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.
The results suggest that the terminal differentiation defects, specifically, increased proliferation and decreased denucleation are responsible for the development of lens opacity in N101D lenses.
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...
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