Figures and data
Overall structure of the GPR30-miniGsqiβ1γ2-scFv16 complex.
A Unsharpened cryo-EM density map of the GPR30-miniGsqiβ1γ2-scFv16-Nb35 complex, with the components individually colored. B The refined structure of the complex is shown as a ribbon representation.
Cryo-EM data collection, refinement and validation statistics
Receptor structure.
A–C Overall structure of the receptor, viewed from the membrane plane (A), intracellular side (B), and extracellular side (C).
Architecture of the extracellular pocket.
A Molecular surface of the extracellular side. B Cross section of the pocket. C–E Residues facing pocket B (C), pocket C (D), and pocket A (E). In panel (E), only residues with reduced bicarbonate responses are highlighted. F Calcium assay using Stable HEK293 cell lines expressing the N-terminal HA-tagged wild-type (WT) and mutant GPR30. The mutants D1112.56A, N2766.52A, Q296ECL3A; E121ECL1A, R122ECL1A, S1343.29A, Q2155.39A, E2185.42A; D210ECL2A, Q1383.33A; and D125ECL1A, C207ECL2A, P711.44A are highlighted in red, blue, green, and purple, respectively. The cells were stimulated by the indicated concentrations of NaHCO3 and 50 µM ATP at the timepoint of t = 20 sec. The Y axis indicates the difference between the maximum and minimum fluorescent values during 15 to 60 sec, normalized by those with ATP stimulation. Nonlinear regression (four-parameter) was used for curve-fitting. G–H X-Y plot of parameters calculated by FACS analysis and Ca assay. D1112.56A; D210ECL2A, Q1383.33A; D125ECL1A, C207ECL2A, P711.44A; and mock are colored in red, green, purple, and grey, respectively(G). cell surface (x-axis) and total (y-axis) expression of GPR30 (H). X-Y plot of cell surface (x-axis) and normalized maximum responses (y-axis) (I). X-Y plot of cell surface (x-axis) and EC50 (y-axis). EC50 was calculated using the data shown in panel (F).
G-protein coupling.
A Hydrogen-bonding interactions between the C-terminal α5-helix residues and the receptor. B Electrostatic and hydrogen-binding interactions between ICL3 and the α5-helix. C–H Interface between ICL2 and Gq, with residues involved in hydrophobic interactions represented by CPK models. G-protein-bound GPCRs used in the comparison are as follows: H1R-Gq (PDB 7DFL, gray), B2R-Gq (PDB 7F6I, yellow-green), MRGPRX2-Gq (PDB 7S8L, green), 5-HT2A-Gq (PDB 6WHA, gray), and GPR103-Gq (PDB 8ZH8, red). I Comparison of the angles and positions of α5h and αN relative to the receptor. J Superimposition of the Gα subunits.
Structural comparison with related GPCRs.
A Structural comparison of GPR30 and AT2R (PDB 5UNF). B Interactions around P711.44 in TM1 of GPR30. C–F Conformational changes of TM1 upon agonist binding in CB1 (C), A2AR (D), and ETB (E). The agonist-bound states are colored with the respective colors, while the inactive states are colored gray. The PDB codes used in this figure are CB1-active (PDB 5XRA), CB1-inactive (PDB 5TGZ), A2AR-active (PDB 6GDG), A2AR-inactive (PDB 3EML), ETB-active (PDB 8IY5), and ETB-inactive (PDB 5X93).
Structural comparison with GPR30 bound to Lys05.
A, B Superimposition of bicarbonate-GPR30, Lys05-binding GPR30 (PDB 8XOF) (A), and apo-GPR30 (PDB 8XOG) (B) structures, viewed from the extracellular side. C Comparison of the angles and positions of α5h and αN relative to the receptor. D Superimposition of the Gα subunits.
