Fig. 4: Structure of hDisp1 bound to native, dually lipidated Shh.

a, b Corresponding views of the cryo-EM density map (a) and atomic model (b) of the hDisp1^NNN–Shh complex. c, d The interaction interfaces between hDisp1 and Shh. Residues near the interfaces are shown as spheres. e WT and mutant Shh-NL constructs were transiently expressed in HEK293T cells together with WT Scube2 or the inactive Scube2 ty97 mutant (negative control). Cells were washed extensively with serum-free media and Shh-NL release was measured after 6 h, for three independent biological replicates. Shh-NL release was normalized to Shh-NL measured in cell lysates, to account for differences in expression level, and specific Scube2-dependent release was determined by subtracting background Shh-NL release for Scube2 ty97. Bars represent mean background-subtracted Shh-NL release, and are plotted as percentage of release for WT Shh-NL. Error bars represent standard error of the mean. Ordinary one-way ANOVA, with Dunnett’s multiple comparisons test, was used to compare WT Shh-NL and each mutant: *, p < 0.05; ****, p < 0.0001; ns, not significant. Mutations are colored by their proximity to the Disp1–Shh interfaces, with yellow bars representing Shh residues close to ECD1 (N50A, V51E, L56A/K), green bars representing Shh residues close to ECD2 (E71A, K74A, Y80A, and N81A), and gray bars representing Shh residues at unrelated sites (P26A, R61A, Q100A, and Q100H). Mutations close to ECD1 and ECD2 show a significant defect (>2-fold reduction) in Shh release, while more distantly located mutations show modest or no reduction in Shh release. f Structural comparison between hDisp1–Shh and dDisp–Hh complexes shows that hDisp1 and dDisp have distinct ligand binding modes, consistent with divergent mechanisms of ligand release in vertebrates and invertebrates. Source data for (e) are provided as a Source Data file.