Structure of a D2 dopamine receptor-G-protein complex in a lipid membrane

a, Energy landscape of three receptor variants with various energy differences in the resting (ligand-free) state ΔG _resting, in the allosteric coupling controlling the inverse agonist efficacy ΔG _{inverse agonist} and the stabilization of the receptor inactive state upon ligand stimulus. b, Active state occupancy as a function of ΔG _resting and ΔG _{inverse agonist} (the energy of inverse agonist binding), as determined by the Boltzmann law. c, Sensitivity of the receptors to inverse agonist binding. d- f, Rational design of DRD2 stabilized in the active state. DRD2 (D2) wild-type active state homology model ( d). The 120-residue-long ICL3 is represented as a dotted blue line. DRD2 designed with residue microswitches shown in spheres selectively stabilizing the active state structure ( e). DRD2 further stabilized in the active state through a de novo designed seven-residue stable ICL3 ( f).

a, Competition binding of dopamine for [ ³H]spiperone on membranes containing hDRD2 ^WT (blue) or hDRD2 modified with nT4L and de novo-designed ICL3 (nT4L-hDRD2(ΔICL3); magenta). The log-transformed inhibition constant ( K_i) ± s.e.m. = −4.6 ± 0.04 for hDRD2 ^WT. log( K_i ± s.e.m.) = −4.6 ± 0.04 for nT4L-hDRD2(ΔICL3). n = 4 biologically independent experiments on Sf9 cell membranes. b, Agonist-stimulated [ ³⁵S]GTPγS binding of nT4L-hDRD2(ΔICL3) (magenta) compared to hDRD2 ^WT (blue). Dose-responses using dopamine (closed magenta circles) or bromocriptine (BCT; open magenta circles) show log(EC ₅₀ ± s.e.m.) = −7.5 ± 0.02 and −7.8 ± 0.06, respectively, for nT4L-hDRD2(ΔICL3) compared to log(EC ₅₀ ± s.e.m.) = −7.0 ± 0.02 for dopamine on hDRD2 ^WT (open blue circles). n = 3 biologically independent experiments on HEK293T cell membranes. c, Saturation binding of [ ³H]spiperone to membranes containing hDRD2 ^WT with or without scFv16. Dissociation constant ( K_d) ± s.e.m. = 0.15 ± 0.03 nM for control. K_d ± s.e.m. = 0.14 ± 0.02 nM with scFv16. n = 4 biologically independent experiments on Sf9 cell membranes. d, Competition binding of dopamine for [ ³H]spiperone on membranes containing DRD2 ^WT with or without scFv16. log( K_high ± s.e.m.) = −6.4 ± 0.3 and log( K_low ± s.e.m.) = −4.3 ± 0.08 for control. log( K_high ± s.e.m.) = −6.5 ± 0.2 and log( K_low ± s.e.m.) = −4.3 ± 0.07 with scFv16. n = 4 biologically independent experiments on Sf 9 cell membranes. In all panels, each data point is displayed as the mean with the error bars showing ± s.e.m., with individual data points for each repeat represented in small symbols.

a, G _i stimulation by dopamine in Sf9 cells transformed with G _i and hDRD2 baculoviruses. log(EC ₅₀ ± s.e.m.) = −6.3 ± 0.1 for hDRD2 ^WT (blue). log(EC ₅₀ ± s.e.m.) = −6.6 ± 0.3 for hDRD2 ^EM (orange). n = 4 biologically independent experiments on Sf9 cell membranes. b, Inhibition of dopamine-stimulated (10 μM) G _i activation by the inverse agonist spiperone. log( IC ₅₀ ± s.e.) = −8.6 ± 0.1 for hDRD2 ^WT (blue). log(IC ₅₀ ± s.e.m.) = −6.3 ± 0.2 for hDRD2 ^EM (orange). n = 4 biologically independent experiments on Sf9 cell membranes. c, Modulation of basal G _i activation by dopamine (open circles), BCT (closed circles) or spiperone (squares) with the hDRD2 ^EM (orange symbols) relative to hDRD2 ^WT (blue symbols). log(EC ₅₀ ± s.e.m.) for dopamine (−6.6 ± 0.2), BCT (−7.6 ± 0.1) and spiperone (−7.6 ± 0.2) for hDRD2 ^EM. log(EC ₅₀ ± s.e.m.) for dopamine (−7.0 ± 0.1) and BCT (−7.8 ± 0.2) for hDRD2 ^WT. n = 3 biologically independent experiments on Sf9 cell membranes. d, Apparent functional stability of agonist-bound hDRD2 constructs assessed by measuring the fraction of partially purified receptor-activating G _i as a function of incubation time at 37 °C. Half-life ± s.e.m. = 27.6 ± 1.0 min for hDRD2 ^WT (blue). Half-life ± s.e.m. = 49.3 ± 1.1 min for hDRD2 with five designed mutations and designed truncated ICL3 (hDRD2(ΔICL3-5mut); orange). n = 3 biologically independent experiments. e, Apparent thermostability of agonist-bound hDRD2 constructs assessed by measuring the fraction of partially purified receptor-binding agonist as a function of temperature. Melting temperature ( T_m) ± s.e.m. = 30.9 ± 0.9 °C for hDRD2 ^WT (blue). T_m ± s.e.m. = 39.6 ± 0.3 °C for hDRD2(ΔICL3-5mut) (orange). n = 3 biologically independent experiments. In all panels, each data point is displayed as the mean with the error bars showing ± s.e.m., with individual data points for each repeat represented in small symbols.

a, Superose 6 gel-filtration profile of hDRD2 ^EM-Gi-scFv16-HDL purified by M1 Flag affinity chromatography. b, Coomassie-stained PAGE of the isolated peak fraction from gel filtration. c, Overlaid Superose 200 gel-filtration profiles (in LMNG detergent) of different hDRD2 constructs purified by M1 Flag chromatography with saturating bromocriptine present. nT4L-hDRD2-5mut has the five thermostabilizing mutations of the EM construct, but has full-length ICL3. nT4L-hDRD2-ΔICL3 has the wild-type transmembrane region (no mutations) but the truncated ICL3. Receptors were purified as in Methods, but in the absence of co-transduced G-protein baculovirus.

a, Representative 2D class averages. Scale bar, 100 Å. b, Gold-standard Fourier shell correlation (FSC) curves of the 3D reconstructions. The dashed lines intercept the y axis at a FSC value of 0.143. c, Image processing procedure with the final maps coloured based on local resolution. Ctf, contrast transfer function.

a, Focused 3D classification subtracting T4L, the AH domain, scFv16 and rHDL/nanodisc density. b, Focused 3D classification subtracting all but the G protein. In a and b, the numbers below the images indicate the particle number in each 3D class. c, FSC curve showing the model-map correlation (focused map without T4L, the AH domain, scFv16 and rHDL/nanodisc density). The dashed line intercepts the y axis at a FSC value of 0.5. d, Representative hDRD2 transmembrane and ligand density.

a, Solvent-accessible surface for the active conformation (purple) bound to bromocriptine (yellow sticks). b, Solvent-accessible surface for the inactive conformation (orange) bound to risperidone (green sticks), showing the deeper subpocket (PDB: 6cm4).

a, Zoomed view of the TM6-TM7 interface on the cytoplasmic side of the receptor. Atomic interactions involving the designed Y6.40 were accurately predicted in the design model. The conformation of R3.50 is displaced in the experimental structure due to the binding of Gα _i, which was absent in the model. The experimental structure is in purple and the predicted DRD2 conformation is in green. b, c, Conformational energy landscape of the wild-type ( b) and designed ( c) DRD2 constructed from multiple all-atom MD simulation trajectories of the receptor active state homology models (orange circle) in the absence of bound agonist. The conformational space is reported along two canonical active state structure metrics (TM3-TM6 distance and NPxxY rmsd to inactive state). The DRD2 wild-type relaxes primarily due to an inactive state conformation, whereas the designed variant remains in partially active state conformation displaying large TM3-TM6 distance. The antagonist-bound DRD2 crystal structure (PDB: 6CM4) is used as a reference for the inactive state (green square).

a, AH domain density in the current structure. b, AH domain density in the rhodopsin-G _i complex (PDB: 6cmo). c, Alignment of the Gα _i subunit in the hDRD2 ^EM-G _i (purple) and the rhodopsin-G _i (blue) complexes. d, Alignment of the AH domain in the hDRD2 ^EM-G _i (purple) and the rhodopsin-G _i (blue) complexes.

a, Surface potential of the hDRD2 (top) and G _i (bottom) components shown separately. b, Surface potential of the complex, with the scale in eV. Calculation was done using the APBS plugin in PyMOL.

Cite this article

Yin, J., Chen, K.M., Clark, M.J. et al. Structure of a D2 dopamine receptor-G-protein complex in a lipid membrane. Nature (2020). https://doi.org/10.1038/s41586-020-2379-5