An official website of the United States government
Abstract Multi-subunit ring-ATPases carry out a myriad of biological functions, including genome packaging in viruses. Though the basic structures and functions of these motors have been well-established, the mechanisms of ATPase firing and motor coordination are poorly understood. Here, using single-molecule fluorescence, we determine that the active bacteriophage T4 DNA packaging motor consists of five subunits of gp17. By systematically doping motors with an ATPase-defective subunit and selecting single motors containing a precise number of active or inactive subunits, we find that the packaging motor can tolerate an inactive subunit. However, motors containing one or more inactive subunits exhibit fewer DNA engagements, a higher failure rate in encapsidation, reduced packaging velocity, and increased pausing. These findings suggest a DNA packaging model in which the motor, by re-adjusting its grip on DNA, can skip an inactive subunit and resume DNA translocation, suggesting that strict coordination amongst motor subunits of packaging motors is not crucial for function.
more »
« less
Function of a viral genome packaging motor from bacteriophage T4 is insensitive to DNA sequence
Abstract 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.
more »
« less
- Award ID(s):
- 1817709
- PAR ID:
- 10275870
- Date Published:
- Journal Name:
- Nucleic Acids Research
- Volume:
- 48
- Issue:
- 20
- ISSN:
- 0305-1048
- Page Range / eLocation ID:
- 11602 to 11614
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Bacteriophage (phage) T4 has served as an extraordinary model to elucidate biological structures and mechanisms. Recent discoveries on the T4 head (capsid) structure, portal vertex, and genome packaging add a significant body of new literature to phage biology. Head structures in unexpanded and expanded conformations show dramatic domain movements, structural remodeling, and a ~70% increase in inner volume while creating high-affinity binding sites for the outer decoration proteins Soc and Hoc. Small changes in intercapsomer interactions modulate angles between capsomer planes, leading to profound alterations in head length. The in situ cryo-EM structure of the symmetry-mismatched portal vertex shows the remarkable structural morphing of local regions of the portal protein, allowing similar interactions with the capsid protein in different structural environments. Conformational changes in these interactions trigger the structural remodeling of capsid protein subunits surrounding the portal vertex, which propagate as a wave of expansion throughout the capsid. A second symmetry mismatch is created when a pentameric packaging motor assembles at the outer “clip” domains of the dodecameric portal vertex. The single-molecule dynamics of the packaging machine suggests a continuous burst mechanism in which the motor subunits adjusted to the shape of the DNA fire ATP hydrolysis, generating speeds as high as 2000 bp/s.more » « less
-
Cingolani, G (Ed.)Large icosahedral viruses and tailed bacteriophages encode a portal protein that assembles into a dodecameric ring and occupies one of the twelve five-fold-symmetric vertices of a viral capsid. This unique symmetry-mismatched and structurally conserved portal vertex is essential for head assembly, genome packaging, neck/tail attachment, and genome ejection, but the underlying mechanisms remain poorly understood. Here, we present evidence that the phage T4 portal functions as a global assembly communicator and signal transducer, with its basket-shaped channel containing twenty-four anti-parallel helices at its core. Disruption of a single inter-helical salt-bridge that connects helices in a circular brace impairs channel movements that might be essential for a DNA grip-release mechanism during genome translocation. Second and third site suppressors that compensate for this defect fall in distant portal and packaging motor domains that together form a sophisticated communication network. Such networks might underlie the structural frameworks of macromolecular assemblies in biological systems.more » « less
-
Cingolani, Gino (Ed.)Background: Genome flow is a fundamental aspect of all biological systems. In viruses, it involves movement of nucleic acid genomes into and out of a proteinaceous capsid. Viruses must recover their newly replicated genomes into a protective capsid shell (packaging) and then safely re-introduce them into a new host (ejection) to initiate infection. While the mechanisms of DNA genome packaging in large icosahedral bacteriophages (phages) and viruses have been extensively investigated, the post-packaging mechanisms involving retention, positioning, and ejection of packaged genome are poorly understood. Aims: Using the tailed phage T4 as a model, we delineated the structural and assembly intermediates involved in transitioning a DNA-full head into an infectious virion particle, and then into a genome delivering supramolecular machine. These include intermediates of neck attachment, virion assembly, and genome release into E. coli. Methods: Various intermediates produced either by mutant phage infection or recombinant protein expression have been purified and biochemically characterized. Molecular genetic approaches were used to analyze the functional significance of amino acids involved in assembly. Structures of the purified particles were determined to near atomic resolution by cryo-electron microscopy and cryo-electron tomography. Results: Following termination of headful packaging, the pressurized T4 capsid containing tightly packed genome is sealed by the assembly of neck proteins gp13 and gp14. A dramatic conformational change in the portal dodecamer is evident, which expels the packaging motor while opening sites in portal’s “clip” domain exposed outside the capsid for binding the gp13 neck protein. Unexpectedly, we discovered that a host protein Hfq, a nucleic acid binding protein, plugs the neck structure. Hfq apparently helps to further stabilize the sealed head as it awaits tail attachment. After tail attachment, a genome end, likely the last packaged DNA, descends into the tail tube and precisely positions through interaction with an N-terminal DNA-binding motif of the tape measure protein (TMP) gp29. Six coiled-coil strands of TMP form the innermost tube of phage T4 tail, connected at the top end with DNA and at the bottom end with gp48 tube and baseplate. When the tail sheath contracts and the baseplate transform from hexagon to star shape, TMP pilots the genome to the tip of the tail tube, poised for delivery. Then, when the baseplate plug is opened fully, TMP is expelled by DNA pressure and remodels into a transmembrane channel and guides the genome to flow smoothly through the E. coli membrane envelope into the cytosol. Conclusion: Our studies describe the structural transitions of a complex and large myophage T4 in unprecedented detail. The mechanisms involve symmetry matches and mismatches, morphing, conformational transitions, and molecular remodeling that lead to genome retention, genome positioning, and genome release, precisely and efficiently.more » « less
-
Many viruses utilize ringed packaging ATPases to translocate double-stranded DNA into procapsids during replication. A critical step in the mechanochemical cycle of such ATPases is ATP binding, which causes a subunit within the motor to grip DNA tightly. Here, we probe the underlying molecular mechanism by which ATP binding is coupled to DNA gripping and show that a glutamate-switch residue found in AAA+ enzymes is central to this coupling in viral packaging ATPases. Using free-energy landscapes computed through molecular dynamics simulations, we determined the stable conformational state of the ATPase active site in ATP- and ADP-bound states. Our results show that the catalytic glutamate residue transitions from an active to an inactive pose upon ATP hydrolysis and that a residue assigned as the glutamate switch is necessary for regulating this transition. Furthermore, we identified via mutual information analyses the intramolecular signaling pathway mediated by the glutamate switch that is responsible for coupling ATP binding to conformational transitions of DNA-gripping motifs. We corroborated these predictions with both structural and functional experimental measurements. Specifically, we showed that the crystal structure of the ADP-bound P74-26 packaging ATPase is consistent with the structural coupling predicted from simulations, and we further showed that disrupting the predicted signaling pathway indeed decouples ATPase activity from DNA translocation activity in the φ29 DNA packaging motor. Our work thus establishes a signaling pathway that couples chemical and mechanical events in viral DNA packaging motors.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1093/nar/gkaa875
