Fig. 4

Fig. 4 The conserved sperm trimer binds to divergent egg proteins in vertebrates (A) Predicted structural model of a tetrameric complex of the egg protein Bouncer (light blue) bound to the zebrafish trimeric complex on sperm. Zoom-in of Bouncer binding the cleft between Izumo1 and Spaca6 is shown in two orientations. The mutated residues are shown as sticks as modeled by AlphaFold-Multimer. Right: PAE plot of the top-scoring tetramer model containing the full-length mature proteins. Index: amino acid residues. (B) Cell-based Bouncer binding assay. HeLa cells were transfected with one plasmid encoding the sperm factors and cytoplasmic EGFP as a transfection marker. Cells were incubated with StrepII-tagged BIR2-Bouncer before fixation and immunofluorescence staining against StrepII under non-permeabilizing conditions. Representative images with StrepII (white) and EGFP (magenta). Quantification of BIR2-Bouncer/BIR2 signal intensity after Ilastik segmentation46,47 normalized to the mock transfection. (C) Bouncer binding assay after mutating predicted interface residues (A) of Spaca6 and Izumo1. Only Spaca6 and Izumo1 were encoded in the transfected plasmid. (D) Quantification of the relative surface expression of Spaca6, Izumo1, and Tmem81 constructs in HeLa cells. Immunofluorescence signal per segmented pixel was normalized to the transfection of wild-type Spaca6 and Izumo1. Representative images are shown in Figure S5G. (E) Predicted structural models of the tetrameric complexes in zebrafish and mice. In mammals, JUNO (purple) binds to IZUMO1,22,23,24 which is predicted as compatible with trimer formation. Bar graphs represent mean ± SD of at least eight images per condition across four independent experiments. Significant adj. p values by Kruskal-Wallis test with Dunn’s multiple comparisons test (B and C). All other comparisons are non-significant (p > 0.9999). See also Figure S5.