Proceedings of Annual Meeting of the Brazilian Biophysical Society
Proceedings of Annual Meeting of the Brazilian Biophysical Society - Vol. 2 2024

Septins are important cytoskeletal proteins involved in many membrane remodelling processes. Some septins have a C-terminal domain (CTD) containing coiled-coil (cc) repeats. In yeast, septins from four groups assemble into linear octameric core particles (Cdc11-Cdc12-cc-Cdc3-Cdc10-Cdc10-Cdc3-cc-Cdc12-Cdc11, cc denotes interfaces where coiled coils are likely present) which polymerize end-to-end to form filaments. Although the CTDs are not essential to core particle formation, they are believed to assist in molecular recognition between septins and mediate septinpartner interactions. Cdc12, essential for yeast survival, is known to interact with itself in vitro [1]. This self-interaction has hindered detailed studies of the heterodimeric cc formed between Cdc12 and Cdc3. Here, we characterized the homomeric states of the Cdc12 CTD. We show that the Cdc12 CTD exhibits a dimertrimer equilibrium in solution. Using SEC-SAXS, we demonstrate that MBP-fused Cdc12 CTD assembles into parallel coiled coils. Alphafold-multimer predicts a highconfidence, in-register Cdc12 CTD homotrimer, likely favored by the presence of a Trp residue (W367) in the core of the cc. This Trp is conserved in all Cdc12 homologues and we show its mutation disrupts both dimer- and trimerization. Additionally, mutation of the adjacent residue upstream of the Trp (E368 in Cdc12) to Val strongly favors trimers. Thermal denaturation experiments by circular dichroism spectroscopy show that the E368V mutant has a significantly increased melting temperature, 24.4 ± 0.4 °C higher than the wild type CTD. These Cdc12 mutants are being tested in yeast for their localization and the resulting yeast phenotype upon overexpression. We hypothesize that the Cdc12 homomerization may potentially create a reservoir of transiently polymerization-incompetent Cdc12 in the cell, which may often be required due to the slow translation rate of Cdc12 observed in vivo [2].

References:
[1] VERSELE, Matthias et al. Protein-Protein Interactions Governing Septin Heteropentamer Assembly and Septin Filament Organization in Saccharomyces cerevisiae. Molecular Biology of the Cell, v. 15, p. 4568-4583, 2004.
[2] HASSELL, Daniel et al. Chaperone requirements for de novo folding of Saccharomyces cerevisiae septins. Molecular Biology of the Cell, v. 33, 2022.

This work was supported by FAPESP (grants 2018/19992-7, 2020/02897-1 and 2022/00262-4).