Abstract
Packaging of viral genomes into preformed procapsids requires the controlled and synchronized activity of an ATPase and a genome-processing nuclease, both located in the large terminase (L-terminase) subunit. In this paper, we have characterized the structure and regulation of bacteriophage P22 L-terminase (gp2). Limited proteolysis reveals a bipartite organization consisting of an N-terminal ATPase core flexibly connected to a C-terminal nuclease domain. The 2.02 Å crystal structure of P22 headful nuclease obtained by in-drop proteolysis of full-length L-terminase (FL-L-terminase) reveals a central seven-stranded β-sheet core that harbors two magnesium ions. Modeling studies with DNA suggest that the two ions are poised for two-metal ion-dependent catalysis, but the nuclease DNA binding surface is sterically hindered by a loop-helix (L(1)-α(2)) motif, which is incompatible with catalysis. Accordingly, the isolated nuclease is completely inactive in vitro, whereas it exhibits endonucleolytic activity in the context of FL-L-terminase. Deleting the autoinhibitory L(1)-α(2) motif (or just the loop L(1)) restores nuclease activity to a level comparable with FL-L-terminase. Together, these results suggest that the activity of P22 headful nuclease is regulated by intramolecular cross-talk with the N-terminal ATPase domain. This cross-talk allows for precise and controlled cleavage of DNA that is essential for genome packaging.
Highlights
The headful nuclease is essential for genome processing during viral DNA packaging
In analogy to T4 L-terminase [21], the L-terminase subunit of bacteriophage P22 contains two folded domains linked by a flexible loop that is readily cleaved by proteases in solution
Both cuts are catalyzed by the nuclease domain of L-terminase, which represents the catalytic core of a viral genome-packaging motor [1,2,3]
Summary
The headful nuclease is essential for genome processing during viral DNA packaging. Results: The crystal structure of P22 nuclease reveals a seven-stranded -sheet core with two magnesium ions poised for catalysis. These results suggest that the activity of P22 headful nuclease is regulated by intramolecular cross-talk with the N-terminal ATPase domain This cross-talk allows for precise and controlled cleavage of DNA that is essential for genome packaging. While no high resolution structure is available for a L-terminase holoenzyme, a pseudoatomic model of T4 L-terminase was proposed based on a 32 Å asymmetric cryo-electron microscopy reconstruction of T4 procapsid bound to L-terminase [6] In this model, the nuclease domain of L-terminase points away from the portal protein, and its ATPase domain forms a pentameric ring bound directly to the portal vertex [6]. This work expands the repertoire of viral nuclease structures solved to date and sheds light on several properties governing the activity and regulation of a prototypical headful nuclease
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