Figure 3: D473^6.54f has different roles in the recognition of Anta XV and LY2940680.

(a–i) The receptor structures in SMO_Anta XV, SMO_SANT1 and SMO_LY2940680 (PDB i.d.: 4JKV, molecule A) complexes are shown in green, yellow and salmon, respectively. Ligand structures of Anta XV, SANT1 and LY2940680 are shown in green, orange and magenta carbons, respectively. (a) Superposition of SMO_LY2940680 and SMO_Anta XV structures near ECL3. The conformational change of F484^ECL3 induced by LY2940680 binding is shown by an arrow. See also Supplementary Fig. 5 for comparison with SMO_SANT1 structure. (b) Superposition of the SMO_SANT1, SMO_Anta XV and SMO_LY2940680 structures reveals a different conformation of ECL3 in LY2940680 bound structure (shown by arrow). (c) Localization of ECL3 (green) and polar residue cluster (R400^5.43f, D473^6.54f and E518^7.38f, red) in SMO. (d) Superposition of the binding poses of LY2940680 and Anta XV reveals distinct orientations (dashed lines parallel to the equatorial bonds of the six-membered chair conformation rings) of the six-membered rings connected to the phthalazine ring. The shift of the phthalazine of LY2940680 compared with that of Anta XV is shown by arrow. (e) Superposition of SMO_Anta XV and SMO_SANT1 structures. (f) Superposition of SMO_LY2940680 and SMO_SANT1 structures. (g) Superposition of SMO_LY2940680 and SMO_Anta XV structures. Ligand-induced shifts of helices are shown by arrows. (h) Superposition of the ligand binding pocket residues of SMO_SANT1, SMO_Anta XV and SMO_LY2940680 structures. Ligand interaction-induced conformation change at D473^6.54f is indicated by arrow. (i) Polar residue cluster in the ligand binding pocket of the SMO_Anta XV structure. Polar interactions are shown as dashed lines. (j) ΔpKi values (pKi wild-type(wt)–pKi mutants (mut)) of the D473^6.54fA and E518^7.38fA mutants for different SMO receptor ligands in ³H-cyclopamine competition experiments. Shown in the graph are mean±s.e.m. of 3–4 independent experiments, see also Supplementary Table 2.