Grupp, B.; Jaeckel, B.; Guenther, A.; Rehberger, J.; Steimle, S.; Gehrke, J. F.; Sieg, C.; Strittmatter, J.; Wunder, T.; Vomhof, T.; Ruhnke, J.; Schleicher, E.; Gerhardt, S.; Johnsson, N.; Gronemeyer, T.

Septins are conserved cytoskeletal GTP-binding proteins that form higher-order structures critical for cytokinesis and polarized growth across opisthokonts. In budding yeast and humans, four homologous septins assemble into hetero-octameric protofilaments through alternating interactions between adjacent G-domains (G-interface) or N- and C-termini (NC-interface), which then polymerize end-to-end into filaments. How nucleotide binding and hydrolysis control filament assembly has remained elusive. We uncover a septin-specific mechanism in which nucleotide binding stabilizes the monomeric G-interface and primes it for protofilament formation. Cryo-EM and DEER spectroscopy reveal nucleotide-induced conformational changes that engage the G-interface and enable an NC-compatible conformation absent in the nucleotide-free state. In vitro binding and reconstitution assays confirm that G-interface formation is strictly nucleotide-dependent and required for subsequent NC-interface assembly. These findings establish unidirectional allosteric signaling from the G- to the NC-interface, revealing the molecular basis for controlled septin protofilament assembly.