Human septins 3, 9 and 12 are the only members of a specific subgroup of septins that display several unusual features, including the absence of a C-terminal coiled coil. This particular subgroup (the SEPT3 septins) are present in rod-like octameric protofilaments but are lacking in similar hexameric assemblies, which only contain representatives of the three remaining subgroups. Both hexamers and octamers can self-assemble into mixed filaments by end-to-end association, implying that the SEPT3 septins may facilitate polymerization but not necessarily function. These filaments frequently associate into higher order complexes which associate with biological membranes, triggering a wide range of cellular events. In the present work, a complete compendium of crystal structures for the GTP-binding domains of all of the SEPT3 subgroup members when bound to either GDP or to a GTP analogue is provided. The structures reveal a unique degree of plasticity at one of the filamentous interfaces (dubbed NC). Specifically, structures of the GDP and GTPγS complexes of SEPT9 reveal a squeezing mechanism at the NC interface which would expel a polybasic region from its binding site and render it free to interact with negatively charged membranes. On the other hand, a polyacidic region associated with helix α5′, the orientation of which is particular to this subgroup, provides a safe haven for the polybasic region when retracted within the interface. Together, these results suggest a mechanism which couples GTP binding and hydrolysis to membrane association and implies a unique role for the SEPT3 subgroup in this process. These observations can be accounted for by constellations of specific amino-acid residues that are found only in this subgroup and by the absence of the C-terminal coiled coil. Such conclusions can only be reached owing to the completeness of the structural studies presented here.

Human septins are guanine nucleotide binding proteins that form membrane associating hetero-filaments which result from the polymerization of core particles composed of either six or eight monomers [1]. The resulting filaments are involved in both barrier formation and in a series of membrane remodelling events including cytokinesis. Human septins can be divided into four groups based on sequence similarity and a representative of each is required to build the octameric core particle to which they all provide two monomers. The most widely studied octamer is that composed of septins 2, 6, 7 and 9 which assemble in the following order: SEPT9-SEPT7-SEPT6-SEPT2-SEPT2-SEPT6-SEPT7-SEPT9 [2] generating two different types of inter-subunit interface (G and NC) which alternate along the filament axis. As such it is obvious that interactions formed between adjacent copies of SEPT9 are fundamental to the polymerization process as they form the interface between successive core particles. We have solved the structure of the GTP binding domain of human SEPT9 complexed to both GDP and GTPγS. Two monomers are observed to be squeezed together at the NC interface in the latter leading to a foreshortening of the homo-filament observed in the crystal. This involves a largely rigid body translation together with limited structural rearrangement of the individual monomers. The result is the reduction of the space available at the interface for accommodating a polybasic helix (absent from the construct used in the present study but observed in previous structures [3]) which is known to be involved in membrane association. Based on both the crystal structures reported here and associated modelling studies, we provide a mechanism by which a hetero-filament would bind to a membrane via its polybasic helix in the presence of GTP but would be expected to bury the helix on GTP hydrolysis thus leading to release of the filament from the membrane. This suggests that the NC interface between successive core particles along a filament may have special properties relevant to the dynamics of membrane association which could be related to membrane remodelling events.