Biochemical and Biophysical Characterization of the dsDNA packaging motor from the<i>Lactococcus lactis</i>bacteriophage asccphi28

Reyes-Aldrete, Emilio; Dill, Erik; Bussetta, Cécile; Szymanski, Michal R.; Diemer, Geoffrey S.; Maindola, Priyank; White, Mark A.; Choi, Kyung Hyun; Morais, Marc C.

doi:10.1101/2020.11.17.383745

Cited by 3 publications

(8 citation statements)

References 48 publications

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“…1 ), indicating that the observed pentamer is the biological assembly. This stoichiometry is consistent with previously reported biochemical and biophysical analysis that indicated gp11 forms pentameric rings in solution 22 . Of note, the kinetic parameters of ATP binding and hydrolysis by isolated gp11 rings are similar to the parameters obtained for φ29 and other bacteriophage DNA packaging motors, but only once these other ATPases are assembled as functional rings on their respective procapsids 22 .…”

Section: Resultssupporting

confidence: 93%

“…This stoichiometry is consistent with previously reported biochemical and biophysical analysis that indicated gp11 forms pentameric rings in solution 22 . Of note, the kinetic parameters of ATP binding and hydrolysis by isolated gp11 rings are similar to the parameters obtained for φ29 and other bacteriophage DNA packaging motors, but only once these other ATPases are assembled as functional rings on their respective procapsids 22 . In the absence of procapsids, other packaging motors negligibly hydrolyze ATP, presumably since they are in monomeric form and therefore cannot efficiently bind or hydrolyze ATP (see also below).…”

Section: Resultssupporting

confidence: 93%

“…This is not surprising, since the φ29 ATPase only assembles functional rings by virtue of binding to the procapsid 17 . In contrast, a close homolog of the φ29 ATPase from the related bacteriophage asccφ28 (gp11) has been shown to form highly soluble functional rings; extensive analytical ultracentrifugation and small-angle X-ray scattering experiments show that the ATPase forms pentameric rings in solution 22 . The 45% sequence similarity between the two proteins assures their structures will be nearly identical.…”

Section: Resultsmentioning

confidence: 99%

“…Cloning, protein purification, kinetic analysis, AUC, SAXS, negative stain TEM, and preliminary crystallization of gp11 has been previously reported 22 . Briefly, two crystal forms of gp11 were obtained: 1) tetragonal crystals belonging to space group P4 3 2 1 2 and 2) trigonal crystals belonging to space group P3 2 2 1 .…”

Section: Resultsmentioning

confidence: 99%

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

Pajak

Dill

Reyes-Aldrete

et al. 2020

Preprint

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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.

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Section: Resultssupporting

confidence: 93%

Section: Resultssupporting

confidence: 93%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

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

Pajak

Dill

Reyes-Aldrete

et al. 2020

Preprint

Self Cite

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show abstract

“…This is not surprising, since the φ29 ATPase only assembles functional rings by virtue of binding to the procapsid ( 18 ). In contrast, a close homolog of the φ29 ATPase from the related bacteriophage asccφ28 (gp11) has been shown to form highly soluble functional rings; extensive analytical ultracentrifugation and small-angle X-ray scattering experiments show that the ATPase forms pentameric rings in solution ( 47 ). The 45% sequence similarity between the two proteins assures their structures will be nearly identical.…”

Section: Resultsmentioning

confidence: 99%

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

Pajak

Dill

Reyes-Aldrete

et al. 2021

Nucleic Acids Research

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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.

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Biophysical and Structural Characterization of a Viral Genome Packaging Motor

Prokhorov

Davis

Maruthi

et al. 2022

Preprint

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Like many dsDNA viruses, bacteriophage λ replicates its genome as a concatemer consisting of multiple copies of covalently linked dsDNA genomes. To encapsidate a single genome within a nascent procapsid, λ must: 1) find its own dsDNA amongst the multitude of host nucleic acids; 2) identify the genomic start site; 3) cut the DNA; 4) bring the excised DNA to a procapsid; 5) translocate DNA into the capsid; 6) cut DNA again at a packaging termination site, 7) disengage from the newly filled capsid; and 8) bring the remainder of the genomic concatemer to fill another empty procapsid. These disparate genome processing tasks are carried out by a single virus-encoded enzyme complex called terminase. While it has been shown that λ terminase initially forms a tetrameric complex to cut DNA, it is not clear whether the same configuration translocates DNA. Here, we describe biophysical and initial structural characterization of a λ terminase translocation complex. Analytical ultracentrifugation (AUC) and small angle X-ray scattering (SAXS) indicate that between 4 and 5 protomeric subunits assemble a cone-shaped terminase complex with a maximum dimension of ~230 and radius of gyration of ~72 Å. Two-dimensional classification of cryoEM images of λ terminase are consistent with these dimensions and show that particles assume a preferred orientation in ice. The orientations appear to be end-on, as terminase rings resemble a starfish with approximate pentameric symmetry. While ~5-fold symmetry is apparent, one of the five arms appears partially displaced with weaker more diffuse density in some classes, suggesting flexibility and/or partial occupancy. Charge detection mass spectrometry (CDMS) is consistent with a pentameric complex, with evidence that one motor subunit is weakly bound. Kinetic analysis indicates that the complex hydrolyzes ATP at a rate comparable to the rates of other phage packaging motors. Together with previously published data, these results suggest that λ terminase assembles conformationally and stoichiometrically distinct complexes to carry out different genome processing tasks. We propose a symmetry resolution pathway to explain how terminase transitions between these structurally and functionally distinct states.

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Biochemical and Biophysical Characterization of the dsDNA packaging motor from theLactococcus lactisbacteriophage asccphi28

Cited by 3 publications

References 48 publications

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

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

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

Biophysical and Structural Characterization of a Viral Genome Packaging Motor

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