The Structure, Evolution, and Assembly Mechanism of the Bacteriophage Tail Tube
Keywords: Structure
Virus

Large multi-component structures play an essential role in many crucial cellular processes. The morphogenetic pathway of the long, non-contractile tail of bacteriophage λ provides a superb paradigm for studying the assembly of macromolecular complexes. This thesis describes the structural and functional characterization of two λ tail proteins, gpU and gpV, with the aim of improving our understanding of phage tail assembly and evolution, while also providing a starting point to answering some of the fundamental questions surrounding the assembly and function of other supramolecular structures. Tail Terminator Proteins (TrPs) play an essential role in regulating the length of phage tails, and serve as the interaction surface for phage heads. To provide insight into the mechanisms by which TrPs exert their functions, I have determined the X-ray crystal structure of gpU, the TrP from phage λ, in its biologically relevant hexameric state. The gpU hexamer displays several flexible loops that are involved in head and tail binding. By comparing the hexameric crystal structure of gpU to its previously determined NMR solution structure I was able to identify large structural rearrangements in the protein, which are likely induced upon oligomerization. In addition, I have shown that the hexameric structure of gpU is very similar to the structure of a putative TrP from a contractile phage tail even though they display no detectable sequence similarity. This finding implies that the TrPs of non-contractile tailed phages are evolutionarily related to those of contractile-tailed phages. To determine the mechanism by which tail tubes self-assemble prior to termination, I have determined the NMR solution structure of the N-terminal domain of gpV (gpVN), the protein comprising the major portion of the phage λ tail tube. I found that approximately 30% of gpVN is disordered in solution and that some of these disordered regions are biologically important. Intriguingly, my gpVN structure is very similar to a previously solved tail tube protein from a contractile-tailed phage, once again suggesting an evolutionary connection between these two distinct tail types. A remarkable structural similarity is also seen to the hexameric structure of Hcp1, a component of the bacterial type VI secretion system. This finding, coupled with other similarities between phage and type VI secretion proteins support an evolutionary relationship between these systems. Using Hcp1 as a model, I proposed a mechanism for the oligomerization and polymerization of gpV involving several disorder-to-order transitions. Further supporting the importance of unstructured regions, I have shown that the unstructured linker between the N- and C-terminal domains of gpV is crucial for protein function and that a complete truncation of the C-terminal domain (gpVC) results in a 100-fold decrease in activity compared to full-length gpV (gpVFL). To provide insight into the role of gpVC, I determined its NMR solution structure and showed that it possesses an Ig-like fold, however the function of gpVC remains unknown. Interestingly, the gpVC structure revealed the location of two residues that when mutated were previously shown to either abrogate (G222D) or restore (G222D/P227L) function of gpVFL. In addition to being inactive, I demonstrated that the G222D mutation also exerts a temperature dependent dominant negative phenotype. My preliminary NMR data suggests that G222D causes gpVC to partially unfold and that this destabilized form of the domain interacts with gpVN in a region that is likely involved in both oligomerization and hexamer-hexamer interactions. To further our understanding of how these mutations exert their effect, I determined the NMR solution structure of gpVC-P227L. My structure reveals that the β7-β8 region of gpVC-P227L is altered compared to gpVC-WT and suggests that the conformational changes in gpVC-P227L may protect the domain from protein-folding defects induced by the G222D mutation.