Structural and biochemical studies of the large terminase protein from thermophilic viruses

In large dsDNA viruses, DNA is translocated into the procapsid by a powerful motor, formed by portal protein, large and small terminase proteins. An essential component of the motor, large terminase protein, is composed of an ATPase domain and a nuclease domain. In this thesis, large terminase proteins from several thermophilic bacteriophages: GVE2; GBSV1; phiOH2; 1-1; G20c and D6E; were cloned, expressed and purified. Of the soluble and functional proteins, the large terminases from G20c and D6E were chosen for further structural and mechanistic studies.
Crystal structures of the large terminase nuclease domain from Thermus thermophilus bacteriophage G20c exhibits the closest similarity with the RuvC endonucleases which belong to the RNase H-like superfamily. Biochemical and mutagenesis studies identified catalytically indispensable residues including D294, D429 and D347 at the nuclease active site. Structures in complex with different metals reveal that only one catalytic metal ion is found at the active site in the absence of DNA. Binding of the second metal ion is likely to be facilitated by the observed conformational variability of loop L1, which may bring the two catalytic aspartic acids D347 and D294 closer to each other. These data support a RuvC like two-metal catalysis mechanism for the G20c large terminase nuclease.
The crystal structure of the large terminase from the Geobacillus stearothermophilus bacteriophage D6E shows a unique inter-domain orientation of the N-terminal ATPase and C-terminal nuclease domains. AUC and SEC data demonstrated the presence of pentamers formed by the ATPase domain and a model pentamer was obtained by molecular docking from which a putative trans-arginine, R158, was predicted- and confirmed experimentally- as functioning as a trans-arginine finger. This residue is in an equivalent position to the trans-finger for the HerA/FtsK superfamily of ATPases, suggesting that large terminase proteins may be more closely related than previously thought.