Structure, function and evolution of phosphoprotein P0 and its unique insert in Tetrahymena thermophila

Phosphoprotein P0 is a highly conserved ribosomal protein that forms the central scaffold of the large ribosomal subunit’s “stalk complex”, which is necessary for recruiting protein elongation factors to the ribosome. Evidence in the literature suggests that P0 may be involved in diseases such as malaria and systemic lupus erythematosus. We are interested in the possibility that the P0 of the “ciliated protozoa” Tetrahymena thermophila may be useful as a model system for vaccine research and drug development. In addition, the P0s of T. thermophila and other ciliated protozoans contain a 15 to 17 amino acid long insert, unique to the N-terminal region. This project sought to further characterize the T. thermophila P0 (TtP0) and its unique insert through structural and functional bioinformatics studies. In order to visualize the three-dimensional structure of TtP0, we created a homology model of the N-terminal region of TtP0 and its insert from available P0 structure and sequence data. When the insert was modeled “in-context” in the presence of a previously published crystal structure of the T. thermophila ribosomal RNA, we discovered a surprising association between the insert and a highly variable portion of the rRNA, termed expansion segment 7, or ES7. When we investigated if this association could occur in other ciliates, we found very little data for the ES7 sequence in other species, meaning that further analysis on the conservation of this association is not possible at this time. Still, the presence of an association in T. thermophila may indicate that the insert has a functional role unique to the ciliates, perhaps in the regulation of P0 function by phosphorylation. In addition, we also investigated whether the highly conserved nature of P0 meant that it could be useful for phylogenetic and evolutionary studies. By studying P0 sequences from ciliates and other closely related clades, we could determine if P0 provides any information on the early evolution of eukaryotic species. We collected P0 sequences representing all of the eukaryotic supergroups, and used them to create phylogenetic alignments and trees based on the whole molecules, as well as the individual functional domains. Overall, we found that the trees did not resolve very well at the basal branches, but terminal branches had much stronger support. The trees also successfully separated the ciliate P0 sequences into groups matching the previously established taxonomy for the ciliates. Finally, we found evidence that the N-terminal domain of P0, called the L10 region, is much more evolutionarily stable than the C-terminal 60S region. Thus, the variability of the 60S region appears to contribute to the diversity of ciliate and eukaryotic P0 sequences. Once additional P0 sequences become available for underrepresented clades, they could be used to provide stronger support for the weaker branches of the tree. Both studies provide a starting framework for further computational-based work on P0, such as homology modeling of P0s from other ciliates or simulations of insert phosphorylation. These studies may also serve as a starting point for in vitro or in vivo experiments on the protein and its ciliate-specific insert.