GTP-induced conformational changes in septins and implications for function

Abstract

Septins constitute a group of GTP-binding proteins involved in cytokinesis and other essential cellular functions. They form heterooligomeric complexes that polymerize into nonpolar filaments and are dynamic during different stages of the cell cycle. Posttranslational modifications and interacting partners are widely accepted regulators of septin filament function, but the contribution of nucleotide is undefined due to a lack of detailed structural information. Previous low-resolution structures showed that the G domain assembles into a linear polymer with 2 different interfaces involving the N and C termini and the G binding sites. Here we report the crystal structure of SEPT2 bound to GppNHp at 2.9 A resolution. GTP binding induces conformational changes in the switch regions at the G interfaces, which are transmitted to the N-terminal helix and also affect the NC interface. Biochemical studies and sequence alignment suggest that a threonine, which is conserved in certain subgroups of septins, is responsible for GTP hydrolysis. Although this threonine is not present in yeast CDC3 and CDC11, its mutation in CDC10 and CDC12 induces temperature sensitivity. Highly conserved contact residues identified in the G interface are shown to be necessary for Cdc3-10, but not Cdc11-12, heterodimer formation and cell growth in yeast. Based on our findings, we propose that GTP binding/hydrolysis and the nature of the nucleotide influence the stability of interfaces in heterooligomeric and polymeric septins and are required for proper septin filament assembly/disassembly. These data also offer a first rationale for subdividing human septins into different functional subgroups.

Fig. 1.

Structure of SEPT2·GppNHp. (A) Ribbon model of the overall structure of the SEPT2 dimerized across the nucleotide-binding site. New elements observed are labeled and the GppNHp and magnesium are colored in brown. (B) Superimposition of the SEPT2·GppNHp structure (gold) with the previous structures of SEPT2·GDP [PDB ID 2QA5 (cyan) and 2QNR (gray)].

Fig. 2.

Sequence alignment and interface analysis. (A) Part of the SEPT2·GppNHp G interface showing residues from the protomers required for tight interface formation, shown as ball and stick, with other structural elements as ribbons. (B) Sequence alignment of the complete set of septins from various organisms, highlighting the residues analyzed and discussed in the text. (C) Role of interface residues in vivo, using yeast complementation to introduce single copies of the corresponding WT and mutant septins described in Materials and Methods.

Fig. 3.

Biochemical analyses of active site residues in SEPT2. (A) Details of the nucleotide-binding site, showing GppNHp and surrounding residues, with the 2 protomers color-coded as in Fig. 1A. (B and C) Affinity for GppNHp (B) and GDP (C) measured by the increase in fluorescence obtained using 500 nM of the corresponding mant nucleotide and adding increasing amounts of WT and mutant SEPT2 as indicated. The data are fitted to a quadratic binding equation to produce the K_d values given in Table S2. (D) The GTPase reaction measured with 10 μM WT and mutant SEPT2 using 150 μM radioactive GTP mixture in a charcoal assay as described in Materials and Methods. The amount of Pi released was measured and plotted against time.

Fig. 4.

Yeast complementation assay. Role of active site residues in vivo, using yeast complementation to introduce single copies of the corresponding WT and mutant septins described in Materials and Methods.