NMR structure of a vestigial nuclease provides insight into the evolution of functional transitions in viral dsDNA packaging motors

Mahler, Bryon; Bujalowski, Paul J.; Mao, Huzhang; Dill, Erik; Jardine, Paul J.; Choi, Kyung Hyun; Morais, Marc C.

doi:10.1093/nar/gkaa874

Cited by 12 publications

(6 citation statements)

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“…Hence, the most tractable path to the atomic structure of a functional viral dsDNA packaging motor is to determine atomic resolution structures of isolated components and then fit them into cryo-EM reconstructions of the entire motor complex assembled on the procapsid ( 42 ). To date, we have determined atomic resolution structures of nearly every component of the phi29 packaging motor including the portal ( 25 ), the prohead-binding domain of the pRNA ( 27 ), the pRNA CCA bulge ( 43 ), part of the A-helix of the pRNA ( 27 , 44 , 45 ), the N-terminal ATPase domain of gp16 ( 31 ), and, most recently, the C-terminal vestigial nuclease domain of gp16 ( 46 ). Further, we have been recently determining the full-length structure of a homologous packaging ATPase from a relative of phi29, bacteriophage asccphi28 ( 47 ).…”

Section: Resultsmentioning

confidence: 99%

A viral genome packaging motor transitions between cyclic and helical symmetry to translocate dsDNA

et al. 2021

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Molecular segregation and biopolymer manipulation require the action of molecular motors to do work by applying directional forces to macromolecules. The additional strand conserved E (ASCE) ring motors are an ancient family of molecular motors responsible for diverse biological polymer manipulation tasks. Viruses use ASCE segregation motors to package their genomes into their protein capsids and provide accessible experimental systems due to their relative simplicity. We show by cryo-EM–focused image reconstruction that ASCE ATPases in viral double-stranded DNA (dsDNA) packaging motors adopt helical symmetry complementary to their dsDNA substrates. Together with previous data, our results suggest that these motors cycle between helical and planar configurations, providing a possible mechanism for directional translocation of DNA. Similar changes in quaternary structure have been observed for proteasome and helicase motors, suggesting an ancient and common mechanism of force generation that has been adapted for specific tasks over the course of evolution.

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Section: Resultsmentioning

confidence: 99%

A viral genome packaging motor transitions between cyclic and helical symmetry to translocate dsDNA

et al. 2021

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“…Asymmetric alignment was similarly observed for the Φ29 motor, where the large terminase channel is tilted at 12.5° to the portal axis (59). However, in contrast, the Φ29 portal-terminase interaction is mediated by a pRNA, which is absent in the HK97 system, while the terminase itself lacks nuclease activity (68).…”

Section: Discussionmentioning

confidence: 99%

Insights into a viral motor: the structure of the HK97 packaging termination assembly

Hawkins

Bayfield

Fung

et al. 2023

Preprint

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Double-stranded DNA viruses utilise machinery, made of terminase proteins, to package viral DNA into the capsid. For cos bacteriophage, a defined signal, recognised by small terminase, flanks each genome unit. Here we present the first structural data for a cos virus DNA packaging motor, assembled from the bacteriophage HK97 terminase proteins, procapsids encompassing the portal protein, and DNA containing a cos site. The cryo-EM structure is consistent with the packaging termination state adopted after DNA cleavage, with DNA density within the large terminase assembly ending abruptly at the portal protein entrance. Retention of the large terminase complex after cleavage of the short DNA substrate suggests that motor dissociation from the capsid requires headful pressure, in common with pac viruses. Interestingly, the clip domain of the 12-subunit portal protein does not adhere to C12 symmetry, indicating asymmetry induced by binding of the large terminase/DNA. The motor assembly is also highly asymmetric, showing a ring of 5 large terminase monomers, tilted against the portal. Variable degrees of extension between N- and C-terminal domains of individual subunits suggest a mechanism of DNA translocation driven by inter-domain contraction and relaxation.

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“…Since φ29 packages a unit-length genome, its packaging motor is accordingly simpler. There is no need for a nuclease function to cut the DNA, and thus the φ29 ATPase lacks a fully functional nuclease domain and is only ~ 60% the size of ATPases/large terminases in genome-cutting packaging systems [ 16 ]. Similarly, the portal protein is only ~60% of the size of the portals in the cutting systems [ 17 ], possibly because it does not have or need any structural components to sense that the capsid is full and/or transmit this signal to the rest of the motor.…”

Section: Introductionmentioning

confidence: 99%

Biochemical and Biophysical Characterization of the dsDNA Packaging Motor from the Lactococcus lactis Bacteriophage Asccphi28

Reyes-Aldrete

Dill

Bussetta

et al. 2020

Viruses

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Double-stranded DNA viruses package their genomes into pre-assembled protein procapsids. This process is driven by macromolecular motors that transiently assemble at a unique vertex of the procapsid and utilize homomeric ring ATPases to couple genome encapsidation to ATP hydrolysis. Here, we describe the biochemical and biophysical characterization of the packaging ATPase from Lactococcus lactis phage asccφ28. Size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), small angle X-ray scattering (SAXS), and negative stain transmission electron microscopy (TEM) indicate that the ~45 kDa protein formed a 443 kDa cylindrical assembly with a maximum dimension of ~155 Å and radius of gyration of ~54 Å. Together with the dimensions of the crystallographic asymmetric unit from preliminary X-ray diffraction experiments, these results indicate that gp11 forms a decameric D5-symmetric complex consisting of two pentameric rings related by 2-fold symmetry. Additional kinetic analysis shows that recombinantly expressed gp11 has ATPase activity comparable to that of functional ATPase rings assembled on procapsids in other genome packaging systems. Hence, gp11 forms rings in solution that likely reflect the fully assembled ATPases in active virus-bound motor complexes. Whereas ATPase functionality in other double-stranded DNA (dsDNA) phage packaging systems requires assembly on viral capsids, the ability to form functional rings in solution imparts gp11 with significant advantages for high-resolution structural studies and rigorous biophysical/biochemical analysis.

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NMR structure of a vestigial nuclease provides insight into the evolution of functional transitions in viral dsDNA packaging motors

Cited by 12 publications

References 50 publications

A viral genome packaging motor transitions between cyclic and helical symmetry to translocate dsDNA

A viral genome packaging motor transitions between cyclic and helical symmetry to translocate dsDNA

Insights into a viral motor: the structure of the HK97 packaging termination assembly

Biochemical and Biophysical Characterization of the dsDNA Packaging Motor from the Lactococcus lactis Bacteriophage Asccphi28

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