X-ray structure of the metastable SEPT14-SEPT7 coiled coil reveals a hendecad region crucial for heterodimerization

Abstract

Septins are membrane-associated, GTP-binding proteins that are present in most eukaryotes. They polymerize to play important roles as scaffolds and/or diffusion barriers as part of the cytoskeleton. α-Helical coiled-coil domains are believed to contribute to septin assembly, and those observed in both human SEPT6 and SEPT8 form antiparallel homodimers. These are not compatible with their parallel heterodimeric organization expected from the current model for protofilament assembly, but they could explain the interfilament cross-bridges observed by microscopy. Here, the first structure of a heterodimeric septin coiled coil is presented, that between SEPT14 and SEPT7; the former is a SEPT6/SEPT8 homolog. This new structure is parallel, with two long helices that are axially shifted by a full helical turn with reference to their sequence alignment. The structure also has unusual knobs-into-holes packing of side chains. Both standard seven-residue (heptad) and the less common 11-residue (hendecad) repeats are present, creating two distinct regions with opposite supercoiling, which gives rise to an overall straight coiled coil. Part of the hendecad region is required for heterodimerization and therefore may be crucial for selective septin recognition. These unconventional sequences and structural features produce a metastable heterocomplex that nonetheless has enough specificity to promote correct protofilament assembly. For instance, the lack of supercoiling may facilitate unzipping and transitioning to the antiparallel homodimeric state.

Keywords: coiled coils; hendecad repeats; heterodimerization; metastability; septins.

Figure 1

Characterization of the oligomeric state and heterodimerization of the CC constructs. (a) Septin protofilament organization according to the cryo-EM structure of the SEPT2G–SEPT6–SEPT7 complex (PDB entry 7m6j). Protofilaments polymerize end to end (adjacent protofilaments are shown semi-transparent) and their NC interfaces are depicted. The CTDs (arrows) are not modelled/present within the structure but are expected to project as indicated from the stubs at the C-termini of the corresponding G domains. (b) ‘Railroad-track’ model in which the CTDs of SEPT6 (and maybe SEPT7) form antiparallel homodimeric coiled coils connecting different filaments. (c) Human septins SEPT14 and SEPT7 have a central Ras-like G domain and a CC sequence within the CTD (boxes). The constructs used and their truncation positions are shown. (d) Septin CC constructs are predominantly dimeric by SEC-MALS. Thin lines represent the normalized UV absorption and differential refractive index, whereas thick lines represent the molar mass. The peptide concentration at injection was 0.8 mM (0.4 mM each in the case of the equimolar mixture). SEPT14CC* elutes as a single peak corresponding to a mixture of dimers and monomers (roughly 2:1, considering the calculated mass) as also demonstrated by the tail associated with the elution profile. To a lesser extent, SEPT7CCext also presents peak tailing and dimeric mass underestimation. The SEPT14CC*–SEPT7CCext mixture behaves essentially as dimers. (e) CD thermal denaturations of the complete CTDs, SEPT14C, SEPT7C (10 µM) and their equimolar mixture (5 µM each, 10 µM total). (f) SEPT14CC*–SEPT7CCext (10 µM total). The presence of residual homodimers and/or monomers cannot be ruled out, potentially explaining the minor transition at around 20°C. (g) SEPT14CC*–SEPT7CC* (10 µM total). There is a considerable gain in the melting temperature in the longer constructs, but not in the case of the SEPT14CC*–SEPT7CC* mixture, as SEPT7CC* is overtruncated (12 residues shorter than SEPT14CC*). (h) Comparison of the melting temperatures (T _m, black bars) and T _m gain (grey bars) found by CD for the different SEPT6-group CTDs when mixed with the SEPT7 CTD (Sala et al., 2016 ▸) and the different constructs involving SEPT14 and SEPT7 (reported here). The complex formed by the SEPT14 and SEPT7 CTDs has the highest thermal stability among them. ‘T _m gain’ refers to the difference between the experimental T _m for the mixture and the predicted T _m for a mixture of the two individual samples (see Section 2).

Figure 2

X-ray structure of the SEPT14CC*–SEPT7CCext heterodimeric CC. (a) Crystal packing viewed roughly along the CC axes and (b) perpendicular to the axes. Chains A, B (purple, SEPT14CC*), C and D (orange, SEPT7CCext) from the asymmetric unit are indicated (labelled in green). The BD dimer is formed using a D chain (labelled in white) which is shifted by a lattice translation with respect to the D chain of the asymmetric unit. The crystal packing is composed of alternate layers of CC heterodimers which are approximately aligned. (c) CC view orthogonal to the plane bisecting the helices and (d) rotated 90° in relation to (c). Blue, SEPT14CC*; yellow, SEPT7CCext. Only dimer AC and the side chains of positions d and h are shown. (e) Periodicities calculated by SamCC-Turbo, smoothed using a seven-residue window. (f) Cumulative supercoiling angle (in degrees); positive and negative first derivatives are related to right- and left-handed structures, respectively (indicated in grey boxes). The values displayed are an average between dimers AC and BD.

Figure 3

Register, core layers and interactions present in the SEPT14CC*–SEPT7CCext structure. (a) Register highlighting the core positions (grey) and genuine hydrophobic residues within the core (bold). Numbers in between sequences identify core layers. Thick and thin horizontal lines indicate hendecads and heptads, respectively. Both register and homologous positions (see the pairwise sequence alignment in Supplementary Fig. S2) have an axial shift of four residues in the same direction, indicated by the diagonal teal lines for selected reference positions. KTK interactions are indicated in core layers 5 and 10 (magenta arrows). (b) Scheme of core layers in the hendecads: core layers 1, 4, 7 and 10 (h-ak cores), 2, 5, 8 and 11 (d-ak cores) and 3, 6 and 9 (d-h cores). For core layers 5 and 10, the KTK interactions are highlighted (magenta) and a fourth site enters the core (light grey). (c) Scheme of core layers (12–18) in the heptads. The C^α–C^β vector is represented for each core position (black lines) as well as that found in comparative structures [φ₀, dashed red lines; from the tetrabrachion structure (PDB entry 1fe6) for (b) and from heptad-based four-helix bundles (Szczepaniak et al., 2018 ▸) for (c)]. Helical wheel representation of the (d) hendecads and (e) heptads. Solid and dashed lines represent π and polar intermolecular interactions, respectively.

Figure 4

Atomistic modelling of the SEC–SAXS data. (a)–(e) Calculated scattering curves of hetero-parallel (hetP, blue) and hetero-antiparallel (hetAP, red) models were computed by MultiFoXS using flexible linkers. The residuals of the fitting to the experimental data (grey dots) are shown at the bottom. For the hetero-parallel solution of 10c–7c (c) (hetP*, purple), an ensemble of hetero-parallel (94.3%, model calculated previously with MultiFoXS) and MBP-fused homo-antiparallel dimers (5.7%, model based on the antiparallel homodimeric X-ray structure of SEPT8C, PDB entry 6wsm) was considered and calculated with FoXS. The χ² of the fit to the 10c–7c data improves slightly with the inclusion of homo-antiparallel dimers (from 2.71 to 2.42).

Figure 5

Register and length differences between homodimeric antiparallel (SEPT8; PDB entry 6wsm) and heterodimeric parallel (SEPT14–SEPT7; PDB entry 8sjj) septin CCs. (a) Comparison of core residues and positions. Core sites with different registers are highlighted in rectangles. Bulky hydrophobic residues (Met344 and Phe348 in SEPT8) are exposed in the homodimer (red). (b) Layers showing register differences in both structures. Nonmodelled residues due to the absence of electron density are indicated by asterisks (*). (c) Scheme representing the longer length of heterodimeric CCs in relation to homodimeric CCs. ‘G’ indicates the position of the G domain. (d) Structural comparison between the SEPT14 helix in the SEPT14–SEPT7 heterodimer and the SEPT8 helix in the SEPT8 homodimer. The region in which the homodimer and heterodimer have identical registers was used for structural alignment (towards the C-terminus). A reorientation of around 30° in the helical axis is necessary to go from one state to the other.

Figure 6

CC periodicities are important for septin recognition. SEPT2–SEPT2 and SEPT6–SEPT7 can assemble due to matching of periodicities (only heptads and hendecads/heptads at the N- and C-termini, respectively). Promiscuous, CC-containing NC interfaces, such as SEPT7–SEPT2 and SEPT6–SEPT2 (with the latter being particularly important, since SEPT2-group members are closely related to SEPT7), are disfavoured because of the different CC periodicities (hendecads versus heptads).