CRYSTALLIZATION OF BAKER’S YEAST CDC11 AND SHS1 SEPTIN C-TERMINAL DOMAINS

Septins were first identified in 1971, by Hartwell. Nowadays it’s known that they are membrane-associated cytoskeletal proteins that are needed in a wide range of essential cellular processes, such as cytokinesis, microtubule dynamics, diffusion barrier formation, and mechanotransduction. These roles 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 help in polymerization and are also 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 Saccharomyces cerevisiae (known as baker's yeast). Those of Cdc11 (Cdc11CC) are expected to form a homodimeric coiled-coil. But Cdc11 can be replaced by its paralog Shs1 that substitutes and competes with Cdc11 for the terminal position and binding with Cdc12. Complexes containing Cdc11 polymerise end-to-end forming long straight filaments, meanwhile complexes with Shs1 form multilayered ringlike structures. It is also known that the C-terminal is not essential for Cdc11’s function but it is necessary for ring formation on Shs1 complexes. Both coiled-coil (CC) regions were expressed and purified. The samples of Cdc11CC were subjected to crystallographic assays using the sitting drop vapor diffusion method. Better characterization of the yeast septin coiled-coils may improve the understanding of its role and impact in the formation of those complex structures formed by complexes with Cdc11 or Shs1.

This work was supported by University of São Paulo