Structure of p22 headful packaging nuclease

Area of Science:

• Structural Biology
• Molecular Virology
• Biochemistry

Background:

• Viral genome packaging into procapsids requires coordinated ATPase and nuclease activities.
• These functions are integrated within the large terminase (L-terminase) subunit of bacteriophages.
• Bacteriophage P22 L-terminase (gp2) is crucial for its DNA packaging mechanism.

Purpose of the Study:

• To characterize the structure and regulatory mechanisms of bacteriophage P22 L-terminase (gp2).
• To elucidate the interplay between the ATPase and nuclease activities within L-terminase.
• To understand how DNA cleavage is precisely controlled during viral genome packaging.

Main Methods:

• Limited proteolysis to determine the domain organization of P22 L-terminase.
• X-ray crystallography to obtain the high-resolution structure of the P22 headful nuclease domain (2.02 Å).
• In vitro biochemical assays to assess nuclease activity, including site-directed mutagenesis.

Main Results:

• P22 L-terminase exhibits a bipartite structure with an N-terminal ATPase core and a C-terminal nuclease domain.
• The crystal structure reveals a magnesium-bound nuclease active site, but its catalytic surface is sterically hindered by an autoinhibitory loop-helix motif (L(1)-α(2)).
• The isolated nuclease is inactive, but regains activity within the full-length L-terminase or upon deletion of the inhibitory motif, indicating intramolecular regulation.

Conclusions:

• The activity of the P22 headful nuclease is tightly regulated by intramolecular cross-talk with the N-terminal ATPase domain.
• An autoinhibitory motif (L(1)-α(2)) prevents premature DNA cleavage, ensuring controlled activity.
• This regulated mechanism is essential for the precise and efficient packaging of viral genomes.