Expression, purification, crystallization and preliminary X-ray diffraction analysis of a lactococcal bacteriophage...

Area of Science:

• Structural Biology
• Virology
• Enzymology

Background:

• Terminases are essential viral enzymes responsible for packaging double-stranded DNA genomes into procapsids.
• These enzymes typically consist of two subunits, TerS (small) and TerL (large), forming a functional complex.
• The precise stoichiometry and oligomeric state of terminase complexes have remained largely undetermined.

Purpose of the Study:

• To elucidate the oligomeric state of the small terminase subunit (TerS) from the lactococcal bacteriophage ASCC454.
• To characterize the structural properties of TerS through crystallization and X-ray diffraction.

Main Methods:

• Cloning, expression, and purification of recombinant TerS from lactococcal bacteriophage ASCC454.
• Crystallization using nanolitre sitting drops and vapor diffusion, optimized with hanging drops under a nitrogen atmosphere.
• X-ray diffraction analysis of crystals at 2.42 Å resolution using synchrotron radiation.

Main Results:

• Crystals of TerS belonged to the P2 space group with specific unit-cell parameters.
• Self-rotation function analysis indicated that TerS oligomerizes into an octamer within the asymmetric unit.
• Size-exclusion chromatography suggested the potential for TerS to form oligomers up to 13 subunits.

Conclusions:

• The study reveals the oligomeric state of the small terminase subunit (TerS) from a lactococcal bacteriophage.
• The findings provide structural insights into the assembly and function of viral DNA packaging machinery.
• Understanding terminase oligomerization is crucial for comprehending viral genome packaging mechanisms.