A CONSERVED NON-CANONICAL G INTERFACE DEFINES FUNGAL GROUP 5 SEPTINS

Septins are GTP-binding cytoskeletal proteins that assemble into filaments and higher-order structures required for membrane remodeling, cell polarity, and cytokinesis. While filament assembly is well characterized for the four canonical septin groups, the structure and function of the evolutionarily distinct Group 5 (non-canonical) septins remain largely unknown.

We present the first comparative structural study of fungal Group 5 septins, from Magnaporthe oryzae (MoSep7) and Aspergillus nidulans (AspE). Four high-resolution crystal structures (1.5–1.7 Å) of MoSep7 and AspE in both GDP- and GTP-bound states provide the first experimental comparison of fungal Group 5 septins across species and nucleotide states. Crystallization was achieved by engineering constructs lacking predicted intrinsically disordered internal loops respectively.

Despite their evolutionary divergence, the two proteins share a highly conserved, non-canonical G interface fundamentally distinct from that of canonical septins. A characteristic α-helical insertion remodels the polymerization interface, replacing the conserved inter-subunit contacts of canonical septins with a novel interaction network stabilized by aromatic residues within a distorted three stranded β-meander. This architecture generates a more open dimer configuration while preserving the overall septin fold. Comparison of the GDP- and GTP-bound structures shows that the G interface is essentially unchanged, indicating that this architecture is an intrinsic and conserved feature of fungal Group 5 septins rather than a nucleotide-dependent conformation.

Biochemical characterization further demonstrates that both Group 5 septins retain intrinsic, albeit slow, GTPase activity despite extensive remodeling of the polymerization interface. Structural superposition with canonical septin filaments suggests that incorporation of a Group 5 dimer into a canonical heterofilament would introduce filament curvature, whereas compensatory rearrangements at the NC interface could instead support linear homomeric filament formation. We are pursuing cryo-EM analysis of Group 5 septin polymer assembly, which will allow direct visualization of filament architecture.

Together, these findings establish the first structural framework for fungal Group 5 septins, revealing a conserved non-canonical polymerization interface that expands the current paradigm of septin assembly and provides new insight into the evolution and functional diversification of the septin cytoskeleton.