Dissecting the Binding Interface of the Septin Polymerization Enhancer Borg BD3.

The molecular basis for septin filament assembly has begun to emerge over recent years. These filaments are essential for many septin functions which depend on their association with biological membranes or components of the cytoskeleton. Much less is known about how septins specifically interact with their binding partners. Here we describe the essential role played by the C-terminal domains in both septin polymerization and their association with the BD3 motif of the Borg family of Cdc42 effector proteins. We provide a detailed description, at the molecular level, of a previously reported interaction between BD3 and the NC-interface between SEPT6 and SEPT7. Upon ternary complex formation, the heterodimeric coiled coil formed by the C-terminal domains of the septins becomes stabilized and filament formation is promoted under conditions of ionic strength/protein concentration which are not normally permissible, likely by favouring hexamers over smaller oligomeric states. This demonstrates that binding partners, such as Borg's, have the potential to control filament assembly/disassembly in vivo in a way which can be emulated in vitro by altering the ionic strength. Experimentally validated models indicate that the BD3 peptide lies antiparallel to the coiled coil and is stabilized by a mixture of polar and apolar contacts. At its center, an LGPS motif, common to all human Borg sequences, interacts with charged residues from both helices of the coiled coil (K368 from SEPT7 and the conserved E354 from SEPT6) suggesting a universal mechanism which governs Borg-septin interactions.

An atomic model for the human septin hexamer by cryo-EM.

In order to form functional filaments, human septins must assemble into hetero-oligomeric rod-like particles which polymerize end-to-end. The rules governing the assembly of these particles and the subsequent filaments are incompletely understood. Although crystallographic approaches have been successful in studying the separate components of the system, there has been difficulty in obtaining high resolution structures of the full particle. Here we report a first cryo-EM structure for a hexameric rod composed of human septins 2, 6 and 7 with a global resolution of ~3.6 Å and a local resolution of between ~3.0 Å and ~5.0 Å. By fitting the previously determined high-resolution crystal structures of the component subunits into the cryo-EM map, we are able to provide an essentially complete model for the particle. This exposes SEPT2 NC-interfaces at the termini of the hexamer and leaves internal cavities between the SEPT6-SEPT7 pairs. The floor of the cavity is formed by the two α0 helices including their polybasic regions. These are locked into place between the two subunits by interactions made with the α5 and α6 helices of the neighbouring monomer together with its polyacidic region. The cavity may serve to provide space allowing the subunits to move with respect to one another. The elongated particle shows a tendency to bend at its centre where two copies of SEPT7 form a homodimeric G-interface. Such bending is almost certainly related to the ability of septin filaments to recognize and even induce membrane curvature.

Orientational Ambiguity in Septin Coiled Coils and its Structural Basis.

Septins are an example of subtle molecular recognition whereby different paralogues must correctly assemble into functional filaments important for essential cellular events such as cytokinesis. Most possess C-terminal domains capable of forming coiled coils which are believed to be involved in filament formation and bundling. Here, we report an integrated structural approach which aims to unravel their architectural diversity and in so doing provide direct structural information for the coiled-coil regions of five human septins. Unexpectedly, we encounter dimeric structures presenting both parallel and antiparallel arrangements which are in consonance with molecular modelling suggesting that both are energetically accessible. These sequences therefore code for two metastable states of different orientations which employ different but overlapping interfaces. The antiparallel structures present a mixed coiled-coil interface, one side of which is dominated by a continuous chain of core hydrophilic residues. This unusual type of coiled coil could be used to expand the toolkit currently available to the protein engineer for the design of previously unforeseen coiled-coil based assemblies. Within a physiological context, our data provide the first atomic details related to the assumption that the parallel orientation is likely formed between septin monomers from the same filament whilst antiparallelism may participate in the widely described interfilament cross bridges necessary for higher order structures and thereby septin function.

A revised order of subunits in mammalian septin complexes.

Septins are GTP binding proteins considered to be novel components of the cytoskeleton. They polymerize into filaments based on hexameric or octameric core particles in which two copies of either three or four different septins, respectively, assemble into a specific sequence. Viable combinations of the 13 human septins are believed to obey substitution rules in which the different septins involved must come from distinct subgroups. The hexameric assembly, for example, has been reported to be SEPT7-SEPT6-SEPT2-SEPT2-SEPT6-SEPT7. Here, we have replaced SEPT2 by SEPT5 according to the substitution rules and used transmission electron microscopy to demonstrate that the resulting recombinant complex assembles into hexameric particles which are inverted with respect that predicted previously. MBP-SEPT5 constructs and immunostaining show that SEPT5 occupies the terminal positions of the hexamer. We further show that this is also true for the assembly including SEPT2, in direct contradiction with that reported previously. Consequently, both complexes expose an NC interface, as reported for yeast, which we show to be more susceptible to high salt concentrations. The correct assembly for the canonical combination of septins 2-6-7 is therefore established to be SEPT2-SEPT6-SEPT7-SEPT7-SEPT6-SEPT2, implying the need for revision of the mechanisms involved in filament assembly.

Correct partner makes the difference: Septin G-interface plays a critical role in amyloid formation.

Septins are members of a group of GTP-binding proteins highly conserved in eukaryotes, being linked to diverse cell processes, such as cytokinesis and membrane association. On the other hand, the malfunction of septins is linked to several pathological processes including neurodegeneration and oncogenesis. Septins interact with each other forming heterocomplexes that polymerize in filaments. Two types of interface between septins alternate along the filament: the G-interface (involving the GTP binding sites), and the NC-interface. This work focuses on the physiological G-interface of SEPT2, used in the SEPT6G-SEPT2G heterodimer assembly, to verify the impact of this interaction on the thermostability and amyloid formation. We found that the SEPT6G-SEPT2G moves to an irreversible state with the ability to bind thioflavin-T at high temperatures, suggesting its amyloid-like nature. Noteworthy, this takes place at a higher temperature than the one observed to the single septins, showing greater thermal/structural stability. Taken together, our results show that in the absence of the partners, the septin becomes unstable and susceptible to amyloid aggregation/formation even in physiological temperatures, and the G-interface appears to have a critical role in this process.