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. 2025 Mar 19;11(12):eadr5744. doi: 10.1126/sciadv.adr5744

Fig. 9. A hydrophobic interaction interface may allow insect SP2Ds to form dimers.

Fig. 9.

(A) (a) SEC-MALS analysis of Sumo-tagged SP2D WT, P817D, or F822D performed at a range of protein concentrations (as indicated). Note how the WT, but not mutant, protein(s) has/have a tendency to form higher MW species at higher concentrations. (b) SEC-MALS analysis of Sumo-tagged Spd-2-CT (697 to 1146 amino acids) WT, P817D, or F822D that has been phosphorylated in vitro with recombinant human PLK1 kinase. The orange horizontal dotted lines indicate the theoretical mass of a monomer or dimer. Note how the WT, but not mutant, protein(s) can form large species when phosphorylated by PLK1. (B) Ribbon diagram showing the packing interactions in the top-ranked AF3 prediction of a DmSP2D dimer (purple) overlaid with the AdSP2D dimer (cyan) observed in crystallo; a similar hydrophobic interface observed in both structures is highlighted in orange and is shown in detail in the inset. (C) CD analysis of SP2D WT, P817D, or F822D shows that each single-point mutation does not strongly affect the protein fold. (D) (a) Confocal images illustrate, and the bar chart below quantifies, the centrosomal fluorescence levels (means ± SD) of WT and Spd-2-P817D or F822D mutant GFP fusions in WT embryos expressing the centriole marker Asl-mCherry and injected with mRNA encoding each protein. Statistical significance was assessed using an unpaired t test in GraphPad Prism (****P < 0.0001). (b) Graphs show the raw (top) or normalized (bottom) centrosomal fluorescence distribution profiles of the WT and Spd-2-P817D or F822D GFP fusions. Scale bar, 2 μm.