Sequential action of six virus-encoded DNA-packaging RNAs during phage phi29 genomic DNA translocation.

Biological motors are ubiquitous in living systems. Currently, how the motor components coordinate the unidirectional motion is elusive in most cases. Here, we report that the sequential action of the ATPase ring in the DNA packaging motor of bacteriophage ϕ29 is regulated by an arginine finger that extends from one ATPase subunit to the adjacent unit to promote noncovalent dimer formation. Mutation of the arginine finger resulted in the interruption of ATPase oligomerization, ATP binding/hydrolysis, and DNA translocation. Dimer formation reappeared when arginine mutants were mixed with other ATPase subunits that can offer the arginine to promote their interaction. Ultracentrifugation and virion assembly assays indicated that the ATPase was presenting as monomers and dimer mixtures. The isolated dimer alone was inactive in DNA translocation, but the addition of monomer could restore the activity, suggesting that the hexameric ATPase ring contained both dimer and monomers. Moreover, ATP binding or hydrolysis resulted in conformation and entropy changes of the ATPase with high or low DNA affinity. Taking these observations together, we concluded that the arginine finger regulates sequential action of the motor ATPase subunit by promoting the formation of the dimer inside the hexamer. The finding of asymmetrical hexameric organization is supported by structural evidence of many other ATPase systems showing the presence of one noncovalent dimer and four monomer subunits. All of these provide clues for why the asymmetrical hexameric ATPase gp16 of ϕ29 was previously reported as a pentameric configuration by cryo-electron microscopy (cryo-EM) since the contact by the arginine finger renders two adjacent ATPase subunits closer than other subunits. Thus, the asymmetrical hexamer would appear as a pentamer by cryo-EM, a technology that acquires the average of many images.

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

10.1101/2020.12.01.406595 ◽

2020 ◽

Author(s):

Joshua Pajak ◽

Rockney Atz ◽

Brendan J. Hilbert ◽

Marc C. Morais ◽

Brian A. Kelch ◽

...

Keyword(s):

Atpase Activity ◽

Signaling Pathway ◽

Bound States ◽

Dna Packaging ◽

Atp Binding ◽

Dna Translocation ◽

Viral Dna ◽

Viral Packaging ◽

Mechanochemical Cycle ◽

Viral Dna Packaging

SummaryMany 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.

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Lattice Transformations and Subunit Conformational Changes in Phage Capsid Maturation

Journal of Theoretical Medicine ◽

10.1080/10273660500149646 ◽

2005 ◽

Vol 6 (2) ◽

pp. 99-105 ◽

Cited By ~ 1

Author(s):

David C. Gossard ◽

Jonathan King

Keyword(s):

Conformational Changes ◽

Genomic Dna ◽

General Feature ◽

Structural Data ◽

Dna Packaging ◽

Double Stranded Dna ◽

Virus Life Cycle ◽

Phage Capsid ◽

Capsid Maturation ◽

Mature Virus

A general feature of the pathways for the assembly of double-stranded DNA phages and viruses is the assembly of coat and scaffolding subunits into a precursor shell or procapsid, followed by packaging of the genomic DNA into the shell. Coupled to this DNA packaging process is the loss of the scaffolding subunits and expansion and re-organization of the procapsid lattice to the lattice of the mature virus. Such lattice transitions have also been observed with adenoviruses and herpesviruses. In re-organizing into the mature capsid lattice, each subunit of the precursor lattice must change its conformation, or its relationship with its neighbours, or both. We briefly review here recent structural data for phages P22 and HK97, and describe the motions and conformational changes associated with this lattice transition. Possible functions of such constrained transformations within the virus life-cycle are discussed.

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A viral genome packaging ring-ATPase is a flexibly coordinated pentamer

10.1101/2021.03.08.434477 ◽

2021 ◽

Author(s):

Li Dai ◽

Digvijay Singh ◽

Suoang Lu ◽

Vishal Kottadiel ◽

Reza Vafabakhsh ◽

...

Keyword(s):

Single Molecule ◽

Viral Genome ◽

Motor Coordination ◽

Bacteriophage T4 ◽

Dna Packaging ◽

Biological Functions ◽

Dna Translocation ◽

Genome Packaging ◽

Dna Packaging Motor ◽

Direct Counting

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, by direct counting using single-molecule fluorescence, we have determined 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 count of active/inactive subunit(s), we found, unexpectedly, that the packaging motor can tolerate an inactive sub-unit. However, motors containing an inactive subunit(s) exhibit fewer DNA engagements, a higher failure rate in encapsidation, reduced packaging velocity, and increased pausing. These findings suggest a new packaging model in which the motor, by re-adjusting its grip on DNA, can skip an inactive subunit and resume DNA translocation, contrary to the prevailing notion of strict coordination amongst motor subunits of other packaging motors.

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Viral packaging ATPases utilize a glutamate switch to couple ATPase activity and DNA translocation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2024928118 ◽

2021 ◽

Vol 118 (17) ◽

pp. e2024928118

Author(s):

Joshua Pajak ◽

Rockney Atz ◽

Brendan J. Hilbert ◽

Marc C. Morais ◽

Brian A. Kelch ◽

...

Keyword(s):

Atpase Activity ◽

Signaling Pathway ◽

Bound States ◽

Atp Hydrolysis ◽

Dna Packaging ◽

Atp Binding ◽

Dna Translocation ◽

Viral Packaging ◽

Mechanochemical Cycle ◽

Dynamics Simulations

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.

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A viral genome packaging ring-ATPase is a flexibly coordinated pentamer

Nature Communications ◽

10.1038/s41467-021-26800-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Li Dai ◽

Digvijay Singh ◽

Suoang Lu ◽

Vishal I. Kottadiel ◽

Reza Vafabakhsh ◽

...

Keyword(s):

Single Molecule ◽

Viral Genome ◽

Motor Coordination ◽

Bacteriophage T4 ◽

Dna Packaging ◽

Biological Functions ◽

Dna Translocation ◽

Single Molecule Fluorescence ◽

Genome Packaging ◽

Dna Packaging Motor

AbstractMulti-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.

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The T4 TerL Prohead Packaging Motor Does Not Drive DNA Translocation by a Proposed Dehydration Mechanism

Viruses ◽

10.3390/v12050522 ◽

2020 ◽

Vol 12 (5) ◽

pp. 522 ◽

Cited By ~ 1

Author(s):

Lindsay W. Black ◽

Bingxue Yan ◽

Krishanu Ray

Keyword(s):

Nucleic Acid ◽

Motor Protein ◽

Linear Motor ◽

Dna Packaging ◽

Dna Translocation ◽

Protein Motion ◽

Previous Evidence ◽

Dehydration Mechanism ◽

Protein Motor

A “DNA crunching” linear motor mechanism that employs a grip-and-release transient spring like compression of B- to A-form DNA has been found in our previous studies. Our FRET measurements in vitro show a decrease in distance from TerL to portal during packaging; furthermore, there is a decrease in distance between closely positioned dye pairs in the Y-stem of translocating Y-DNA that conforms to B- and A- structure. In normal translocation into the prohead the TerL motor expels all B-form tightly binding YOYO-1 dye that cannot bind A-form. The TerL motor cannot package A-form dsRNA. Our work reported here shows that addition of helper B form DNA:DNA (D:D) 20mers allows increased packaging of heteroduplex A-form DNA:RNA 20mers (D:R), evidence for a B- to A-form spring motor pushing duplex nucleic acid. A-form DNA:RNA 25mers, 30mers, and 35mers alone are efficiently packaged into proheads by the TerL motor showing that a proposed hypothetical dehydration motor mechanism operating on duplex substrates does not provide the packaging motor force. Taken together with our previous studies showing TerL motor protein motion toward the portal during DNA packaging, our present studies of short D:D and D:R duplex nucleic acid substrates strongly supports our previous evidence that the protein motor pushes rather than pulls or dehydrates duplex substrates to provide the translocation into prohead packaging force.

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Only one pRNA hexamer but multiple copies of the DNA-packaging protein gp16 are needed for the motor to package bacterial virus phi29 genomic DNA

Virology ◽

10.1016/s0042-6822(03)00011-4 ◽

2003 ◽

Vol 309 (1) ◽

pp. 108-113 ◽

Cited By ~ 26

Author(s):

Dan Shu ◽

Peixuan Guo

Keyword(s):

Genomic Dna ◽

Dna Packaging ◽

Bacterial Virus ◽

Multiple Copies

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

10.1101/2020.07.27.223032 ◽

2020 ◽

Cited By ~ 3

Author(s):

Joshua Pajak ◽

Erik Dill ◽

Mark A. White ◽

Brian A. Kelch ◽

Paul Jardine ◽

...

Keyword(s):

Bound States ◽

Helical Structure ◽

Dna Packaging ◽

Dna Translocation ◽

Genome Packaging ◽

Viral Dna ◽

Dna Viruses ◽

Dynamics Simulations ◽

Viral Dna Packaging ◽

Viral Dna Packaging Motor

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-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 φ29 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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