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.

The Structural Biology of Septins and Their Filaments: An Update.

In order to fully understand any complex biochemical system from a mechanistic point of view, it is necessary to have access to the three-dimensional structures of the molecular components involved. Septins and their oligomers, filaments and higher-order complexes are no exception. Indeed, the spontaneous recruitment of different septin monomers to specific positions along a filament represents a fascinating example of subtle molecular recognition. Over the last few years, the amount of structural information available about these important cytoskeletal proteins has increased dramatically. This has allowed for a more detailed description of their individual domains and the different interfaces formed between them, which are the basis for stabilizing higher-order structures such as hexamers, octamers and fully formed filaments. The flexibility of these structures and the plasticity of the individual interfaces have also begun to be understood. Furthermore, recently, light has been shed on how filaments may bundle into higher-order structures by the formation of antiparallel coiled coils involving the C-terminal domains. Nevertheless, even with these advances, there is still some way to go before we fully understand how the structure and dynamics of septin assemblies are related to their physiological roles, including their interactions with biological membranes and other cytoskeletal components. In this review, we aim to bring together the various strands of structural evidence currently available into a more coherent picture. Although it would be an exaggeration to say that this is complete, recent progress seems to suggest that headway is being made in that direction.

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.

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.