Portal-Large Terminase Interactions of the Bacteriophage T4 DNA Packaging Machine Implicate a Molecular Lever Mechanism for Coupling ATPase to DNA Translocation

Abstract

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...

Single-molecule packaging initiation in real time by a viral DNA packaging machine from bacteriophage T4

“…The combined effect of the two sources of randomness gives rise to the bursting behavior and large heterogeneity in the initiation rate. These dynamics are consistent with the hypothesis that fast motors such as the T4 motor that package at a rate of up to ∼2,000 bp/s might be prone to errors because the timing of interactions between the motor components (gp17 subunits, portal subunits, ATP, and DNA) must be synchronized at submillisecond timescale (12,24). When mistakes occur, for instance if DNA binds to a motor subunit before ATP does (e.g., at low ATP concentration), the motor enters a quiescent state during which it slowly recovers and resets the motor to an active state, thus creating a lag between bursts of packaging initiations.…”

“…To investigate the interactions involved in the assembly of a functional packaging machine, we prepared the packaging complexes using the same concentration of heads and gp17 but omitting ATPγS or priming DNA in the assembly step. Previous experiments showed that motor assembly per se on the prohead portal does not require ATP or DNA (12,24). Unexpectedly, most of the motors assembled in the absence of ATPγS could not initiate DNA packaging.…”

Large Terminase Conformational Change Induced by Connector Binding in Bacteriophage T7

“…5B) by an outward rotation and a stand-up rigid body movement of the gp19 monomer (see supplemental Movie S1). The topology of the monomers in the complex shows the two domains almost parallel to the longitudinal axis of the complex, specifically restricting the connector interaction area to the amino domain of gp19, as proposed for the large terminase of phage T4 (79). The different topology of the gp19 monomers in the two structures generates different electrostatic environments in the channel surface (Fig.…”

Structure and mechanism of the ATPase that powers viral genome packaging

“…Although many ATPase rings are asymmetrical during function (12, 37), we assume fivefold symmetry for simplicity. Regardless, most ringed ATPase structures were first modeled as symmetrical assemblies (32,38), and these models were refined later to show asymmetry during function (9,10,12,37). After docking of the ATPase ring, we used the full-length crystal structure of T4-TerL (8) to orient a homology model of the P74-26 nuclease domain, which results in minimal steric clashes between the two domains.…”

“…Cryoelectron microscopy (cryo-EM) studies indicate that a pentamer of TerL subunits attaches to the capsid by binding to a dodecameric assembly called portal (8). However, there are conflicting reports as to the orientation of TerL relative to portal during packaging (8)(9)(10).…”