Structural Studies on Bacteriophage Portal Proteins

In tailed bacteriophages and evolutionarily related herpes viruses, the portal protein is a central component of the DNA packaging molecular motor, which translocates viral genomic DNA into a preformed procapsid. The motor is the most powerful molecular machine discovered in nature, generating forces reaching ~50 pN and translocating DNA with a speed of several hundred bp/sec using ATP as an energy source. The oligomeric portal protein ring is situated at a unique vertex of the procapsid forming a conduit for DNA entry and exit. Although the three-dimensional structure has already been determined for portal proteins from bacteriophages P22, SPP1 and phi29, several important questions about the role of individual protein segments in DNA translocation and their interaction with other components of the motor remain unanswered. Structural information on portal proteins from other bacteriophages, like T4 for which a wealth of biochemical information is already available, will help to answer at least some of these questions. The portal protein of bacteriophage SPP1 (gp6) can form circular oligomers containing 12 or 13 subunits. It is found as a 12-subunit oligomer when incorporated into the viral capsid and as a 13-subunit assembly in its isolated form. The X-ray structure of the SPP1 portal protein is available only for the isolated 13-subunit assembly of the N365K mutant form. Because this mutation results in a reduction in the length of packaged DNA, determining the structure of the wild type portal protein would shed light on the mechanism of DNA translocation. Elucidation of the mechanism of DNA packaging depends also on the availability of accurate structural information on the SPP1 portal protein in its 12-subunit biologically active state. Such structural knowledge would be particularly useful in future, for designing a stable molecular machine that can function in vitro. In this thesis, experiments were designed to promote the formation of the dodecameric gp6: viz fusing gp6 with TRAP protein that forms a stable circular dodecamer as well as the co-expression of gp6 with the SPP1 scaffolding protein gp11. The protein targets were cloned, expressed and purified, and the oligomeric state of gp6 was characterised by a combination of biochemical, biophysical and structural approaches. The structure of the wild type gp6 was solved at 2.8 Å resolution, revealing a 13-fold symmetrical molecule. The protein’s fold is the same as for the N365K mutant, with most significant conformational differences observed in the tunnel loop and in segments of the clip and crown domains. Comparison with the structure of N365K mutant reveals significant differences in subunit-subunit interactions formed by tunnel loops, including different hydrogen bonding and van der Waals interactions. It is likely that these differences account for the different amount of packaged DNA, indicating involvement of tunnel loops in DNA packaging. The portal protein of bacteriophage CNPH82, cn3, was also successfully cloned, expressed and purified. Promising crystallisation conditions have been identified that yield crystals diffracting to 4.2 Å. Further optimisation should lead to determination of the X-ray structure of this protein in not too distant future. Self-rotation function calculations and SEC-MALLS analysis indicate that the cn3 protein forms 13-subunit assemblies, in common with the SPP1 portal protein. Foundation work has been carried out for the bacteriophage T4 portal protein, aimed at identifying suitable production and purification conditions. In addition, the full-length bacteriophage SPP1 scaffolding protein gp11 has been cloned, purified and crystallised. Degradation was observed in the full length gp11 protein and therefore a series of truncations were designed, cloned and purified aiming to improve the stability. Further studies on limited proteolysis of the full-length gp11 should lead to a stable gp11 tuncation that will form crystals with better diffraction.