Structure of the human smoothened receptor bound to an antitumour agent

Abstract

The smoothened (SMO) receptor, a key signal transducer in the hedgehog signalling pathway, is responsible for the maintenance of normal embryonic development and is implicated in carcinogenesis. It is classified as a class frizzled (class F) G-protein-coupled receptor (GPCR), although the canonical hedgehog signalling pathway involves the GLI transcription factors and the sequence similarity with class A GPCRs is less than 10%. Here we report the crystal structure of the transmembrane domain of the human SMO receptor bound to the small-molecule antagonist LY2940680 at 2.5 Å resolution. Although the SMO receptor shares the seven-transmembrane helical fold, most of the conserved motifs for class A GPCRs are absent, and the structure reveals an unusually complex arrangement of long extracellular loops stabilized by four disulphide bonds. The ligand binds at the extracellular end of the seven-transmembrane-helix bundle and forms extensive contacts with the loops.

Figure 1. Overall structure of the human SMO receptor 7TM domain in complex with LY2940680

a, Overall structure of the SMO receptor bound to ligand LY2940680. The SMO receptor is crystallized as a dimer in an asymmetric unit with molecule A colored light blue and molecule B colored yellow. LY2940680 is shown in wheat carbon. The four disulfide bonds are shown in orange sticks. The lipids are shown in sticks with green carbons. Membrane boundaries are shown in dotted lines. b and c, Extracellular (EC) and intracellular (IC) view of the SMO receptor dimer.

Figure 2. Comparison of the 7TM bundle of the SMO receptor with class A GPCRs

a, Side view of superimposed structures of the SMO receptor (magenta) and class A GPCRs (light green). b, Extracellular view of the superimposed structures. c, Superposition of ICL1 and helix VIII. The residues involved in the packing interface of helix VIII and helix I in the SMO receptor are shown in sticks with magenta carbons. d–f, Superposition of Helix V–VII. The P^5.50, P^6.50 and P^7.50 residues conserved in class A GPCRs are shown in sticks. In d, P407^5.46 in the SMO receptor is shown in sticks. The α carbons of glycines in the SMO receptor are shown as yellow spheres. Structures of class A GPCRs used (PDB accessions): 1U19, 2RH1, 2YCW, 3RZE, 3PBL, 3UON, 4DAJ, 3EML, 3V2W, 3ODU, 4DJH, 4EA3, 4DKL, 4EJ4, 3VW7, 4GRV.

Figure 3. Ligand binding pocket for LY2940680

a, The ligand binding pocket surface is colored according to binding properties (green: hydrophobic; blue: hydrogen-bond donor; red: hydrogen-bond acceptor). LY2940680 is shown by sticks with magenta carbons. The membrane boundary is shown as a red line. b, |2F_O|-|F_C| map (blue mesh) is shown for LY2940680 (contoured at 1.0σ, 0.10 e/ Å). Binding pocket residues (4.0 Å cut-off distance from LY2940680) are shown in sticks with colors indicating the location (white: TM helices; cyan: ECD linker domain; yellow: ECL1; pink: ECL2; light blue: ECL3). The hydrogen bond interactions between R400^5.39, N219 and LY2940680 are shown as black dashed lines. c, Diagram of ligand interactions in the binding pocket. The color scheme of the boxes for the residues is the same as b.

Figure 4. The structure of the ECD linker domain and ECLs

a and b, Different views of the cartoon presentation of the structure of the ECD linker domain and ECLs. ECD linker domain is colored cyan. ECL1 is colored yellow. ECL2 is colored pink. ECL3 is colored light blue. Disulfide bonds are shown in orange sticks. c, Side chain interactions that stabilize the structure of ECL1. The hydrogen bond network among residues N202, S205, D298 and T300 and the ionic interaction between R302 and E305 are shown in dashed lines. d, Side chain interactions that stabilize the structure of ELC3. The ionic interactions between E208 and R485, E479 and R482, and hydrogen bond interaction between Q491 and V195, R512 and L221 are shown in dashed lines.

Figure 5. Comparison of ECL2 of the SMO receptor with class A GPCRs

a–f, Cartoon presentation of the extracellular part of the structures for (a) the SMO receptor (white), (b) rhodopsin (PDB accession: 1U19) (dark grey), (c) CXCR4 chemokine receptor (3ODU) (yellow), (d) κ-opioid receptor (4DJH) (light green, representing the opioid receptor family), (e) neurotensin receptor NTSR1 (4GRV) (cyan), (f) protease activated receptor 1 (PAR1) (3VW7) (light blue). The ECL2 of each structure is shown in magenta. All ligands are shown in wheat. The approximate position of the extracellular membrane boundary is shown in orange dotted lines. The disulfide bonds connecting ECL2 and helix III are shown in orange sticks.

Figure 6. Structural insight for the Frizzled (FZD) receptors based on their sequence homology with the SMO receptor

a and b, Fully conserved residues in class F members are highlighted on the SMO structure. The side chains of all these conserved residues are shown in sticks with green carbons. c, Alignment of the KTxxxW motif in the FZD receptors to the helix VIII of the SMO receptor. The similar α-helical structure could exist for the KTxxxW motif of the FZD receptors which is stabilized by the hydrogen bond interactions between the hydroxyl group of the conserved residue T541 and the main chain carbonyl group of V536, the indole nitrogen of the conserved residue W545 and the hydroxyl group of the conserved residue T251^1.56, shown in dashed lines. The non-conserved residues are colored yellow.