Heterotypic Coiled-Coil Formation is Essential for the Correct Assembly of the Septin Heterofilament

Abstract

Protein-protein interactions play a critical role in promoting the stability of protein quaternary structure and in the assembly of large macromolecular complexes. What drives the stabilization of such assemblies is a central question in biology. A limiting factor in fully understanding such systems is the transient nature of many complexes, making structural studies difficult. Septins comprise a conserved family of guanine nucleotide binding proteins that polymerize in the form of heterofilaments. In structural terms, they have a common organization: a central GTPase domain, an N-terminal domain, and a C-terminal domain; the latter is predicted to form a coiled coil. Currently, even for the best characterized human septin heterocomplex (SEPT2/SEPT6/SEPT7), the role of C-terminal domain is not fully established, and this is partly due to the absence of electron density for the C-terminal domains in the x-ray structure. Here we present results on the homo/heterotypical affinity for the C-terminal domains of human septins belonging to the SEPT6 and SEPT7 groups (SEPT6C/8C/10C/11C and SEPT7C, respectively) and provide clear evidence that this domain determines the preference for heterotypic interactions at one specific interface during the assembly of the heterofilament. This observation has wider implications where macromolecular assemblies are defined by coiled-coil protein interactions.

Septin structure and filament assembly

“…For example, human septins of the SEPT6 and SEPT7 groups present a long C-terminal domain with more than 100 residues, while this region presents only an intermediate length in members of the SEPT2 group and is smallest of all in the SEPT3-like septins. As mentioned above, with the exception of the SEPT3 group, the sequences of the C-terminal domains are predicted to form coiled coils, and biophysical studies have shown that the C-termini of septins of the SEPT6 group associate with high affinity with those of SEPT7, forming stable hetero-dimeric coiled coils (Marques et al 2012;Sala et al 2016). The pairing of the coiled coil regions of SEPT6-like septins with SEPT7 is consistent with the predicted number of heptad repeats, which is greater than that expected for SEPT2 and its group members.…”

“…Two types of generic interface, G and NC, alternate along the filament. These can be further subdivided into NC, NC het and NC hom for the NC-interfaces and G DD and G DT for the G-interfaces (further explanation is given in the text) the C-domain may play a dominant role in selective assembly (Marques et al 2012;Sala et al 2016).…”

A revised order of subunits in mammalian septin complexes

“…Each septin monomer participates in two interfaces which alternate along the filament: the NC interface, involving the N and C terminal helices of the G domains; and the G interface, including the region directly involved in guanine‐nucleotide‐binding (Sirajuddin et al, ; Valadares et al, ). The remaining domains (particularly the coiled coil region of the C‐domain) also contribute to the affinity and specificity of the NC interface (Barth, Schoeffler, & Alber, ; de Marques et al, ; Sala, Valadares, Macedo, Borges, & Garratt, ; Versele et al, ).…”

Repositioning septins within the core particle

“…Each septin monomer participates in two interfaces which alternate along the filament: the NC interface, involving the N and C terminal helices of the G domains; and the G interface, including the region directly involved in guanine-nucleotide-binding [3,13]. The remaining domains (particularly the coiled coil region of the C-domain) also contribute to the affinity and specificity of the NC interface [15,16]. Sirajuddin et al, 2007, using negative-stain electron microscopy and employing a version of SEPT2 fused to MBP, proposed that SEPT2 occupies the central position of the core complex and SEPT7 lies at its extremities, leading to the following arrangement for the hexamer: SEPT7-SEPT6-SEPT2-SEPT2-SEPT6-SEPT7 (which, for convenience, we will abbreviate to 7-6-2-2-6-7).…”