Morphogenesis of the T4 tail and tail fibers

Abstract

Remarkable progress has been made during the past ten years in elucidating the structure of the bacteriophage T4 tail by a combination of three-dimensional image reconstruction from electron micrographs and X-ray crystallography of the components. Partial and complete structures of nine out of twenty tail structural proteins have been determined by X-ray crystallography and have been fitted into the 3D-reconstituted structure of the "extended" tail. The 3D structure of the "contracted" tail was also determined and interpreted in terms of component proteins. Given the pseudo-atomic tail structures both before and after contraction, it is now possible to understand the gross conformational change of the baseplate in terms of the change in the relative positions of the subunit proteins. These studies have explained how the conformational change of the baseplate and contraction of the tail are related to the tail's host cell recognition and membrane penetration function. On the other hand, the baseplate assembly process has been recently reexamined in detail in a precise system involving recombinant proteins (unlike the earlier studies with phage mutants). These experiments showed that the sequential association of the subunits of the baseplate wedge is based on the induced-fit upon association of each subunit. It was also found that, upon association of gp53 (gene product 53), the penultimate subunit of the wedge, six of the wedge intermediates spontaneously associate to form a baseplate-like structure in the absence of the central hub. Structure determination of the rest of the subunits and intermediate complexes and the assembly of the hub still require further study.

Figure 1

Structure of bacteriophage T4. (A) Schematic representation; CryoEM-derived model of the phage particle prior to (B) and upon (C) host cell attachment. Tail fibers are disordered in the cryoEM structures, as they represent the average of many particles each having the fibers in a slightly different conformation.

Figure 2

Assembly of the tail. Rows A, B and C show the assembly of the wedge; the baseplate and the tail tube with the sheath, respectively.

Figure 3

Assembly of (gp27-gp5*-gp5C)₃; reprinted from [13]. A, Domain organization of gp5. The maturation cleavage is indicated with the dotted line. Initial and final residue numbers are shown for each domain. B, Alignment of the octapeptide units composing the intertwined part of the C-terminal β-helix domain of gp5. Conserved residues are in bold print; residues facing the inside are underlined. The main chain dihedral angle configuration of each residue in the octapeptide is indicated at the top by κ (kink), β (sheet), and α (helix). C Assembly of gp5 and gp27 into the hub and needle of the baseplate.

Figure 4

Structure of the gp5-gp27 complex. A, The gp5-gp27 trimer is shown as a ribbon diagram in which each chain is shown in a different color. B, Domains of gp27. The two homologous domains are colored in light green and cyan. C, Side and end on views of the C-terminal β-helical domain of gp5. D, The pseudohexameric feature of the gp27 trimer is outlined with a hexamer (domains are colored as in B).

Figure 5

Crystal structures of the baseplate proteins. The star (*) symbol after the protein name denotes that the crystal structure is available for the C-terminal fragment of the protein. Residue numbers comprising the solved structure are given in parentheses.

Figure 6

Comparison of gp10 with other baseplate proteins; reprinted from [11]. A, Stereo view of the superposition of gp10, gp11, and gp12. For clarity, the finger domain of gp11 and the insertion loop between β-strands 2 and 3 of gp12 are not shown. The β-strands are numbered 1 through 6 and the α-helix is indicated by "A". B, The structure-based sequence alignment of the common flower motifs of gp10, gp11, and gp12. The secondary structure elements are indicated above the sequences. The insertions between the common secondary structure elements are indicated with the number of inserted residues. The residues and their similarity are highlighted using the color scheme of the CLUSTAL program [89]. The alignment similarity profile, calculated by CLUSTAL, is shown below the sequences. C, The topology diagrams of the flower motif in gp10, gp11, and gp12. The circular arrows indicate interacting components within each trimer. The monomers are colored red, green, and blue. The numbers indicate the size of the insertions not represented in the diagram.

Figure 7

CryoEM reconstructions of the T4 tube-baseplate complex (A, B) and the tail in the extended (C) and contracted (D) conformation. Constituent proteins are shown in different colors and identified with the corresponding gene names. reprinted from [5,47] and [6].

Figure 8

Details of the T4 baseplate structure; reprinted from [5]. Proteins are labeled with their respective gene numbers. A, The garland of short tail fibers gp12 (magenta) with gp11 structures (light blue C_α trace) at the kinks of the gp12 fibers. The six-fold axis of the baseplate is shown as a black line. B, The baseplate "pins", composed of gp7 (red), gp8 (dark blue C_α trace), gp10 (yellow), and gp11(light blue C_α trace). Shown also is gp9 (green C_α trace), the long tail fiber attachment protein, with a green line along its three-fold axis, representing the direction of the long tail fibers. C, Gp6, gp25, and gp53 density.

Figure 9

Arrangement of gp6, gp25 and gp53 in the baseplate; reprinted from [7]. A, B, Gp6 is shown in magenta for the "hexagonal" dome-shaped baseplate (left) and in blue for the star-shaped baseplate (right). The C-terminal part of gp6 corresponds to the crystal structure and is shown as a Cα trace with spheres representing each residue. The N-terminal part of gp6 was segmented from the cryo-EM map. The densities corresponding to gp53 and gp25 are shown in white. C, D, The densities of gp53 and gp25 after the density for the whole of gp6 was zeroed out. E, F, The N-terminal gp6 dimers as found in the baseplate wedge. The C-terminal domain is shown as a Cα trace, whereas the N-terminal domain, for which the structure remains unknown, is shown as a density mesh. G, A stereo view of the four neighboring gp6 molecules from the two neighboring wedges of the dome-shaped baseplate. The N-terminal part of gp6 is shown as a density mesh and the C-terminal part corresponds to the crystal structure. H, Schematic of the four gp6 monomers using the same colors as in G. The N-terminal part is shown as a triangle and the C-terminal part as a rectangle.

Figure 10

Comparison of the baseplate in the two conformations; reprinted from [5]. A and B, Structure of the periphery of the baseplate in the hexagonal and star conformations, respectively. Colors identify different proteins as in the other figures: gp7 (red), gp8 (blue), gp9 (green), gp10 (yellow), gp11 (cyan) and gp12 (magenta). Directions of the long tail fibers are indicated with gray rods. The three domains of gp7 are labeled with letters A, B and C. The four domains of gp10 are labeled with Roman numbers I through IV. The C-terminal domain of gp11 is labeled with a black hexagon or black star in the hexagonal or star conformations, respectively. The baseplate sixfold axis is indicated by a black line. C and D, Structure of the proteins surrounding the hub in the hexagonal and star conformations, respectively. The proteins are colored as follows: spring green, gp5; pink, gp19; sky blue, gp27; violet, putative gp48 or gp54; beige, gp6-gp25-gp53; orange, unidentified protein at the tip of gp5. A part of the tail tube is shown in both conformations for clarity.

Figure 11

Structures of the gp18 deletion mutants reprinted from [53]. A, A ribbon diagram of the gp18PR mutant. The N terminus is shown in blue, the C terminus in red and the intermediate residues change color in spectral order. B, C, A ribbon diagram of the gp18M mutant (¾ of the total protein length). The three domains are shown in blue (domain I), olive green (domain II) and orange red (domain III); the β-hairpin (residues 454-470) and the last 14 C-terminal residues of gp18M are shown in cyan. D, Domain positions on the amino acid sequence, using the same color scheme as in (B) and (C). Brown indicates the part of gp18 for which the structure remains unknown.

Figure 12

Arrangement of the gp18 domains in the extended (A) and the contracted (B) tail reprinted from [53]. Domains I, II and III of gp18M are colored blue, olive green and orange red, respectively. The same color scheme is used in (C) the linear sequence diagram of the full-length gp18 and on the ribbon diagram of the gp18M structure. In (B) a part of the domain II from the next disk that becomes inserted between the subunits is shown in bright green. In both extended and contracted sheaths the additional density corresponds to domain IV of gp18 and the tail tube.

Figure 13

Connectivity of the sheath subunits in the extended (A) and contracted (B) tail sheath reprinted from [53]. The cryoEM map of the entire tail is shown on the far left. Immediately next to it, the three adjacent helices (in pink, blue and green) are shown to permit a better view of the internal arrangement. The successive hexameric discs are numbered 1, 2, 3, 4 and 5 with disc number 1 being closest to the baseplate. In the middle panels are the three helices formed by domains I, II and III. On the right is the arrangement of domain IV, for which the crystal structure is unknown. This domain retains the connectivity between neighboring subunits within each helix in both conformations of the sheath. C, One sixth of the gp18 helix - one strand - is shown for the extended (green) and contracted (golden brown) sheath conformations.

Figure 14

The structure of the collar and whiskers; reprinted from [5]. A, Cutaway view of the tail neck region. B, The structure of the gp15 hexameric ring in the extended and contracted tail. C, and D, Side and top views of the collar structure. For clarity, only one long tail fiber (LTF) is shown. The uninterpreted density between the fibritin molecules is indicated with brown color and labeled "NA".

Figure 15

Gene structure, assembly pathway and domain organization of the bacteriophage T4 long tail fibers. Chaperone interactions are shown as grey arrows. Domains of the proximal tail fiber are named P1-5 and of the distal half D1-11; gp35, or the knee-cap (KC) is represented as a green triangle.

Figure 16

Baseplate conformational switch schematic reprinted from [6]. A and B, The phage is free in solution. The long tail fibers are extended and oscillate around their midpoint position. The movements of the fibers are indicated with black arrows. The proteins are labeled with their corresponding gene numbers and colored as in other figures. C and D, The long tail fibers attach to their surface receptors and adapt the "down" conformation. The fiber labeled "A" and its corresponding attachment protein gp9 interact with gp11 and with gp10, respectively. These interactions, labeled with orange stars, probably initiate the conformational switch of the baseplate. The black arrows indicate tentative domain movements and rotations, which have been derived from the comparison of the two terminal conformations. The fiber labeled "B" has advanced along the conformational switch pathway so that gp11 is now seen along its threefold axis and the short tail fiber is partially extended in preparation for binding to its receptor. The thick red arrows indicate the projected movements of the fibers and the baseplate. E and F, The conformational switch is complete; the short tail fibers have bound their receptors and the sheath has contracted. The phage has initiated DNA transfer into the cell.