Functional Dissection of a Viral DNA Packaging Machine's Walker B Motif

Dill

Reyes-Aldrete

et al. 2020

Preprint

Self Cite

SummaryDouble-stranded DNA viruses package their genomes into pre-assembled protein capsids using virally-encoded ATPase ring motors. While several structures of isolated monomers (subunits) from these motors have been determined, they provide little insight into how subunits within a functional ring coordinate their activities to efficiently generate force and translocate DNA. Here we describe the first atomic-resolution structure of a functional ring form of a viral DNA packaging motor and characterize its atomic-level dynamics via long timescale molecular dynamics simulations. Crystal structures of the pentameric ATPase ring from bacteriophage asccφ28 show that each subunit consists of a canonical N-terminal ASCE ATPase domain connected to a ‘vestigial’ nuclease domain by a small lid subdomain. The lid subdomain closes over the ATPase active site and engages in extensive interactions with a neighboring subunit such that several important catalytic residues are positioned to function in trans. The pore of the ring is lined with several positively charged residues that can interact with DNA. Simulations of the ATPase ring in various nucleotide- and DNA-bound states provide information about how the motor coordinates sequential nucleotide binding, hydrolysis, and exchange around the ring. Simulations also predict that the ring adopts a helical structure to track DNA, consistent with recent cryo-EM reconstruction of the φ28 packaging ATPase. Based on these results, an atomistic model of viral DNA packaging is proposed wherein DNA translocation is powered by stepwise helical-to-planar ring transitions that are tightly coordinated by ATP binding, hydrolysis, and release.

Section: Resultsmentioning

confidence: 99%

Section: Atp Binding and Regulation Of Hydrolysismentioning

confidence: 97%

Atomistic Basis of Force Generation, Translocation, and Coordination in a Viral Genome Packaging Motor

Dill

Reyes-Aldrete

et al. 2020

Preprint

Self Cite

“…These pre-tight-binding poses are characterized by the WA arginine pointing out of the binding pocket and an increased distance between the WA and WB motif backbones ( Fig. S5 ), based on our two previous studies of tight-binding conformational changes (9, 10). We find that for φ29 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Other related ASCE families include AAA+ (ATPases Associated with diverse cellular Activities) and ABC (ATP Binding Cassette) transporter proteins (6, 7). A common feature among ASCE enzymes is the presence of a P-loop/Walker A (WA) and Walker B (WB) motifs which bind ATP and catalyze hydrolysis, respectively (8, 9). The feature that distinguishes ASCE enzymes from other superfamilies of ATPases is the presence of a conserved glutamate residue downstream of the WB motif that is necessary for catalytic activity (6, 10, 11).…”

Section: Introductionmentioning

confidence: 99%

Viral Packaging ATPases Utilize a Glutamate Switch to Couple ATPase Activity and DNA Translocation

Atz

Hilbert

et al. 2020

Preprint

Self Cite

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 apo, ATP-bound, and ADP-bound states. Our results show that the catalytic glutamate residue transitions from an inactive to an active pose upon ATP binding, and that a residue assigned as the glutamate switch is necessary for regulating the transition. Further, 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 data. Specifically, we showed that the crystal structure of the ADP-bound P74-26 packaging ATPase is consistent with the predicted structural coupling 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 in viral DNA packaging motors that ensures coordination between chemical and mechanical events involved in viral DNA packaging.

“…The simulations predicted that Mg 2+ -ATP binds to the cis -acting side of the inter-subunit active site via canonical interactions with the Walker A motif: (i) the β-phosphate of ATP forms several hydrogen bonds with the backbone nitrogens of the Walker A motif; (ii) the critical P-loop Lys32 coordinates the β- and γ-phosphates and (iii) Thr33 chelates the Mg 2+ ion (Figure 2A ). The cis- acting Walker B motif residues engage in similar conserved canonical interactions: (a) Asp146 chelates the Mg 2+ ion through a water molecule and (b) Asp146 hydrogen bonds to the Walker A Thr33, which has been previously predicted to be a key interaction that helps close the active site as part of the tight-binding transition ( 52 , 53 ).…”

Section: Resultsmentioning

confidence: 99%

Atomistic basis of force generation, translocation, and coordination in a viral genome packaging motor

Dill

Reyes-Aldrete

et al. 2021

Nucleic Acids Research

Self Cite

Double-stranded DNA viruses package their genomes into pre-assembled capsids using virally-encoded ASCE ATPase ring motors. We present the first atomic-resolution crystal structure of a multimeric ring form of a viral dsDNA packaging motor, the ATPase of the asccφ28 phage, and characterize its atomic-level dynamics via long timescale molecular dynamics simulations. Based on these results, and previous single-molecule data and cryo-EM reconstruction of the homologous φ29 motor, we propose an overall packaging model that is driven by helical-to-planar transitions of the ring motor. These transitions are coordinated by inter-subunit interactions that regulate catalytic and force-generating events. Stepwise ATP binding to individual subunits increase their affinity for the helical DNA phosphate backbone, resulting in distortion away from the planar ring towards a helical configuration, inducing mechanical strain. Subsequent sequential hydrolysis events alleviate the accumulated mechanical strain, allowing a stepwise return of the motor to the planar conformation, translocating DNA in the process. This type of helical-to-planar mechanism could serve as a general framework for ring ATPases.