Nucleotide-dependent DNA gripping and an end-clamp mechanism regulate the bacteriophage T4 viral packaging motor.
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ABSTRACT:
SUBMITTER: Ordyan M
PROVIDER: S-EPMC6303390 | biostudies-literature | 2018 Dec
REPOSITORIES: biostudies-literature
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Project description:Many viruses employ ATP-powered motors during assembly to translocate DNA into procapsid shells. Previous reports raise the question if motor function is modulated by substrate DNA sequence: (i) the phage T4 motor exhibits large translocation rate fluctuations and pauses and slips; (ii) evidence suggests that the phage phi29 motor contacts DNA bases during translocation; and (iii) one theoretical model, the 'B-A scrunchworm', predicts that 'A-philic' sequences that transition more easily to A-form would alter motor function. Here, we use single-molecule optical tweezers measurements to compare translocation of phage, plasmid, and synthetic A-philic, GC rich sequences by the T4 motor. We observed no significant differences in motor velocities, even with A-philic sequences predicted to show higher translocation rate at high applied force. We also observed no significant changes in motor pausing and only modest changes in slipping. To more generally test for sequence dependence, we conducted correlation analyses across pairs of packaging events. No significant correlations in packaging rate, pausing or slipping versus sequence position were detected across repeated measurements with several different DNA sequences. These studies suggest that viral genome packaging is insensitive to DNA sequence and fluctuations in packaging motor velocity, pausing and slipping are primarily stochastic temporal events.
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Project description:The speed at which a molecular motor operates is critically important for the survival of a virus or an organism but very little is known about the underlying mechanisms. Tailed bacteriophage T4 employs one of the fastest and most powerful packaging motors, a pentamer of gp17 that translocates DNA at a rate of up to ?2000-bp/s. We hypothesize, guided by structural and genetic analyses, that a unique hydrophobic environment in the catalytic space of gp17-adenosine triphosphatase (ATPase) determines the rate at which the 'lytic water' molecule is activated and OH- nucleophile is generated, in turn determining the speed of the motor. We tested this hypothesis by identifying two hydrophobic amino acids, M195 and F259, in the catalytic space of gp17-ATPase that are in a position to modulate motor speed. Combinatorial mutagenesis demonstrated that hydrophobic substitutions were tolerated but polar or charged substitutions resulted in null or cold-sensitive/small-plaque phenotypes. Quantitative biochemical and single-molecule analyses showed that the mutant motors exhibited 1.8- to 2.5-fold lower rate of ATP hydrolysis, 2.5- to 4.5-fold lower DNA packaging velocity, and required an activator protein, gp16 for rapid firing of ATPases. These studies uncover a speed control mechanism that might allow selection of motors with optimal performance for organisms' survival.
| S-EPMC5737356 | biostudies-other
Project description:Viral DNA packaging motors are among the most powerful molecular motors known. A variety of structural, biochemical, and single-molecule biophysical approaches have been used to understand their mechanochemistry. However, packaging initiation has been difficult to analyze because of its transient and highly dynamic nature. Here, we developed a single-molecule fluorescence assay that allowed visualization of packaging initiation and reinitiation in real time and quantification of motor assembly and initiation kinetics. We observed that a single bacteriophage T4 packaging machine can package multiple DNA molecules in bursts of activity separated by long pauses, suggesting that it switches between active and quiescent states. Multiple initiation pathways were discovered including, unexpectedly, direct DNA binding to the capsid portal followed by recruitment of motor subunits. Rapid succession of ATP hydrolysis was essential for efficient initiation. These observations have implications for the evolution of icosahedral viruses and regulation of virus assembly.
| S-EPMC4210281 | biostudies-literature
Project description:Tailed bacteriophages and herpes viruses use powerful molecular motors to translocate DNA into a preassembled prohead and compact the DNA to near crystalline density. The phage T4 motor, a pentamer of 70-kDa large terminase, gp17, is the fastest and most powerful motor reported to date. gp17 has an ATPase activity that powers DNA translocation and a nuclease activity that cuts concatemeric DNA and generates the termini of viral genome. An 18-kDa small terminase, gp16, is also essential, but its role in DNA packaging is poorly understood. gp16 forms oligomers, most likely octamers, exhibits no enzymatic activities, but stimulates the gp17-ATPase activity, and inhibits the nuclease activity. Extensive mutational and biochemical analyses show that gp16 contains three domains, a central oligomerization domain, and N- and C-terminal domains that are essential for ATPase stimulation. Stimulation occurs not by nucleotide exchange or enhanced ATP binding but by triggering hydrolysis of gp17-bound ATP, a mechanism reminiscent of GTPase-activating proteins. gp16 does not have an arginine finger but its interaction with gp17 seems to position a gp17 arginine finger into the catalytic pocket. gp16 inhibits DNA translocation when gp17 is associated with the prohead. gp16 restricts gp17-nuclease such that the putative packaging initiation cut is made but random cutting is inhibited. These results suggest that the phage T4 packaging machine consists of a motor (gp17) and a regulator (gp16). The gp16 regulator is essential to coordinate the gp17 motor ATPase, translocase, and nuclease activities, otherwise it could be suicidal to the virus.
| S-EPMC2782041 | biostudies-literature
Project description:How viral packaging motors generate enormous forces to translocate DNA into viral capsids remains unknown. Recent structural studies of the bacteriophage T4 packaging motor have led to a proposed mechanism wherein the gp17 motor protein translocates DNA by transitioning between extended and compact states, orchestrated by electrostatic interactions between complimentarily charged residues across the interface between the N- and C-terminal subdomains. Here we show that site-directed alterations in these residues cause force dependent impairments of motor function including lower translocation velocity, lower stall force and higher frequency of pauses and slips. We further show that the measured impairments correlate with computed changes in free-energy differences between the two states. These findings support the proposed structural mechanism and further suggest an energy landscape model of motor activity that couples the free-energy profile of motor conformational states with that of the ATP hydrolysis cycle.
| S-EPMC4157569 | biostudies-literature
Project description:Complex viruses are assembled from simple protein subunits by sequential and irreversible assembly. During genome packaging in bacteriophages, a powerful molecular motor assembles at the special portal vertex of an empty prohead to initiate packaging. The capsid expands after about 10%-25% of the genome is packaged. When the head is full, the motor cuts the concatemeric DNA and dissociates from the head. Conformational changes, particularly in the portal, are thought to drive these sequential transitions. We found that the phage T4 packaging machine is highly promiscuous, translocating DNA into finished phage heads as well as into proheads. Optical tweezers experiments show that single motors can force exogenous DNA into phage heads at the same rate as into proheads. Single molecule fluorescence measurements demonstrate that phage heads undergo repeated initiations, packaging multiple DNA molecules into the same head. These results suggest that the phage DNA packaging machine has unusual conformational plasticity, powering DNA into an apparently passive capsid receptacle, including the highly stable virus shell, until it is full. These features probably led to the evolution of viral genomes that fit capsid volume, a strikingly common phenomenon in double-stranded DNA viruses, and will potentially allow design of a novel class of nanocapsid delivery vehicles.
| S-EPMC3039672 | biostudies-literature
Project description:Bacteriophage T4 is a large-tailed Escherichia coli virus whose capsid is 120x86 nm. ATP-driven DNA packaging of the T4 capsid results in the loading of a 171-kb genome in less than 5 min during viral infection. We have isolated 50-mg quantities of uniform (15)N- and [epsilon-(15)N]lysine-labeled bacteriophage T4. We have also introduced (15)NH(4)(+) into filled, unlabeled capsids from synthetic medium by exchange. We have examined lyo- and cryoprotected lyophilized T4 using (15)N{(31)P} and (31)P{(15)N} rotational-echo double resonance. The results of these experiments have shown that (i) packaged DNA is in an unperturbed duplex B-form conformation; (ii) the DNA phosphate negative charge is balanced by lysyl amines (3.2%), polyamines (5.8%), and monovalent cations (40%); and (iii) 11% of lysyl amines, 40% of -NH(2) groups of polyamines, and 80% of monovalent cations within the lyophilized T4 capsid are involved in the DNA charge balance. The NMR evidence suggests that DNA enters the T4 capsid in a charge-unbalanced state. We propose that electrostatic interactions may provide free energy to supplement the nanomotor-driven T4 DNA packaging.
| S-EPMC2633174 | biostudies-literature
Project description:Many viruses utilize molecular motors to package their genomes into preformed capsids. A striking feature of these motors is their ability to generate large forces to drive DNA translocation against entropic, electrostatic, and bending forces resisting DNA confinement. A model based on recently resolved structures of the bacteriophage T4 motor protein gp17 suggests that this motor generates large forces by undergoing a conformational change from an extended to a compact state. This transition is proposed to be driven by electrostatic interactions between complementarily charged residues across the interface between the N- and C-terminal domains of gp17. Here we use atomistic molecular dynamics simulations to investigate in detail the molecular interactions and residues involved in such a compaction transition of gp17. We find that although electrostatic interactions between charged residues contribute significantly to the overall free energy change of compaction, interactions mediated by the uncharged residues are equally if not more important. We identify five charged residues and six uncharged residues at the interface that play a dominant role in the compaction transition and also reveal salt bridging, van der Waals, and solvent hydrogen-bonding interactions mediated by these residues in stabilizing the compact form of gp17. The formation of a salt bridge between Glu309 and Arg494 is found to be particularly crucial, consistent with experiments showing complete abrogation in packaging upon Glu309Lys mutation. The computed contributions of several other residues are also found to correlate well with single-molecule measurements of impairments in DNA translocation activity caused by site-directed mutations.
| S-EPMC4376518 | biostudies-literature
Project description:The packaging motor of bacteriophage T4 translocates DNA into the capsid at a rate of up to 2000 bp/s. Such a high rate would require coordination of motor movements at millisecond timescale. Designing a cysteine-less gp17 is essential to generate fluorescently labeled motors and measure distance changes between motor domains by FRET analyses. Here, by using sequence alignments, structural modeling, combinatorial mutagenesis, and recombinational rescue, we replaced all nine cysteines of gp17 and introduced single cysteines at defined positions. These mutant motors retained in vitro DNA packaging activity. Single mutant motors translocated DNA molecules in real time as imaged by total internal reflection fluorescence microscopy. We discovered, unexpectedly, that a hydrophobic or nonpolar amino acid next to Walker B motif is essential for motor function, probably for efficient generation of OH(-) nucleophile. The ATPase Walker B motif, thus, may be redefined as "β-strand (4-6 hydrophobic-rich amino acids)-DE-hydrophobic/nonpolar amino acid".
| S-EPMC4438746 | biostudies-literature
Project description:Tailed bacteriophages and herpes viruses use powerful ATP-driven molecular motors to translocate their viral genomes into a preformed capsid shell. The bacteriophage T4 motor, a pentamer of the large terminase protein (gp17) assembled at the portal vertex of the prohead, is the fastest and most powerful known, consistent with the need to package a ~170-kb viral genome in approximately 5 min. Although much is known about the mechanism of DNA translocation, very little is known about how ATP modulates motor-DNA interactions. Here, we report single-molecule measurements of the phage T4 gp17 motor by using dual-trap optical tweezers under different conditions of perturbation. Unexpectedly, the motor pauses randomly when ATP is limiting, for an average of 1 s, and then resumes translocation. During pausing, DNA is unpackaged, a phenomenon so far observed only in T4, where some of the packaged DNA is slowly released. We propose that the motor pauses whenever it encounters a subunit in the apo state with the DNA bound weakly and incorrectly. Pausing allows the subunit to capture ATP, whereas unpackaging allows scanning of DNA until a correct registry is established. Thus, the "pause-unpackaging" state is an off-translocation recovery state wherein the motor, sometimes by taking a few steps backward, can bypass the impediments encountered along the translocation path. These results lead to a four-state mechanochemical model that provides insights into the mechanisms of translocation of an intricately branched concatemeric viral genome.
| S-EPMC3523870 | biostudies-other