Bacteriophage N4 large terminase: expression, purification and X-ray crystallographic analysis

Area of Science:

• Structural biology
• Molecular microbiology
• Virology

Background:

• Genome packaging in double-stranded DNA (dsDNA) bacteriophages is essential for viral assembly.
• The large terminase enzyme powers this process, acting as a molecular motor.
• Limited structural data exists for wild-type large terminase proteins, hindering mechanistic understanding.

Purpose of the Study:

• To obtain structural information on the large terminase from bacteriophage N4.
• To investigate the biochemical activity of the N4 large terminase, particularly its interaction with the small terminase.
• To contribute to understanding the dsDNA bacteriophage genome packaging mechanism.

Main Methods:

• Cloning, expression, and purification of large and small terminase proteins from bacteriophage N4.
• Biochemical assays to assess the ATPase activity of the large terminase, alone and with the small terminase.
• X-ray crystallography of the purified large terminase protein using sitting-drop vapour-diffusion.
• X-ray diffraction data collection and analysis to determine crystal space group and unit-cell parameters.

Main Results:

• The purified N4 large terminase protein exhibits ATP hydrolysis activity.
• ATP hydrolysis is enhanced in the presence of the N4 small terminase.
• A crystal of the N4 large terminase was obtained, diffracting to 2.8 Å resolution.
• The crystal belongs to space group P2₁2₁2₁ with specific unit-cell parameters.

Conclusions:

• The study successfully purified and characterized the N4 large terminase, demonstrating its ATPase activity.
• The interaction with the small terminase enhances the enzyme's function, suggesting a cooperative mechanism.
• The obtained crystal structure provides a foundation for further detailed structural analysis of the N4 terminase, advancing the understanding of dsDNA bacteriophage genome packaging.