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.
The bacterial FtsK motor harvests energy from ATP to translocate double-stranded DNA during cell division. Here, we probe the molecular mechanisms underlying coordinated DNA translocation in FtsK by performing long timescale simulations of its hexameric assembly and individual subunits. From these simulations we predict signaling pathways that connect the ATPase active site to DNA-gripping residues, which allows the motor to coordinate its translocation activity with its ATPase activity. Additionally, we utilize well-tempered metadynamics simulations to compute free-energy landscapes that elucidate the extended-to-compact transition involved in force generation. We show that nucleotide binding promotes a compact conformation of a motor subunit, whereas the apo subunit is flexible. Together, our results support a mechanism whereby each ATP-bound subunit of the motor conforms to the helical pitch of DNA, and ATP hydrolysis/product release causes a subunit to lose grip of DNA. By ordinally engaging and disengaging with DNA, the FtsK motor unidirectionally translocates DNA.
more » « less- NSF-PAR ID:
- 10370047
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Nucleic Acids Research
- Volume:
- 50
- Issue:
- 15
- ISSN:
- 0305-1048
- Format(s):
- Medium: X Size: p. 8459-8470
- Size(s):
- ["p. 8459-8470"]
- Sponsoring Org:
- National Science Foundation
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