C-TERMINAL DOMAINS OF SEPTINS FROM RICE BLAST FUNGUS

Septins are membrane-associated cytoskeletal proteins that are relevant in a wide range of essential cellular processes, such as cytokinesis, microtubule dynamics, diffusion barrier formation, and mechanotransduction. These attributes are, at least in part, due to their ability to self-assemble into heterofilaments and highly organized polymers. In the C-terminal region, septins may possess coiled-coil forming sequences that appear to aid in polymerization and also be involved in interfilament contacts, membrane and partner interactions. Despite several septin structures already being resolved, both by X-ray crystallography and cryo-electron microscopy, the C-terminal domains do not generate interpretable signal due to their intrinsic dynamics, which seem to be independent of the central domain. Thus, the present work aims at the biophysical and structural characterization of the C-terminal domains of septins from Magnaporthe oryzae (rice blast fungus). Those of Sep3 (Sep3C) and Sep6 (Sep6C), which are expected to form a heterodimeric coiled-coil, were expressed and purified. Experiments to characterize the oligomeric state of the samples and the formation of the Sep3C-Sep6C heterodimer were carried out, including analysis using SEC-MALS, circular dichroism spectroscopy and chemical crosslinking. Homo- and heterodimeric samples were subjected to crystallographic assays using the sitting drop vapor diffusion method. Currently, the obtained results  for Sep3C suggest a fully monomeric sample, meanwhile the results for Sep6C and the combination of Sep3C-Sep6C indicate that the sample has different oligomeric states, meaning that crystallizing this protein can be challenging. Better characterization of the fungal septin coiled-coils may improve the understanding of septin filament interactions with other filaments, partner proteins, and biological membranes and could contribute to new forms to combat the blast disease in rice plants.