Conservation and divergence of the G‐interfaces of Drosophila melanogaster septins

Abstract

Septins possess a conserved guanine nucleotide‐binding (G) domain that participates in the stabilization of organized hetero‐oligomeric complexes which assemble into filaments, rings and network‐like structures. The fruit fly, Drosophila melanogaster, has five such septin genes encoding Sep1, Sep2, Sep4, Sep5 and Pnut. Here, we report the crystal structure of the heterodimer formed between the G‐domains of Sep1 and Sep2, the first from an insect to be described to date. A G‐interface stabilizes the dimer (in agreement with the expected arrangement for the Drosophila hexameric particle) and this bears significant resemblance to its human counterparts, even down to the level of individual amino acid interactions. On the other hand, a model for the G‐interface formed between the two copies of Pnut which occupy the centre of the hexamer, shows important structural differences, including the loss of a highly favourable bifurcated salt‐bridge network. Whereas wild‐type Pnut purifies as a monomer, the reintroduction of the salt‐bridge network results in stabilizing the dimeric interface in solution as shown by size exclusion chromatography and thermal stability measurements. Adaptive steered molecular dynamics reveals an unzipping mechanism for dimer dissociation which initiates at a point of electrostatic repulsion within the switch II region. Overall, the data contribute to a better understanding of the molecular interactions involved in septin assembly/disassembly.

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“…With a classic septin fold based on a three layered αβα sandwich (Fig. 2) (Cavini et al, 2021), we observed that Cdc3G, Cdc10G and Cdc10 ∆1-10 are all very similar to other septin structures already described in other species (de Freitas Fernandes et al, 2022;Mendonça et al, 2021;Rosa et al, 2020), reinforcing that many of the structural characteristics are highly conserved.…”

“…S2A) confirms the co-purification of Cdc3G-Cdc10G, demonstrating the positive influence of the correct G-interface partner (Cdc3) on the stability and solubility of Cdc10 (as has also been observed for their full-length versions (Baur et al, 2019)). In addition, the absence of the N-and C-terminal domains suggests that complex formation via the G-interface is viable as reported for septins from other organisms (de Freitas Fernandes et al, 2022;Rosa et al, 2020). The SEC profile shows two oligomeric states in solution (Fig.…”

“…2A). Non-physiological NC-interfaces have been observed in crystal structures of heterodimers from other species, demonstrating that the G-domain alone is insufficient to guarantee the formation of the correct NC pairs (de Freitas Fernandes et al, 2022;Rosa et al, 2020). On the other hand, the Cdc3G-Cdc10 ∆1-10 structure has two dimers in the asymmetric unit (both forming G-interfaces).…”

“…Since the Cdc3 construct used here is limited to the Gdomain and the heterodimeric interface is intact, our structure is unable to shed light on the details of the predicted interaction in cis between the N-terminal domain of Cdc3 and its own Ginterface which is believed to control important aspects of oligomer assembly (Weems and McMurray, 2017). For descriptive purposes we will use the structure with the higher resolution (Cdc3G-Cdc10G), and to facilitate the interpretation, we will divide the interface into three sections: upper, middle, and lower regions in accordance with de Freitas Fernandes et al,2022 (Fig. 5A).…”

“…On the other hand, the NC-interface is characterized by interactions which involve the N and C termini of each subunit including the PB1 region (between the N-and G-domains) and the coiled coils formed by the C-terminal extensions (Cavini et al, 2021;Mendonça et al, 2021;Sirajuddin et al, 2007). Recently, several papers have described in detail how specific amino acids in human and fly septins determine the necessary specificity to guarantee the interaction of each subunit with its correct partner at each of these two interfaces (de Freitas Fernandes et al, 2022;Mendonça et al, 2021;Rosa et al, 2020;Sala et al, 2016). However, similar information concerning the specific interactions which govern the correct assembly of the yeast octamer is currently lacking even though the order in which the four canonical septins (Cdc3, Cdc10, Cdc11 and Cdc12) associate with one another to generate the full particle appears to have been established (Weems and McMurray, 2017).…”