The structure and properties of septin 3: a possible missing link in septin filament formation.

The human genome codes for 13 members of a family of filament-forming GTP-binding proteins known as septins. These have been divided into four different subgroups on the basis of sequence similarity. The differences between the subgroups are believed to control their correct assembly into heterofilaments which have specific roles in membrane remodelling events. Many different combinations of the 13 proteins are theoretically possible and it is therefore important to understand the structural basis of specific filament assembly. However, three-dimensional structures are currently available for only three of the four subgroups. In the present study we describe the crystal structure of a construct of human SEPT3 which belongs to the outstanding subgroup. This construct (SEPT3-GC), which includes the GTP-binding and C-terminal domains, purifies as a nucleotide-free monomer, allowing for its characterization in terms of GTP-binding and hydrolysis. In the crystal structure, SEPT3-GC forms foreshortened filaments which employ the same NC and G interfaces observed in the heterotrimeric complex of human septins 2, 6 and 7, reinforcing the notion of 'promiscuous' interactions described previously. In the present study we describe these two interfaces and relate the structure to its tendency to form monomers and its efficiency in the hydrolysis of GTP. The relevance of these results is emphasized by the fact that septins from the SEPT3 subgroup may be important determinants of polymerization by occupying the terminal position in octameric units which themselves form the building blocks of at least some heterofilaments.

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.