The structure and assembly of bacteriophage T4 has been extensively studied. However, the detailed structure of the portal protein remained unknown. Here we report the structure of the bacteriophage T4 portal assembly, gene product 20 (gp20), determined by cryo-electron microscopy (cryo-EM) to 3.6 Å resolution. In addition, analysis of a 10 Å resolution cryo-EM map of an empty prolate T4 head shows how the dodecameric portal assembly interacts with the capsid protein gp23 at the special pentameric vertex. The gp20 structure also verifies that the portal assembly is required for initiating head assembly, for attachment of the packaging motor, and for participation in DNA packaging. Comparison of the Myoviridae T4 portal structure with the known portal structures of φ29, SPP1 and P22, representing Podo- and Siphoviridae, shows that the portal structure probably dates back to a time when self-replicating microorganisms were being established on Earth.

The 3.3-Å cryo-EM structure of the 860-Å-diameter isometric mutant bacteriophage T4 capsid has been determined. WT T4 has a prolate capsid characterized by triangulation numbers (T numbers) T = 13 for end caps and T = 20 for midsection. A mutation in the major capsid protein, gp23, produced T=13 icosahedral capsids. The capsid is stabilized by 660 copies of the outer capsid protein, Soc, which clamp adjacent gp23 hexamers. The occupancies of Soc molecules are proportional to the size of the angle between the planes of adjacent hexameric capsomers. The angle between adjacent hexameric capsomers is greatest around the fivefold vertices, where there is the largest deviation from a planar hexagonal array. Thus, the Soc molecules reinforce the structure where there is the greatest strain in the gp23 hexagonal lattice. Mutations that change the angles between adjacent capsomers affect the positions of the pentameric vertices, resulting in different triangulation numbers in bacteriophage T4. The analysis of the T4 mutant head assembly gives guidance to how other icosahedral viruses reproducibly assemble into capsids with a predetermined T number, although the influence of scaffolding proteins is also important.

DNA packaging by double-stranded DNA bacteriophages and herpesviruses is driven by a powerful molecular machine assembled at the portal vertex of the empty prohead. The phage T4 packaging machine consists of three components: dodecameric portal (gp20), pentameric large terminase motor (gp17), and 11-or 12-meric small terminase (gp16). These components dynamically interact and orchestrate a complex series of reactions to produce a DNA-filled head containing one viral genome per head. Here, we analyzed the interactions between the portal and motor proteins using a direct binding assay, mutagenesis, and structural analyses. Our results show that a portal binding site is located in the ATP hydrolysis-controlling subdomain II of gp17. Mutations at key residues of this site lead to temperature-sensitive or null phenotypes. A conserved helix-turn-helix (HLH) that is part of this site interacts with the portal. A recombinant HLH peptide competes with gp17 for portal binding and blocks DNA translocation. The helices apparently provide specificity to capture the cognate prohead, whereas the loop residues communicate the portal interaction to the ATPase center. These observations lead to a hypothesis in which a unique HLH-portal interaction in the symmetrically mismatched complex acts as a lever to position the arginine finger and trigger ATP hydrolysis. Transiently connecting the critical parts of the motor; subdomain I (ATP binding), subdomain II (controlling ATP hydrolysis), and C-domain (DNA movement), the portal-motor interactions might ensure tight coupling between ATP hydrolysis and DNA translocation.T ailed bacteriophages and herpesviruses use powerful ATPdriven machines to package their genomes into preformed capsid shells (36). They generate forces greater than 60 pN, ϳ20 times that of myosin motor, in order to compact a highly negatively charged, relatively rigid double-stranded DNA (dsDNA) to near-crystalline density (ϳ500 g/ml) (22, 39). The phage T4 employs one of the fastest and most powerful packaging machines reported to date. Packaging at a rate of up to ϳ2,000 bp/s, the T4 machine is estimated to generate twice the power (ϳ5,000 kW/ m 3 ) of an automobile engine (12, 36). The T4 packaging machine consists of three components ( Fig. 1): (i) an empty prohead containing a dodecameric portal protein, gp20 (25, 38); (ii) a pentameric large terminase motor protein, gp17 (5, 24, 42); and (iii) an 11-or 12-mer small terminase regulatory protein, gp16 (2, 24, 35). The cone-shaped portal links the head to the motor and DNA, with its wider end inside the capsid and the narrower end protruding outside. It has a central channel with a diameter of ϳ35 Å, through which DNA is threaded into the capsid. The portal works in conjunction with the motor, but its exact role is unclear. Various models such as the portal acting as a valve, DNA cruncher, or packaging sensor have been proposed (8,9,10,38,43).Five molecules of gp17 assemble on the portal into a packaging motor (Fig. 1). gp17 consists of an N-terminal ATPase domai...