PMC

A ceSAS-6 spiral can support ninefold symmetry. (A and B) Orthogonal views of a single ninefold symmetric ceSAS-6 spiral modeled with the full-length coiled coil. The individual ceSAS-6 dimers are numbered. The relative position of SAS-5 interaction sites (purple patches) is indicated. (C) Side view of two ninefold symmetric intertwined spirals in red and cyan with the ceSAS-6 dimers of the cyan spiral numbered. Note that the top view of two intertwined spirals would not differ from the view of a single spiral as shown in B. (D) Simplified representation of a C. elegans centriole showing our proposed model, where four turns of an intertwined ceSAS-6 double spiral account for the central tube ().

Functional analysis of ceSAS-6[I154W]. (A–C) Still images at the end of the second cell cycle from representative differential interference contrast recordings of embryos treated with sas-6(RNAi) and expressing (B) GFP-SAS-6RR or (C) GFP-SAS-6RR[I154W]; complete cytokinesis movies are provided (Movies S1, S2, and S3). Elapsed time after the onset of cytokinesis is shown in minutes and seconds. In every panel, anterior is to the left; arrowheads indicate cells. (Scale bar: 10 μm.) (D) Quantification of experiments illustrated in A–C. (E) Western blot analysis of GFP-SAS-6RR and GFP-SAS-6RR[I154W] worm lysates probed with SAS-6 antibodies to reveal both endogenous protein and the GFP fusion variant. Note that the substituted construct expresses at lower levels but functions as well as the WT, likely because of the enhanced N-N affinity.

Crystal structure of the coiled coil dimer of ceSAS-6. (A) Superposition of the three ceSAS-6 dimer structures: Shown in blue is ceN-CC[Δ102–130/I154G], which was solved with one dimer per asymmetric unit. In orange and magenta are the two dimer copies per the asymmetric unit of ceN-CC[Δ102–130/Δ151–156]. The first three heptad repeats of the coiled coil were resolved in all structures (residues 1–191), except for ceN-CC[Δ103–130/I154G], where additional segments of the coiled coil are stabilized through contacts with a symmetry-related subunit. The N-N dimerization interfaces of the two subunits face in opposite directions as indicated, thereby allowing formation of large oligomers. (B and C) Six ceN-CC dimers were associated such that their N-terminal domains interact as observed in the N-N dimer structure (). B presents a side-on view of the spiral. The two dimer interfaces (N-N and C-C) used for this model are indicated. C shows a top-down view that emphasizes the approximately fivefold symmetry of the model. The individual ceN-CC dimers are numbered. The outer diameter of the spiral was calculated using the ceN-CC[Δ102–130/I154G] model, where almost the entire coiled coil length is visible. (D) Superposition of the N-terminal domains of ceN-CC (blue) and the equivalent structure of crSAS-6 (green), showing the displacement of the coiled coil helix-α3.

AUC analysis and visualization of ceSAS-6 oligomers. (A) AUC velocity analysis of ceN-CC[S123E/I154E] (blue) at 50 μM concentration shows a narrow sedimentation distribution corresponding to the molecular mass of a dimer. ceN-CC[S123E], which can oligomerize through its coiled coil and the low-affinity N-N interface, shows a distribution of small oligomers at 50 μM concentration (red). In contrast, ceN-CC[S123E/I154W], which exhibits high N-N dimerization affinity, forms larger oligomers at 25 μM concentration (green). (B and C) Electron micrographs of negatively stained ceN-CC[S123E/I154W] (B) after or (C) before sample centrifugation. The stabilized ceN-CC forms spirals at higher concentrations, which in turn, give rise to micrometer-long filaments that show evidence of spiral intertwining. The white arrowhead in C indicates the point where the two strands separate. (D) Close-up view of a representative ceN-CC[S123E/I154W] micrograph from B compared with our crystallographic model of ceN-CC spirals. (Scale bar: 10 nm.) (E and F) Histograms of spiral diameter and pitch, respectively. The values are consistent with the dimensions of our crystallographic model of ceN-CC spirals (black solid lines; 17-nm diameter, 33-nm pitch) but fit better to the dimensions of a 4.5-fold symmetric spiral (red lines; 15-nm diameter, 31-nm pitch). Experimental mean values and SDs are shown by dotted lines. (G) Histogram of filament diameter from C and similar migrographs (Fig. S7B).

I154W increases the ceSAS-6 N-N dimerization affinity. (A) Fluorescence polarization experiments of WT- (purple squares), I154E- (orange crosses), or I154W-substituted (green triangles) ceN constructs to determine the N-N dimerization affinity. The I154E substitution disrupts N-N dimerization (), and the K_D of WT ceN matches the value estimated from isothermal titration calorimetry experiments (110 ± 30 μM) (). The I154W substitution leads to a 20-fold increase in affinity compared with the WT. Error bars derive from five independent measurements. (B) Superposition of the four N-N dimer models from the two crystal structures of ceN[S123E/I154W]. The average rmsd for all C_α atoms is 0.51 Å. The N-N dimer interface is essentially unchanged compared with the interface of ceN[S123E], with the β6-β7 loop from one subunit contacting a hydrophobic patch between α1 and α2 of the second molecule. (C) Superposition of one copy of ceN[S123E/I154W] (gold) onto ceN[S123E] (red). The main difference in the two structures is the slight displacement of α1 in ceN[S123E/I154W]. Note that the relative position and orientation of the head domains are unchanged. (D and E) Close-up view of the area around residue 154 reveals the reason for α1 displacement. Whereas I154 in ceN[S123E] fits tightly into a cavity formed between primarily hydrophobic residues (E, green), the aromatic ring of W154 sits on top of this hydrophobic patch, leading to a partial collapse of the cavity (D).