Structural investigation of two viral proteins involved in DNA-packaging

Viral DNA-packaging motors are molecular machines that enable viruses to replicate by providing the means of storing the viral genome into empty procapsids. In double stranded DNA bacteriophages the molecular motor is comprised by three elements: the portal protein and the small and large terminases. The portal protein nucleates polymerisation of the capsid and scaffold proteins, initiating procapsid assembly, besides allowing passage of DNA. The small terminase recognises the viral genome and presents it to the large terminase which possesses both nuclease activity to cleave concatemeric DNA at the initiation of the packaging, and ATPase activity to drive translocation.
Although currently several X-ray structures for the different components of the motor from different bacteriophages are available fundamental questions regarding the DNA recognition mechanism, stoichiometry and orientation of the motor components in vivo and the mechanism of ATP-driven DNA-translocation remain. This project focused on elucidating the X-ray crystal structures of (i) the major capsid protein, from Bacillus subtilis bacteriophage SPP1 and (ii) the small terminase protein from Thermus thermophilus bacteriophage G20C.
Several constructs of the SPP1 capsid protein, including truncations at the N- and C-termini, single and double mutants and engineered proteins were generated in order to produce a protein suitable for crystallisation. Mutant uG13P;T104Y;A261W was the only construct that produced native and Se-Met crystals. The X-ray structure of the SPP1 capsid protein was determined at 3.0 Å resolution by single wavelength anomalous diffraction. The structure exhibited the HK97-fold consisting of the axial and peripheral domains and the extended E-loop.
The X-ray structure of the small terminase from phage G20C was solved at 2.5 Å resolution by single wavelength anomalous diffraction. The structure consisted of circular nine-mers where the N- terminal domains of each subunit reside at the periphery of the assembly and the C-terminal oligomerisation domains form a central channel. The conserved structural features between small terminases suggest that the DNA-recognition mechanism might be conserved. DNA-binding experiments demonstrated that the G20C small terminase binds to viral and non-viral DNA with both the N- and C- terminal domains playing an important role.
The structural information generated from these two elements of SPP1 and G20C bacteriophages provides insight into two different aspects of the assembly process: (i) how the capsid protein’s conformational plasticity may assist the assembly and (ii) DNA-recognition during virus particle construction. This information is critical for understanding similar processes in other viruses, in particular, in the evolutionarily related herpes viruses.