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Role of ?29 connector channel loops in late-stage DNA packaging.

ABSTRACT: Double-stranded DNA bacteriophages and their eukaryotic virus counterparts have 12-fold head-tail connector assemblages embedded at a unique capsid vertex. This vertex is the site of assembly of the DNA packaging motor, and the connector has a central channel through which viral DNA passes during genome packaging and subsequent host infection. Crystal structures of connectors from different phages reveal either disordered residues or structured loops that project into the connector channel. Given the proximity to the translocating DNA substrate, these loops have been proposed to play a role in DNA packaging. Previous models have proposed structural motions in either the packaging ATPase or the connector channel loops as the driving force that translocates the DNA into the prohead. Here, we mutate the channel loops of the Bacillus subtilis bacteriophage ?29 connector and show that these loops have no active role in translocation of DNA. Instead, they appear to have an essential function near the end of packaging, acting to retain the packaged DNA in the head in preparation for motor detachment and subsequent tail assembly and virion completion.

SUBMITTER: Grimes S

PROVIDER: S-EPMC3140409 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Role of φ29 connector channel loops in late-stage DNA packaging.

Grimes Shelley S Ma Shuhua S Gao Jiali J Atz Rockney R Jardine Paul J PJ

Journal of molecular biology 20110505 1

Double-stranded DNA bacteriophages and their eukaryotic virus counterparts have 12-fold head-tail connector assemblages embedded at a unique capsid vertex. This vertex is the site of assembly of the DNA packaging motor, and the connector has a central channel through which viral DNA passes during genome packaging and subsequent host infection. Crystal structures of connectors from different phages reveal either disordered residues or structured loops that project into the connector channel. Give ...[more]

PMID: 21570409

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Project description:The DNA packaging motor of the bacteriophage ϕ29, comprising head-tail connector, ATPase, and pRNA, transports the viral DNA inside the procapsid against pressure differences of up to ∼60 atm during replication. Several models for the DNA packaging mechanism have been proposed, which attribute different roles to the connector, and require specific mechanical properties of the connector. To characterize these properties at the atomic level, and to understand how the connector withstands this large pressure, we have carried out molecular dynamics simulations of the whole connector both in equilibrium and under mechanical stress. The simulations revealed a quite heterogeneous distribution of stiff and soft regions, resembling that of typical composite materials that are also optimized to resist mechanical stress. In particular, the conserved middle α-helical region is found to be remarkably stiff, similar only to structural proteins forming viral shell, silk, or collagen. In contrast, large parts of the peripheral interface to the ϕ29 procapsid turned out to be rather soft. Force probe and umbrella sampling simulations showed that large connector deformations are remarkably reversible, and served to calculate the free energies required for these deformations. In particular, for an untwisting deformation by 12°, as postulated by the untwist-twist model, more than four times' larger energy is required than is available from hydrolysis of one ATP molecule. Combined with previous experiments, this result is incompatible with the untwist-twist model. In contrast, our simulations support the recently proposed one-way revolution model and suggest in structural terms how the connector blocks DNA leakage. In particular, conserved loops at the rim of the central channel, which are in direct contact with the DNA, are found to be rather flexible and tightly anchored to the rigid central region. These findings suggest a check-valve mechanism, with the flexible loops obstructing the channel by interacting with the viral DNA.

Dataset Information

Role of ?29 connector channel loops in late-stage DNA packaging.

Publications

Role of φ29 connector channel loops in late-stage DNA packaging.

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