	Wedge	Attachment of STF
			gp7, gp8, gp11, gp12
gp11	219	Trimer	Baseplate periphery	Bending and attachment of STF
			gp10, gp12
gp12 (STF)	527	Trimer	Baseplate periphery	Irreversible binding to host cell receptor
			gp10, gp11
gp25	132	Monomer	Wedge	Initiation of the tail sheath polymerization
			gp6, gp18, gp48–gp54, gp53
gp27	391	Trimer	Hub	Wedge to hub interaction
			gp5, gp6, gp48–gp54
gp29	590	ND	Hub	Tail length determination
			gp3, gp19, gp48–gp54
gp48	364	ND	Hub	Termination of baseplate assembly
			gp19, gp25, gp27, gp29, gp54
gp53	196	Monomer	Wedge	Holding of inter wedge junction
			gp6, gp7, gp25
gp54	320	ND	Hub	Initiation of the tail tube polymerization
			gp19, gp27, gp29, gp48

Open in a separate windowLTF, long-tail fiber; STF, short tail fiber; ND, not determined.The assembly of a wedge had been shown to follow a strictly ordered sequence. First, an initial complex is formed by a monomer of gp7 and a trimer of gp10, followed sequentially by binding of a dimer of gp8 and a dimer of gp6 to the complex (13, 14). In the absence of a central hub, at least five proteins (gp7, gp10, gp8, gp6, and gp53) are required for assembly of wedges in vitro into a star-shaped, low-energy baseplate-like structure (14). Assembly of the high-energy, dome-shaped structure requires the presence of the central hub. However, how the sequential wedge assembly events are regulated remained unknown. In particular, the question remained how the high-energy dome-shaped baseplate could assemble.We report here a 3.8-Å resolution 3D cryo-EM map of a ∼3.3-MDa in vitro-assembled star-shaped, hubless, baseplate-like complex (Fig. 2). The component proteins of this in vitro-assembled baseplate were gp7, gp10, gp8, gp6, and gp53. We show that gp7 provides the primary control of the sequential assembly events that regulate the conformational changes of the baseplate during assembly and during infection. We also show that interaction between gp6 and gp27 in associating the wedges around the central hub is the critical nucleation step to form the high-energy dome-shaped baseplate. Furthermore, we describe that the transition of the baseplate from the dome-shaped to the star-shaped conformations probably occurs in two steps.Open in a separate windowFig. 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.