(A) Ribbon diagram of zSmo CRD in rainbow coloring from blue (N-terminus) to red (C-terminus) with the secondary structure elements numbered. The four disulfide bridges (black sticks) conserved in all Fz-like CRDs are depicted with Roman numerals, and the non-conserved disulfide bridge is marked with an asterisk (*). N- and C-termini are labeled. (B) Structural phylogenetic analysis of the CRDs. Structural superposition of CRDs from zSmo, Frizzled 8 (Fz8, PDB ID 4F0A, Janda et al., 2012), secreted Frizzled-related protein 3 (sFRP3, PDB ID 1IJX, Dann et al., 2001), muscle-specific kinase (MuSK, PDB ID 3HKL, Stiegler et al., 2009), Niemann-Pick C1 protein (NPC1, PDB ID 3GKI, Kwon et al., 2009), riboflavin-binding protein (RFBP, Monaco, 1997), and folate receptor α (FRα, PDB ID 4LRH, Chen et al., 2013) were superimposed using SHP (Stuart et al., 1979; Riffel et al., 2002). CRDs that form ligand-binding pockets (red background) or grooves (blue background) form two distinct evolutionary branches. In addition, CRDs show distant structural similarity to the extracellular domains of glypicans (Pei and Grishin, 2012). However, analysis of the crystal structures of glypicans Dally-like protein and glypican 1 revealed no apparent grooves or pockets that could accommodate small molecules (Kim et al., 2011; Svensson et al., 2012) and thus were not included in our structural analyses. (C–H) Ribbon diagrams of superimposed Fz-like CRD domains from the structural phylogenetic analysis in (B). (C) Fz8-palmitoleyl complex, (D) sFRP3, (E) MuSK, (F) NPC1-cholesterol complex, (G) RFBP-riboflavin complex, (H) FRα-folate complex. Color coding and labeling follows (A). Ligands are shown as spheres in atomic coloring (carbon: slate; oxygen: red; nitrogen: blue). In (F–H) the conserved disulfide bridges are highlighted with a circle. NPC1 (F) does not contain disufide bridge IV.