Viral genomes are packaged into "procapsids" by powerful molecular motors. We report the crystal structure of the DNA packaging motor protein, gene product 17 (gp17), in bacteriophage T4. The structure consists of an N-terminal ATPase domain, which provides energy for compacting DNA, and a C-terminal nuclease domain, which terminates packaging. We show that another function of the C-terminal domain is to translocate the genome into the procapsid. The two domains are in close contact in the crystal structure, representing a "tensed state." A cryo-electron microscopy reconstruction of the T4 procapsid complexed with gp17 shows that the packaging motor is a pentamer and that the domains within each monomer are spatially separated, representing a "relaxed state." These structures suggest a mechanism, supported by mutational and other data, in which electrostatic forces drive the DNA packaging by alternating between tensed and relaxed states. Similar mechanisms may occur in other molecular motors.

An ATP-powered DNA translocation machine encapsidates the viral genome in the large dsDNA bacteriophages. The essential components include the empty shell, prohead, and the packaging enzyme, terminase. During translocation, terminase is docked on the prohead's portal protein. The translocation ATPase and the concatemer-cutting endonuclease reside in terminase. Remarkably, terminases, portal proteins, and shells of tailed bacteriophages and herpes viruses show conserved features. These DNA viruses may have descended from a common ancestor. Terminase's ATPase consists of a classic nucleotide binding fold, most closely resembling that of monomeric helicases. Intriguing models have been proposed for the mechanism of dsDNA translocation, invoking ATP hydrolysis-driven conformational changes of portal or terminase powering DNA motion. Single-molecule studies show that the packaging motor is fast and powerful. Recent advances permit experiments that can critically test the packaging models. The viral genome translocation mechanism is of general interest, given the parallels between terminases, helicases, and other motor proteins.

The head of bacteriophage T4 is a prolate icosahedron with one unique portal vertex to which the phage tail is attached. The three-dimensional structure of mature bacteriophage T4 head has been determined to 22-Å resolution by using cryo-electron microscopy. The T4 capsid has a hexagonal surface lattice characterized by the triangulation numbers Tend ‫؍‬ 13 laevo for the icosahedral caps and Tmid ‫؍‬ 20 for the midsection. Hexamers of the major capsid protein gene product (gp)23* and pentamers of the vertex protein gp24*, as well as the outer surface proteins highly antigenic outer capsid protein (hoc) and small outer capsid protein (soc), are clearly evident in the reconstruction. The size and shape of the gp23* hexamers are similar to the major capsid protein organization of bacteriophage HK97. The binding sites and shape of the hoc and soc proteins have been established by analysis of the soc ؊ and hoc ؊ soc ؊ T4 structures. Bacteriophage T4 is a large, tailed, double-stranded DNA (dsDNA) virus (family Myoviridae) that uses Escherichia coli as a host. The mature T4 virion, which contains Ϸ50 different proteins, consists of a prolate capsid, 172-kbp genomic DNA, and a tail with a contractile sheath terminating in a base plate to which are attached six long tail fibers. The architecture and the molecular composition of the T4 head, tail, and fibers have been characterized extensively by using a variety of techniques (1-3) leading to a structural model (4). Recent studies of the T4 components by cryo-electron microscopy (cryo-EM) and x-ray crystallography extended the structural knowledge to higher resolution (5).T4 has one of the most complex structures of any virus that has been studied. There are Ͼ2,000 protein molecules of at least five different gene products (gps) in the head alone. The molecular mass of the DNA-filled head is 194 MDa and of the capsid alone is 82 MDa (6). The T4 head assembly proceeds via a number of intermediate stages. First, a DNA-free precursor, or prohead, is assembled that is processed proteolytically. Next, the genomic DNA is packaged into the prohead in a process that requires ATP energy (1). The prohead assembly is initiated by the portal protein gp20. The prohead contains an internal core made up of the major core protein, gp22, the minor core proteins, gpalt, a serine-type protease, gp21, and other internal proteins (1). The major capsid protein, gp23, is assembled around the scaffolding core together with the minor capsid protein, gp24. After completion of prohead assembly, the inactive gp21 enzyme is converted to the active protease, which cleaves the scaffold proteins into small peptides. A 65-residue-long amino-terminal ''⌬-piece'' is also cleaved from the 56-kDa gp23 molecule, thus yielding 48.7-kDa gp23* (1) ʈ . In addition, a 2.2-kDa amino-terminal piece of the 48.4-kDa gp24 is cleaved, giving rise to gp24* during head maturation. Most of the small peptides produced by the gp21 protease are expelled from the prohead, thus providing space necessary to accommodate the geno...