Structure and assembly of bacteriophage T4 head

Abstract

The bacteriophage T4 capsid is an elongated icosahedron, 120 nm long and 86 nm wide, and is built with three essential proteins; gp23*, which forms the hexagonal capsid lattice, gp24*, which forms pentamers at eleven of the twelve vertices, and gp20, which forms the unique dodecameric portal vertex through which DNA enters during packaging and exits during infection. The past twenty years of research has greatly elevated the understanding of phage T4 head assembly and DNA packaging. The atomic structure of gp24 has been determined. A structural model built for gp23 using its similarity to gp24 showed that the phage T4 major capsid protein has the same fold as that found in phage HK97 and several other icosahedral bacteriophages. Folding of gp23 requires the assistance of two chaperones, the E. coli chaperone GroEL and the phage coded gp23-specific chaperone, gp31. The capsid also contains two non-essential outer capsid proteins, Hoc and Soc, which decorate the capsid surface. The structure of Soc shows two capsid binding sites which, through binding to adjacent gp23 subunits, reinforce the capsid structure. Hoc and Soc have been extensively used in bipartite peptide display libraries and to display pathogen antigens including those from HIV, Neisseria meningitides, Bacillus anthracis, and FMDV. The structure of Ip1*, one of the components of the core, has been determined, which provided insights on how IPs protect T4 genome against the E. coli nucleases that degrade hydroxymethylated and glycosylated T4 DNA. Extensive mutagenesis combined with the atomic structures of the DNA packaging/terminase proteins gp16 and gp17 elucidated the ATPase and nuclease functional motifs involved in DNA translocation and headful DNA cutting. Cryo-EM structure of the T4 packaging machine showed a pentameric motor assembled with gp17 subunits on the portal vertex. Single molecule optical tweezers and fluorescence studies showed that the T4 motor packages DNA at a rate of up to 2000 bp/sec, the fastest reported to date of any packaging motor. FRET-FCS studies indicate that the DNA gets compressed during the translocation process. The current evidence suggests a mechanism in which electrostatic forces generated by ATP hydrolysis drive the DNA translocation by alternating the motor between tensed and relaxed states.

Figure 1

Structure of the bacteriophage T4 head. A) Cryo-EM reconstruction of phage T4 capsid [5]; the square block shows enlarged view showing gp23 (yellow subunits), gp24 (purple subunits), Hoc (red subunits) and Soc (white subunits); B) Structure of RB49 Soc; C) Structural model showing one gp23 hexamer (blue) surrounded by six Soc trimers (red). Neighboring gp23 hexamers are shown in green, black and magenta [28]; D) Structure of gp24 [6]; E) Structural model of gp24 pentameric vertex.

Figure 2

Models of packaged DNA structure. a) T4 DNA is packed longitudinally to the head-tail axis [91], unlike the transverse packaging in T7 capsids [16](b). Other models shown include spiral fold (c), liquid-crystal (d), and icosahedral-bend (e). Both packaged T4 DNA ends are located in the portal [79]. For references and evidence bearing on packaged models see [19].

Figure 3

Structure and function of T4 internal protein I*. The NMR structure of IP1*, a highly specific inhibitor of the two-subunit CT (gmrS/gmrD) glucosyl-hmC DNA directed restriction endonuclease (right panel); shown are DNA modifications blocking such enzymes. The IPI* structure is compact with an asymmetric charge distribution on the faces (blue are basic residues) that may allow rapid DNA bound ejection through the portal and tail without unfolding-refolding.

Figure 4

In vitro display of antigens on bacteriophage T4 capsid. Schematic representation of the T4 capsid decorated with large antigens, PA (83 kDa) and LF (89 kDa), or hetero-oligomeric anthrax toxin complexes through either Hoc or Soc binding [39,41]. See text for details. The insets show electron micrographs of T4 phage with the anthrax toxin complexes displayed through Soc (top) or Hoc (bottom). Note the copy number of the complexes is lower with the Hoc display than with the Soc display.

Figure 5

Domains and motifs in phage T4 terminase proteins. Schematic representation of domains and motifs in the small terminase protein gp16. A) and the large terminase protein gp17 (B). The functionally critical amino acids are shown in bold. Numbers represent the number of amino acids in the respective coding sequence. For further detailed explanations of the functional motifs, refer to [46] and [51].

Figure 6

Structures of the T4 packaging motor protein, gp17. Structures of the ATPase domain: A) nuclease/translocation domain; B), and full-length gp17; C). Various functional sites and critical catalytic residues are labeled. See references [68] and [74] for further details.

Figure 7

Structure of the T4 DNA packaging machine. A) Cryo-EM reconstruction of the phage T4 DNA packaging machine showing the pentameric motor assembled at the special portal vertex. B-D) Cross section, top and side views of the pentameric motor respectively, by fitting the X-ray structures of the gp17 ATPase and nuclease/translocation domains into the cryo-EM density.

Figure 8

A model for the electrostatic force driven DNA packaging mechanism. Schematic representation showing the sequence of events that occur in a single gp17 molecule to translocate 2 bp of DNA (see the text and reference [74] for details).

Figure 9

A model for the torsional compression portal-DNA-grip-and-release packaging mechanism. A-C) Short nicked or other abnormal structure containing DNA substrates are released from the motor. D) Leader containing Y-DNA substrates are retained by the motor and are anchored in the procapsid in proximity to portal GFP fusions; and E) compression of the Y-stem B segment in the stalled complex is observed by FRET [88,89]