Structure of the dopamine D₂ receptor in complex with the antipsychotic drug spiperone

Abstract

In addition to the serotonin 5-HT_2A receptor (5-HT_2AR), the dopamine D₂ receptor (D₂R) is a key therapeutic target of antipsychotics for the treatment of schizophrenia. The inactive state structures of D₂R have been described in complex with the inverse agonists risperidone (D₂R_ris) and haloperidol (D₂R_hal). Here we describe the structure of human D₂R in complex with spiperone (D₂R_spi). In D₂R_spi, the conformation of the extracellular loop (ECL) 2, which composes the ligand-binding pocket, was substantially different from those in D₂R_ris and D₂R_hal, demonstrating that ECL2 in D₂R is highly dynamic. Moreover, D₂R_spi exhibited an extended binding pocket to accommodate spiperone’s phenyl ring, which probably contributes to the selectivity of spiperone to D₂R and 5-HT_2AR. Together with D₂R_ris and D₂R_hal, the structural information of D₂R_spi should be of value for designing novel antipsychotics with improved safety and efficacy.

The dopamine D₂ receptor (D₂R) is a GPCR and an important drug target for schizophrenia treatment. Here, the authors present the crystal structure of human D₂R in complex with the antipsychotic drug spiperone, which is of interest for designing antipsychotics with improved receptor selectivity.

Introduction

Dopamine is a neurotransmitter that controls numerous physiologic functions in the brain and peripheral nervous system via dopamine receptors of the G-protein-coupled receptor (GPCR) superfamily. In humans, five dopamine receptors (D₁R–D₅R) have been identified and have been classified according to their sequence, intracellular signaling, pharmacology, and localization as D₁-class receptors (D₁R and D₅R) or D₂-class receptors (D₂R, D₃R, and D₄R)^1–3. Thus, D₂R is similar to both D₃R and D₄R, with 80 and 54% sequence identities, respectively, in their transmembrane helices^4–6. D₂R is highly distributed in the striatum, nucleus accumbens, and olfactory tubercle^7,8, and it plays important pharmacologic roles in numerous human disorders related to dopaminergic dysfunction, including schizophrenia^9–11 and Parkinson’s disease^12,13.

D₂R antagonists have been developed as antipsychotics to block dopaminergic transmission for the treatment of schizophrenia¹⁴. Antipsychotics are either typical or atypical; typical antipsychotics generally antagonize D₂R, whereas atypical antipsychotics antagonize both D₂R and the serotonin 5-HT_2A receptor (5-HT_2AR). Both of these groups at least improve the positive symptoms of schizophrenia¹⁵. However, they also are associated with a wide range of severe side effects, such as extrapyramidal symptoms, weight gain, metabolic disorders, and constipation^16,17. Extrapyramidal symptoms are caused by excessive inhibition of D₂R in the nigrostriatal pathway. Other side effects are primarily due to the undesired binding of antipsychotics to other aminergic receptors, such as the serotonin 5-HT_2C receptor (5-HT_2CR), which exhibits 46% sequence identity with 5-HT_2AR.

GPCR structures have been successfully utilized for the structure-guided discovery of new ligands^18,19. In D₂-class receptors, D₃R and D₄R structures were determined in complex with the benzamide antipsychotics eticlopride and nemonapride (D₃R_eti and D₄R_nem), respectively^20,21. The inactive conformations of D₂R have been described in complexes with risperidone (D₂R_ris), a pyridopyrimidine antipsychotic²², and with haloperidol (D₂R_hal), a butyrophenone antipsychotic²³. D₂R_ris and D₂R_hal are practically identical, as shown by the RMSD values of Cα atoms between them (Supplementary Table 1). A G-protein-bound active conformation of D₂R also was reported in complex with an agonist, bromocriptine (D₂R_bro)²⁴. Interestingly, the conformation of the extracellular loop (ECL) 2 in D₂R_ris and D₂R_hal is entirely different from those of D₃R_eti, D₄R_nem, and D₂R_bro (Supplementary Fig. 1). Additionally, while the conformation of ECL1 is relatively conserved among the structures of D₂-class receptors, Trp100^23.50 on ECL1 of D₂R_ris is uniquely directed to the binding pocket (Supplementary Fig 1).

In this study, we describe the structure of D₂R in complex with spiperone (D₂R_spi), a butyrophenone typical antipsychotic that binds with high affinity to D₂R, D₃R, D₄R, and 5-HT_2AR²⁵. We also present a structural comparison of D₂R_spi with other D₂R structures, D₃R_eti, and D₄R_nem, in addition to 5-HT_2AR complexed with risperidone (5-HT_2AR_ris)²⁶ and 5-HT_2CR with ritanserin (5-HT_2CR_rit)²⁷. The structure of D₂R_spi given herein provides valuable information for the rational design of antipsychotics with improved receptor selectivity.

Results

Overall structure of D₂R_spi

Because wild-type D₂R is not expressed in Spodoptera frugiperda (Sf9) insect cells, we prepared a stable construct for crystallization trials. D₂R was stabilized by the truncation of 34 N-terminal residues and the replacement of the intracellular loop (ICL) 3 with the thermostabilized apocytochrome b562RIL²⁸ (D₂R-bRIL). D₂R-bRIL was further stabilized by the mutations S121K^3.39 and L123W^3.41 (here, superscripts indicate residue numbers according to the Ballesteros–Weinstein scheme¹⁵) and the replacement of bRIL with mbIIG, the loop-modified cytochrome b₅₆₂IIG²⁹ (D₂R-mbIIG S121K^3.39/L123W^3.41, see Methods). The use of mbIIG instead of bRIL was essential to obtain D₂R crystals. S121K^3.39 is a mutation of the allosteric sodium ion binding site of class A GPCRs that mimics the presence of the sodium ion, therefore stabilizing the inactive state^30,31. The S121K^3.39, L123W^3.41, and S121K^3.39/L123W^3.41 mutants, in addition to the stabilized construct, showed similar affinities for spiperone to the wild-type human D₂R (Supplementary Table 2 and Supplementary Fig. 2), suggesting that these mutations did not substantially affect the binding of spiperone. Additionally, the L123W^3.41 mutant showed similar antagonist activity to that of the wild-type human D₂R by a transforming growth factor alpha (TGFα) shedding assay³², which measured the antagonist activities of wild-type and mutant D₂R for spiperone against a fixed concentration of dopamine  (Supplementary Table 3 and Supplementary Fig. 3). However, because the S121K^3.39 mutation stabilizes the inactive state of D₂R, the antagonist activities of the mutants with S121K^3.39 could not be determined (Supplementary Table 3 and Supplementary Fig. 3). Eticlopride and sulpiride enhance the affinity for D₂R in the presence of the sodium ion, whereas spiperone does not³³. This enhancement is ascribed to an interaction network from the bound sodium ion in the allosteric binding site³³. The S121K^3.39 mutation decreased the affinity for eticlopride (Supplementary Table 2), suggesting that the side chain of Lys121^3.39 does not sufficiently mimic the sodium ion in the allosteric binding site for the binding of eticlopride.

For crystallization, we generated an antibody recognizing the D₂R structure (IgG3089) and prepared a novel Fab fragment (Fab3089) of IgG3089. Using the stabilized construct and Fab3089, we successfully obtained crystals of D₂R (Supplementary Fig. 4a). The structure of D₂R was determined in complex with spiperone at 3.1 Å resolution using an X-ray free-electron laser (Fig. 1, Table 1, and Supplementary Fig. 4). D₂R_spi bound to Fab3089 at the extracellular region (Fig. 1a and Supplementary Fig. 4c), exhibiting a canonical GPCR fold with seven transmembrane helices (TM1–7) and an intracellular amphipathic helix 8 (H8) (Fig. 1). The D₂R_spi structure demonstrated the inactive conformation because the seven helical bundle structure and the conformations of the four activation microswitches are more similar to those of the inactive state conformation in D₂R_ris than those of the active state conformation in D₂R_bro (Supplementary Fig. 5 and Supplementary Table 1). We also compared the PIF motif of D₂R_spi with those of the inactive state and the active state structures of the β₂-adrenergic receptor to confirm D₂R_spi demonstrated the inactive conformation, because D₂R_ris contains the I122A^3.40 mutation in the motif.

Fig. 1. Structure of D₂R_spi in complex with spiperone.

a Overall structure of D₂R_spi-Fab 3089 complex. Extracellular (b) and side (c) views of D₂R_spi. Spiperone, D₂R, Fab3089, and mbIIG are indicated in orange, green, cyan, and gray, respectively. The disulfide bond and the C-terminal segment of ECL2 are shown in yellow and magenta, respectively, in (b) and (c). Ext, extracellular; Int, intracellular.

Table 1.

Data collections and structure refinement statistics.

	D2Rspi (PDB 7DFP)
Data collection
Space group	C2
Cell dimensions
a, b, c (Å)	161.9, 40.5, 165.9
α, β, γ (°)	90, 116.5, 90
Resolution (Å)	43.1-3.1 (3.2-3.1)a
Rsplit (%)	18.8 (73.2)
CC1/2	0.97 (0.58)
I / σ(I)	4.4 (1.5)
Completeness (%)	100 (100)
Redundancy	99.8 (45.4)
Refinement
Resolution (Å)	43.1-3.1 (3.2-3.1)
No. reflections	18,048
Rwork / Rfree	18.5 / 21.7 (26.7/31.7)
No. atoms
Protein	5896
Ligand	29
B factors
Protein	97.1
Ligand	105.9
R.M.S. deviations
Bond lengths (Å)	0.002
Bond angles (°)	0.56

^aValues in parentheses are for highest-resolution shell.

There were no secondary structures in the ECLs and ICL1; ICL2 in D₂R_spi was disordered. Spiperone was bound to the orthosteric binding site (Fig. 1 and Supplementary Fig. 4d). Like other class A GPCRs, the ligand-binding pocket was covered by the C-terminal segment of ECL2, which is stabilized by a disulfide bond between Cys107^3.25 on TM3 and Cys182^45.50 on ECL2³⁴ (Fig. 1).

Binding mode of spiperone

In D₂R_spi, spiperone was surrounded by residues from TM2, 3, 5, 6, and 7 and ECL2 (Figs. 1 and 2a, b). The binding mode of spiperone was consistent with the results of the TGFα shedding assay (Supplementary Table 3) and of the ligand-binding assay for spiperone using the mutants^35–38.

Fig. 2. The ligand-binding pocket of D₂R_spi.

a Close-up view of the ligand-binding pocket of D₂R_spi. Spiperone and D₂R are indicated in orange and green, respectively. The side chains of the contact residues within 4.5 Å of spiperone are shown as green sticks. The side chain of W100^23.50 is indicated in magenta. The side chains of the residues in the PIF motif are shown as blue sticks. b Diagram of the interactions between D₂R and spiperone. c Fab3089 binding site. The side chains of I183^45.51 of D₂R_spi and Y55 of Fab3089 are shown as green and cyan sticks, respectively. d Vertical cross section of (a). Red and blue circles indicate the EBP and the bottom hydrophobic cleft, respectively.

The tertiary amine in the triazaspiro ring formed a salt bridge with Asp114^3.32; this is strictly conserved in aminergic receptor structures (Fig. 2a, b). In D₂R, the mutant of Asp114^3.32 loses its affinities to both agonists and antagonists^22,36. This interaction may be stabilized by the conserved hydrogen bond between Asp114^3.32 and Tyr416^7.43 39. On the opposite side of spiperone from Asp114^3.32, there is hydrophobic contact between spiperone and Phe389^6.51. This contact is likely essential for spiperone binding, given that F389A^6.51 showed a 34-fold reduction in affinity for spiperone compared to that of wild-type D₂R³⁵. Additionally, there was contact between the triazaspiro ring and Ile183^45.51, Ile184^45.52, and Cys182^45.50 on ECL2 (Fig. 2a, b). The loss of this contact with the I184A^45.52 mutation significantly reduced the antagonist activity for spiperone (Supplementary Table 3), suggesting that the contact with Ile184^45.52 is crucial for the antagonist activity of spiperone. The contact between Ile183^45.51 and spiperone was influenced by the binding of Fab3089, because the side-chain conformation of Ile183^45.51 was stabilized by Tyr55 of Fab3089 (Fig. 2c). The I183A^45.51 mutant slightly increased the antagonist activity for spiperone (Supplementary Table 3).

On the extracellular side of the salt bridge, the phenyl ring of spiperone was bound in an extended binding pocket (EBP) between TM2 and TM3 (Fig. 2a, d). The EBP was formed by residues Val87^2.57, Trp90^2.60, Val91^2.61, Leu94^2.64, Trp100^23.50, Phe110^3.28, Val111^3.29, and Cys182^45.50. The drastic reduction of the antagonist activity of W100A^23.50 for spiperone indicates that Trp100^23.50 is crucial for maintaining EBP conformation (Supplementary Table 3). Because of the triazaspiro ring rigidity and the direction of the conserved salt bridge between the tertiary amine and Asp114^3.32, the EBP is likely essential for spiperone binding. In the EBP of D₂R_spi, spiperone’s phenyl ring forms hydrophobic contacts with Trp90^2.60, Val91^2.61, Leu94^2.64, Phe110^3.28, and Val111^3.29 (Fig. 2a, b). Mutations of most of these residues resulted in considerably decreased antagonist activity for spiperone (Supplementary Table 3).

On the intracellular side of the salt bridge, spiperone’s fluorophenyl ring penetrated deeply into the ligand-binding pocket, binding in the bottom hydrophobic cleft (Fig. 2a, b, d). Similar bottom hydrophobic clefts have been observed in the structures of the histamine H₁ receptor⁴⁰, 5-HT_2AR²⁶, and 5-HT_2CR²⁷. In the cleft, the fluorophenyl ring formed a CH–π interaction with Cys118^3.36; hydrophobic interactions with Thr119^3.37, Ile122^3.40, Ser197^5.46, Phe198^5.47, and Phe382^6.44; and edge-to-face π interactions with Trp386^6.48 and Phe390^6.52 (Fig. 2a, b). Trp386^6.48 is a microswitch in the CWxP motif. Indeed, W386L^6.48 affected activation by dopamine and showed no antagonist activity for spiperone (Supplementary Table 3 and Supplementary Fig. 3). The interaction with Phe390^6.52 is essential for the binding of spiperone, because F390A^6.52 drastically decreases the affinity for spiperone³⁵. By contrast, contact with Phe198^5.47 is not essential for the binding of spiperone, given that F198A^5.47 showed a similar affinity for spiperone to that of wild-type D₂R³⁵. The S197A^5.46 mutant also showed an affinity for spiperone similar to that of the wild-type D₂R^36–38, thus strengthening contact between spiperone and the Cβ atom of Ser197^5.46.

Ile122^3.40 and Phe382^6.44 belong to the PIF motif located at the bottom of the ligand-binding pocket in aminergic receptors⁴¹. When activated, the PIF motif conformationally rearranges, with the outward movement of the cytoplasmic side of TM6. The PIF motif conformation in D₂R_spi is that of the inactive state (Supplementary Fig. 5). Thus, the direct interactions of spiperone with Ile122^3.40 (3.7 Å distance) and Phe382^6.44 (3.6 Å distance) in the PIF motif may block the conformational rearrangements of the PIF motif and help to stabilize the inactive conformation, as has been observed in the structural studies of 5-HT_2AR²⁶ and 5-HT_2CR²⁷. Of the 21 contact residues, 20 were conserved between D₂R and D₃R (Supplementary Table 4), reflecting the similarly high affinity of these receptors for spiperone⁴².

Comparison with D₂R_ris, D₂R_hal, and D₂R_bro

There are striking structural differences in the ligand-binding pocket of D₂R_spi and the other inactive state structures of D₂R: D₂R_ris and D₂R_hal (Fig. 3a, b and Supplementary Table 1). In D₂R_spi, the ligand-binding pocket was covered by the C-terminal segment of ECL2 (Fig. 3a–c), on which the side chains Ile183^45.51 and Ile184^45.52 pointed to the entrance and the bottom of the ligand-binding pocket, respectively. This conformation was conserved in the structures of other aminergic receptors, including D₃R, D₄R, and 5-HT_2AR (Supplementary Fig. 6). In D₂R_ris and D₂R_hal, however, ECL2 extended away from the top of the receptor core (Fig. 3a, b, d). In this conformation, Ile183^45.51 was buried in the hydrophobic core outside the ligand-binding pocket, and Ile184^45.52 reached the top of the ligand-binding pocket (Fig. 3a, b). Thus, Ile184^45.52 did not contact with risperidone in D₂R_ris, while Ile184^45.52 contacted with spiperone in D₂R_spi. These findings were consistent with I184A^45.52 showing similar antagonist activity for risperidone with the wild-type (Supplementary Table 3).

Fig. 3. Comparison of D₂R structures.

a Extracellular view of the superposition of D₂R_spi and D₂R_ris. The side chains of disulfide bridge, I183^45.51 and I184^45.52 are shown as sticks. Red arrows indicate the shift of helices in D₂R_ris with the distance relative to D₂R_spi. b Extracellular view of ECL1 and ECL2 of D₂R_spi, D₂R_ris, D₂R_hal and D₂R_bro. The side chains of the disulfide bridge, W100^23.50, I183^45.51, and I184^45.52 are shown as sticks. Surface representation of D₂R_spi (c) and D₂R_ris (d) viewed from the extracellular side. ECL1 and ECL2 are pink and yellow, respectively. e The EBP of D₂R_spi and the corresponding part of D₂R_ris, D₂R_hal and D₂R_bro. The side chains of W90^2.60 and F110^3.28 are shown as sticks. f Side view of the superposition of D₂R_spi, D₂R_ris, D₂R_hal and D₂R_bro around TM5 and ECL2. Red arrows indicate the shift of the extracellular half of TM5 in D₂R_ris and D₂R_hal relative to D₂R_spi. In (b), (e), and (f), D₂R_spi (green), D₂R_ris (cyan), D₂R_hal (purple), D₂R_bro (olive), spiperone (orange), risperidone (magenta), haloperidol (ivory), and bromocriptine (lightblue) are shown.

On ECL1, D₂R demonstrated the diverse side-chain conformation of Trp100^23.50. In D₂R_spi, Trp100^23.50 interacted with the conserved disulfide bond (Fig. 3b). The conformation was highly conserved in the structures of class A GPCRs (Supplementary Fig. 6). The interaction between a disulfide bond and tryptophan is often observed in protein structures; this may contribute to protecting the disulfide bond and stabilizing the structure^43,44. Trp100^23.50 in D₂R_hal exhibited similar side-chain conformation with that of D₂R_spi, although it did not contact with the disulfide bond because of ELC2 flipping (Fig. 3b). In D₂R_ris, Trp100^23.50 moves to the ligand-binding pocket and forms a T-stacking interaction with risperidone’s tetrahydropyridopyrimidinone ring²² (Fig. 3b). Trp100^23.50 in D₂R_ris forms a hydrophobic patch with Leu94^2.64 and Ile184^45.52, covering the ligand-binding pocket. Despite this, the ligand-binding pocket in D₂R_ris was more exposed to the extracellular solution compared with that in D₂R_spi (Fig. 3c, d). W100A^23.50 in D₂R has been shown to reduce the residence times of several antipsychotics²². Based on these results, it was hypothesized that the hydrophobic patch in D₂R_ris contributes to the slow dissociation of antipsychotics²². However, the results were also consistent with the conformation observed in D₂R_spi, in which Trp100^23.50 stabilized the conformation of ECL2 and EBP.

The EBP is uniquely observed in D₂R_spi among the inactive state structures of D₂R. In D₂R_spi, the side chain of Phe110^3.28 that creates the EBP was flipped compared with those of D₂R_ris and D₂R_hal (Fig. 3e), D₃R_eti, and the 5-HT₂ receptors. In D₂R_spi, the flipped Phe110^3.28 side chain formed a stacking interaction with Trp90^2.60 (Fig. 3e).

D₂R_ris and D₂R_hal also possessed the bottom hydrophobic cleft (Fig. 3f). However, the conformation of this cleft in D₂R_ris and D₂R_hal was altered by the shift of the extracellular half of TM5 in the ligand-binding pocket relative to that in D₂R_spi (Fig. 3a, f)_. Resultantly, Phe189^5.38 and Ser193^5.42 contacted risperidone in D₂R_ris, though these residues did not contact haloperidol in D₂R_hal (Fig. 3f). The shift of TM5 observed in D₂R_ris and D₂R_hal is likely inhibited in D₂R_spi by steric contact between the extracellular end of TM5 and ECL2. Indeed, Phe189^5.38 in D₂R_ris and D₂R_hal occupied a similar position with Ile184^45.52 in D₂R_spi in the ligand-binding pocket (Fig. 3f). The shift can also be affected by the I122A^3.40 mutation introduced to stabilize the receptor in D₂R_ris and D₂R_hal^22,23. In D₂R_ris and D₂R_hal, the Cβ atom of Ala122^3.40 was in contact (3.8 Å distance) with the carbonyl oxygen atom of Ser197^5.46 (Supplementary Fig. 7a); when the side chain of Ala122^3.40 was replaced by isoleucine using Coot⁴⁵, the resulting side chain formed steric contacts (less than 3.0 Å distance) with the surrounding residues, including Ser197^5.46 and Pro201^5.50 on TM5, and with risperidone or haloperidol in any of the seven allowed side-chain rotamers for isoleucine (Supplementary Fig. 7b).

Unlike inactive state structures, D₂R_bro showed a typical active state conformation in the microswitches and the seven helical bundles (Supplementary Fig. 5). Conformations of ECL2 and the extracellular end of TM5 in D₂R_bro were more similar to those of D₂R_spi than those of D₂R_ris and D₂R_hal. (Fig. 3b, f). Trp100^23.50 of D₂R_bro existed at a similar position with those in D₂R_spi and D₂R_hal but with a different side-chain rotamer (Fig. 3b). EBP in D₂R_spi was not observed in D₂R_bro (Fig. 3e).

Comparison with 5-HT_2AR_ris and 5-HT_2CR_rit

The conformations of the extracellular end of TM5, the conserved Trp^23.50 on ECL1, the C-terminal segment of ECL2, and the disulfide bridge between ECL2 and TM3 in 5-HT_2AR_ris and 5-HT_2CR_rit were similar to those of D₂R_spi (Fig. 4a, b and Supplementary Fig. 6). On the C-terminal segment of ECL2, the Leu228^45.51 and Leu229^45.52 residues of 5-HT_2AR contacted risperidone and zotepine, respectively²⁶, and the Val208^45.52 residue of 5-HT_2CR interacted with ritanserin²⁷. The high conservation of the residues in the ligand-binding pocket (Supplementary Table 4) and the structural similarity of 5-HT_2AR_ris, 5-HT_2CR_rit, and D₂R_spi explain why antipsychotics often bind to D₂R, 5-HT_2AR, and 5-HT_2CR with high affinity.

Fig. 4. Comparison of D₂R_spi and 5-HT₂ receptors.

Extracellular view of the superpositions of D₂R_spi and either 5-HT_2AR_ris (a) or 5-HT_2CR_rit (b). c The EBP of D₂R_spi and the corresponding part of 5-HT_2AR_ris. d The EBP of D₂R_spi and the corresponding part of 5-HT_2CR_rit. e Vertical cross sections of D₂R_spi and 5-HT_2AR_ris. In this figure, D₂R_spi (green), 5-HT_2AR_ris (blue), 5-HT_2CR_rit (brown), spiperone (orange), risperidone (yellow), and ritanserin (gray) are shown.

Phe110^3.28 and Trp90^2.60 forming EBP in D₂R_spi corresponded with Trp151^3.28 and Val130^2.60 in 5-HT_2AR and Trp130^3.28 and Leu109^2.60 in 5-HT_2CR, respectively. In D₂R, W90L^2.60 reduced the antagonist activity more than F110W^3.28 and similarly with W90L^2.60/F110W^3.28 (Supplementary Table 3). Spiperone shows a high affinity to D₂R and 5-HT_2AR but a low affinity to 5-HT_2CR²⁵. A potential explanation of this difference in affinity is that while Trp151^3.28 in 5-HT_2AR_ris can be flipped to form the EBP without any steric hindrance, Trp130^3.28 in 5-HT_2CR_rit is challenging to flip because of the steric contact with Leu109^2.60 (Fig. 4c, d). Thus, the EBP of D₂R_spi and the putative EBP of 5-HT_2AR_ris may contribute to spiperone’s higher selectivity for these receptors than for 5-HT_2CR. The binding mode of spiperone in the EBP may be useful for designing selective D₂R and 5-HT_2AR antipsychotics.

A unique side-extended cavity was previously observed in the structure of 5-HT_2AR between TM4 and TM5 that was suggested to contribute to the binding site of 5-HT_2AR-selective drugs²⁶. D₂R_spi did not possess the side-extended cavity between TM4 and TM5 (Fig. 4e).

Comparison with D₃R_eti and D₄R_nem

The conformation of D₂R_spi was similar to that of D₃R_eti and D₄R_nem, except for the extracellular half of TM6 (Fig. 5a, b). The conformation of the C-terminal segment of ECL2 in D₂R_spi was almost identical to that of D₃R_eti and D₄R_nem (Fig. 5a, b). On ECL2, Ile183^45.52 in D₃R_eti and Leu187^45.52 in D₄R_nem contacted eticlopride²⁰ and nemonapride²¹, respectively, similar to Ile184^45.52 in D₂R_spi, which contacts spiperone. A previous study showed that eticlopride and nemonapride bind just above the bottom hydrophobic cleft in D₃R_eti and D₄R_nem²² (Supplementary Fig. 8a), but this is different from the binding of spiperone in D₂R_spi, risperidone in D₂R_ris, and haloperidol in D₂R_hal. To interact with these benzamide antipsychotics, the extracellular half of TM6 exhibited a greater tilt toward TM3 in D₃R_eti and D₄R_nem than in D₂R_spi, D₂R_ris, and D₂R_hal (Fig. 5a, b and Supplementary Fig. 8b). Thus, His349^6.55 in D₃R_eti and His414^6.55 in D₄R_nem interacted with eticlopride and nemonapride, respectively, whereas no contact was made between spiperone and His393^6.55 in D₂R_spi (Supplementary Fig. 8b). Due to the large tilt of TM6, the distances between Cys^3.36 and Phe^6.52 in D₃R_eti and D₄R_nem were approximately 1.5 and 2.0 Å closer, respectively, than in D₂R_spi (Fig. 5a, b and Supplementary Fig. 8b). Together, the side-chain flip of Cys118^3.36 and the tilt of TM6 resulted in the closure of the bottom hydrophobic cleft in D₃R_eti and D₄R_nem (Fig. 5a, b and Supplementary Fig. 8a, b). Conversely, the insertion of spiperone’s fluorophenyl ring between Cys^3.36 and Phe^6.52 created the bottom hydrophobic cleft and inhibited the large tilt of TM6 in D₂R_spi. D₂-class receptors show high affinities for eticlopride, nemonapride, spiperone, and risperidone²⁵, and display conserved residues in the ligand-binding pocket. Therefore, it is likely that these receptors show a similar conformation with D₃R_eti and D₄R_nem when they bind to benzamide antipsychotics and with D₂R_spi, D₂R_ris, and D₂R_hal when they bind to butyrophenone or pyridopyrimidine antipsychotics (Supplementary Fig. 8c).

Fig. 5. Comparison of D₂R_spi, D₃R_eti and D₄R_nem.

Extracellular view of the superpositions of D₂R_spi and either D₃R_eti (a) or D₄R_nem (b). c The EBP of D₂R_spi and the corresponding part of D₃R_eti. d Superposition of the EBP of D₂R_spi and D₄R_nem. In this figure, D₂R_spi (green), D₃R_eti (yellow), D₄R_nem (pink), spiperone (orange), eticlopride (blue), and nemonapride (red) are shown.

Spiperone and nemonapride show high affinities for D₂-class receptors²⁵. In D₃R, the putative EBP was closed by the side chain of Phe106^3.28 (Fig. 5c). If the Phe106^3.28 is flipped, D₃R can form an EBP similar to that of D₂R_spi with the conserved residues around this region. The EBP in D₂R_spi was observed at a position similar to that of the EBP in D₄R_nem that binds the phenyl ring of nemonapride²¹ (Fig. 5d), although the contact residues were not conserved between D₂R and D₄R (Supplementary Table 4).

Discussion

D₂R_spi was observed to differ substantially from the other inactive state structures of D₂R, D₂R_ris, and D₂R_hal, especially in ECL2, forming the entrance part of the ligand-binding pocket. This suggests that ECL2 is highly dynamic in the inactive state of D₂R. The residues on ECL2 in D₂R have been mapped by the substituted-cysteine accessibility method⁴⁶. In that study, the sulfhydryl groups of I183C^45.51 and I184C^45.52 reacted with charged sulfhydryl-specific reagents, indicating that these residues are water-accessible. The binding of N-methylspiperone to I183C^45.51 and I184C^45.52 was inhibited by the reaction with sulfhydryl-specific reagents, indicating that Ile183^45.51 and Ile184^45.52 were directed toward the ligand-binding pocket. Ile183^45.51 is less likely to contact the ligand because N-methylspiperone binding to I183C^45.51 was inhibited only by a bulkier sulfhydryl-specific reagent, MTSET. I184C^45.52 reduced the affinities for nemonapride and N-methylspiperone, suggesting that Ile184^45.52 contacts nemonapride and N-methylspiperone. These results are consistent with the conformation of ECL2 observed for D₂R_spi but not with that of D₂R_ris or D₂R_hal.

The difference of the ECL2 conformation can be caused by the bound ligand, although D₂R_ris and D₂R_hal show very similar ECL2 conformation. Indeed, ECL2 in 5-HT_2AR moves slightly to bind a different ligand²⁶. An MD simulation study also suggested these dynamics of the ECL2 of D₂R⁴⁷ and reported that the helical conformation of ECL2 observed in D₂R_ris tended to unwind toward an extended conformation, similar to that of D₃R_eti, regardless of the bound ligand, including spiperone, risperidone, or eticlopride⁴⁷. The unwinding involves a drastic rearrangement of the side chain of Ile183^45.51, dissociating from a hydrophobic pocket. The study also suggested that the ECL2 conformation observed in D₂R_ris represents a higher energy state than the extended conformation. Considering the structural similarity between D₂R_spi and D₃R_eti, the conformation of ECL2 in D₂R_spi likely corresponds to a lower energy state conformation.

Currently, there is a need for novel, safer antipsychotics that bind selectively to 5-HT_2AR and D₂R. In this study, we revealed that the ligand-binding pocket of D₂R forms more than two different conformations in the inactive state. Moreover, we showed that the EBP in D₂R_spi and the putative EBP in 5-HT_2AR_ris could be used as the binding site for selective atypical antipsychotics. D₂R_hal was used for the structure-based discovery of selective ligand²³. The use of multiple different conformations in the structure-based design instead of a single conformation clearly increased the possibility of finding high-affinity compounds. Together with D₂R_ris, D₂R_hal, and 5-HT_2AR_ris, the structure of D₂R_spi can be utilized for a rational, structure-based design of new antipsychotics with low side effects.

Methods

Protein engineering for structure determination

The coding sequence of human D₂R (UniProt ID P14416) was synthesized by TAKARA Bio. D₂R was stabilized by removing the N-terminal 34 residues, by introducing two mutations (S121K^3.39 and L123W^3.41)^30,48 and by replacing ICL3 (Lys221^5.70 to Leu363^6.25) with the loop-modified cytochrome b₅₆₂IIG mutant (D₂R-mbIIG S121K^3.39/L123W^3.41)²⁹. mbIIG contains four mutations (M7W, R98I, H102I, and R106G), and residues 41–65 have been replaced with the Gly-Ser-Gly-Ser-Gly linker to increase thermostability and reduce conformational variation. All the constructs were prepared by high-throughput fluorescent-based screening in Saccharomyces cerevisiae. A 30 ng of SmaI-linearized plasmid pDDGFP2 and 3 µl of the PCR reaction mixtures were co-transformed into S. cerevisiae strain FGY217⁴⁹. Transformants harboring the plasmid encoding the receptor were selected on an agar plate without uracil [0.192% (w/v) yeast synthetic dropout media without uracil (Sigma), 0.67% (w/v) yeast nitrogen base without amino acids (BD), 2% (w/v) agar and 2% (w/v) glucose]. The S. cerevisiae transformant was cultured in 5 ml of a medium [0.192% (w/v) yeast synthetic dropout media without uracil, 0.671% (w/v) yeast nitrogen base without amino acids and 2% (w/v) glucose] at 30 °C for 24 h. The generated plasmid encoding the receptor was isolated from S. cerevisiae with the Miniprep Kit (Qiagen) by disrupting cells with 0.5 mm glass beads⁵⁰. The construct was subcloned into the pFastBac1 vector (Invitrogen), with a C-terminus tobacco etch virus (TEV) protease cleavage site, green fluorescent protein (GFP), and an octa-histidine tag.

Protein expression and purification

The stabilized D₂R was expressed in Sf9 cells using a Bac-to-Bac baculovirus expression system (Invitrogen). The Sf9 cells were infected at 1.5 × 10⁶ cells/ml, at a multiplicity of infection (MOI) of 0.05, and were harvested 84 h later. Cell pellets were resuspended with hypotonic buffer (10 mM HEPES, pH 7.5, 20 mM KCl, and 10 mM MgCl₂) and were repeatedly washed and centrifuged in high osmotic buffer (10 mM HEPES, pH 7.5, 1 M NaCl, 20 mM KCl, and 10 mM MgCl₂) containing EDTA-free complete protease inhibitor cocktail (Roche) to purify the cell membranes. The purified membranes were solubilized for 2 h at 4 °C in solubilization buffer (50 mM HEPES, pH 7.5, 500 mM NaCl, 1% (w/v) n-dodecyl-ß-D-maltopyranoside (DDM, Anatrace), 0.2% (w/v) cholesteryl hemisuccinate (CHS, Sigma-Aldrich), and 20% (v/v) glycerol) supplemented with 2 mg/ml iodoacetamide (Wako Pure Chemical Industries, Ltd), 200 µM spiperone (Sigma-Aldrich), and the protease inhibitor cocktail. Insoluble materials were removed by centrifugation, and the supernatants were incubated with TALON metal affinity resin (Clontech) for 10 h at 4 °C. The resin was washed with 10 column volumes (CV) of wash buffer I (50 mM HEPES, pH 7.5, 500 mM NaCl, 10% (v/v) glycerol, 0.05% (w/v) DDM, 0.01% (w/v) CHS, 20 mM imidazole, 10 mM MgCl₂, 8 mM ATP, and 100 µM ligand) and 10 CV of wash buffer II (50 mM HEPES, pH 7.5, 500 mM NaCl, 10% (v/v) glycerol, 0.05% (w/v) DDM, 0.01% (w/v) CHS, 20 mM imidazole, and 100 µM ligand). The protein was eluted in 4 CV of elution buffer (50 mM HEPES, pH 7.5, 500 mM NaCl, 10% (v/v) glycerol, 0.05% (w/v) DDM, 0.01% (w/v) CHS, 200 mM imidazole, and 100 µM ligand) and concentrated to 2.5 ml with a 100-kDa molecular weight cutoff Amicon Ultra-15 concentrator (Millipore). The imidazole was removed using a PD-10 column (GE Healthcare). The desalted protein was loaded onto Ni-NTA Superflow resin (Qiagen) and incubated for 10 h. The resin was washed with 10 CV of Ni wash buffer (50 mM HEPES, pH 7.5, 500 mM NaCl, 10% (v/v) glycerol, 0.03% (w/v) DDM, 0.006% (w/v) CHS, 20 mM imidazole, and 100 µM ligand) and eluted with 3 CV of Ni elution buffer (50 mM HEPES, pH 7.5, 500 mM NaCl, 10% (v/v) glycerol, 0.03% (w/v) DDM, 0.006% (w/v) CHS, 400 mM imidazole, and 100 µM ligand). The imidazole was removed using a PD-10 column, and the sample was then incubated with His-tagged TEV protease (expressed and purified in-house) for 10 h. TEV protease, cleaved His-tagged GFP and uncleaved protein were removed by passing the suspension through Ni Sepharose High Performance resin (GE Healthcare).

Antibody generation

All the animal experiments conformed to the guidelines of the Guide for the Care and Use of Laboratory Animals of Japan and were approved by the Kyoto University Animal Experimentation Committee (approval no. Medkyo16043). As the antigen, we used a stabilized D₂R (D₂R-mbIIG S121K^3.39/L123W^3.41). Purified antigen was reconstituted into liposomes containing chicken egg yolk phosphatidylcholine (Avanti) and monophosphoryl lipid A (Sigma-Aldrich). MRL/lpr mice were immunized three times at two-week intervals with 0.1 mg of the proteoliposome D₂R antigen. Single cells were harvested from mice spleens and were fused with NS-1 myeloma cells. To select antibodies that recognized the 3D structure of human D₂R, we performed a multi-step screening method, using D₂R-i3d, which lacks residues of the N-terminal and ICL3, at each step, which included liposome-ELISA, denatured ELISA, and fluorescence size-exclusion chromatography. The collected clones were evaluated using a Biacore T100 protein interaction analysis system (GE Healthcare) and were subsequently isolated by limiting dilution to establish monoclonal hybridoma cell lines. The resulting immunoglobulin-G (IgG3089) was purified with HiTrap Protein G HP (GE Healthcare) followed by proteolytic cleavage with papain (Nacalai Tesque). The Fab fragment (Fab3089) was then purified by size-exclusion chromatography (Superdex 200 10/300 GL, GE Healthcare) and affinity chromatography with a Protein A Sepharose 4 Fast-Flow column (GE Healthcare). The sequence of Fab3089 was determined via standard 5′-RACE using total RNA isolated from hybridoma cells.

Crystallization

The D₂R–Fab3089 complex was prepared by mixing the purified D₂R-mbIIG S121K^3.39/L123W^3.41 and Fab3089 at a molar ratio of 1:1.2 for 1 h on ice. The mixture was injected onto a Superdex 200 10/300 GL column (GE Healthcare), and the fractions containing the complex were concentrated to approximately 30 mg/ml with a 50-kDa molecular weight cutoff Amicon Ultra-15 concentrator (Millipore). The D₂R–Fab3089 in complex with spiperone was reconstituted in LCP by mixing approximately 30 mg/ml protein solution with monoolein and 10% w/w cholesterol at a volume ratio of 2:3 (protein:lipid) using two 100-μl Hamilton syringes and a syringe coupler. One syringe and a coupler were then removed, and a cleaning wire was inserted into the protein-laden LCP in the other syringe^51,52. Approximately 10 μl of the protein-laden LCP was extruded from the syringe with the wire and was soaked in a 0.6-ml tube filled with precipitant solution (0.1 M Tris-HCl, pH 8.0, 0.1 M CH₃COOLi, 28–32% PEG400, 5% dimethyl sulfoxide, 0.01 M ATP, and 1 mM spiperone) and incubated at 20 °C. Microcrystals appeared after 2 days, growing to a maximum size of 20 × 2 × 2 µm³ within a week.

Data collection using an X-ray free-electron laser

The data were collected at beamline BL3 of the SPring-8 Angstrom Compact Free-Electron Laser (SACLA)⁵³ (Hyogo, Japan) by the serial femtosecond crystallography technique using 1.5 × 1.5 µm² microbeams focused by Kirkpatrick–Baez mirrors⁵⁴ with a short-working-distance octal multiport CCD detector with eight sensor modules⁵⁵. The data were collected at 7 keV with a pulse duration of approximately 10 fs and a repetition rate of 30 Hz. To inject the microcrystals, LCP was loaded into a sample cartridge through a needle connected to a syringe.

After centrifugation at 2000g for 10 s, the cartridge was mounted in a high-viscosity micro-extrusion injector with a nozzle diameter of 100 µm^51,52,56. The injector was set in a chamber filled with helium gas in the Diverse Application Platform for Hard X-ray Diffraction in SACLA (DAPHNIS) set-up⁵⁷ and was maintained at a constant 20 °C. A total volume of 60 μl of LCP was injected at a flow rate of 420 nl/min. Data collection was guided by a real-time data processing pipeline⁵⁸ based on Cheetah⁵⁹. Data processing and indexing were performed using CrystFEL 0.8.0^60,61 and XGANDALF (https://onlinelibrary.wiley.com/iucr/doi/10.1107/S2053273319010593), respectively. The total number of collected, hit, and indexed images were 351,326, 11,373, and 9,464, respectively. The dataset was merged by process_hkl in the CrystFEL suite, without scaling. A per-image resolution cutoff was applied by using the –push-res = 1.2 option to account for variations in the crystal quality.

Structure determination and refinement

The structure was determined by molecular replacement with Phaser⁶² software using the structures of the transmembrane region of D₃R (PDB ID: 3PBL), cytochrome b₅₆₂RIL (PDB ID: 1M6T), and the Fab fragment (PDB ID: 1NGZ) as the search models. Refinements were performed using phenix.refine⁶³ in reciprocal space against experimental structure factors, followed by manual examination and rebuilding of the refined coordinates in Coot⁴⁵. Fourteen TLS groups were used that were chosen by the phenix.find_tls_groups tool⁶³. Spiperone was modeled using 2Fo-Fc map, Fo-Fc map, and polder map⁶⁴ (Supplementary Fig. 4d). Statistics for the data collection and refinement are shown in Table 1. The Ramachandran statistics analyzed using MolProbity⁶⁵ were as follows: 98.0% in the favored region, 2.0% allowed, with no outliers. Figures were prepared using Cuemol (http://www.cuemol.org/) and PyMOL (https://www.pymol.org/).

Radioligand-binding assay

The mutants were prepared using the primers listed in Supplementary Table 5. The radioligand-binding assay was performed using HEK293 cell or Sf9 cell membranes that expressed the receptor. The wild-type or a mutant D₂R was transfected with a pCAGGS plasmid into HEK293 cells using a FuGENE HD transfection reagent (10 μg of plasmid, 50 μl of FuGENE HD solution per 10-cm culture dish). The membranes were prepared as described in the “Protein expression and purification” section. The protein concentration of the membrane was determined by the bicinchoninic acid (BCA) method (Thermo Fisher Scientific) with bovine serum albumin as a standard. The membranes were stored at –80 °C until use. All the experiments were performed in triplicate (with independent expressions) in a total volume of 200 µl. The membranes were dispersed with binding assay buffer (50 mM Tris-HCl, pH 7.5, and 150 mM NaCl). Then, 0.5–5 µg of the membranes were incubated for 2 h at room temperature with [³H]-spiperone (Perkin Elmer) at concentrations of 0.31–20 nM or [³H]-raclopride (Perkin Elmer) at concentrations of 0.20–100 nM. Unifilter-96 GF/B filter plates (Perkin Elmer) were pre-soaked in 0.3% polyethyleneimine (PEI, Nacalai Tesque) for 30 min to reduce non-specific binding. Non-specific binding was determined in the presence of 100 μM spiperone (Sigma) or raclopride (Tocris). For the competition-binding assay, 2 μg of membranes were incubated for 2 h at room temperature with 30 nM of [³H]-raclopride and unlabeled eticlopride at concentrations ranging from 0.01 nM to 1.0 μM. Samples were harvested with Unifilter-96 GF/B filter plates and the unbound ligand was washed three times with distilled water using a FilterMate Cell Harvester system (Perkin Elmer). After adding 20 μl of MicroScint-20 (PerkinElmer), the bound [³H]-spiperone or [³H]-raclopride was quantified with a MicroBeta2 scintillation counter (PerkinElmer). The data were analyzed by nonlinear curve fitting using GraphPad Prism 5 software. To determine Kd value of the receptors for spiperone, we used the equation accounting for ligand depletion. Binding data are reported as the mean ± SEM.

TGFα shedding assay

The antagonist activity of spiperone for the mutant D₂Rs was determined by the TGFα shedding assays³². Briefly, a pCAGGS plasmid encoding the human wild-type or a mutant D₂R (full-length, untagged), together with pCAGGS plasmids that encoded the chimeric Gα_q/i3 subunit and alkaline phosphatase-tagged TGFα (AP-TGFα; human codon optimized), were transfected into HEK293A cells that were negative for mycoplasma contamination (MycoAlert Mycoplasma detection kit, Lonza) by using a PEI transfection reagent (PEI MAX MW 40,000; Polysciences). The chimeric Gα_q/i3 subunit comprises the Gα_q backbone and the Gα_i3-derived 6-amino acid C-terminus, and it couples with G_i-coupled D₂R, but induces a G_q-dependent TGFα shedding response³². For each 10-cm culture dish, we used 1 µg of D₂R plasmid, 0.5 µg of Gα_q/i3 plasmid, 2.5 µg of AP-TGFα plasmid and 25 µl of 1 mg/ml PEI solution. After culturing for one day at 37 °C in a 5% CO₂ incubator, the transfected cells were harvested by trypsinization, washed once with Hank’s balanced salt solution (HBSS) containing 5 mM HEPES (pH 7.4), and resuspended in 30 ml of the HBSS-containing HEPES. The cell suspension was seeded in a 96-well culture plate (“cell plate”) at a volume of 80 µl per well and incubated for 30 min in the CO₂ incubator. To determine the antagonist activity of spiperone or risperidone, cells were pretreated with 3.2-fold-titrated concentration of the antagonists (final concentrations of 32 pM–1 µM for spiperone or 320 pM–10 µM for risperidone; 10 µl per well) for 5 min and stimulated with dopamine (final concentration of 1 µM; 10 µL per well). To determine the agonist activity of dopamine, vehicle (10 µL per well) were predispensed before cell seeding and 3.2-fold-titrated concentration of dopamine (final concentrations of 1 nM–32 µM; 10 µl per well) was added to the cells. For all the experiments, the compounds were diluted in 0.01% bovine serum albumin- and HBSS-containing HEPES. After incubation with dopamine for 1 h, the cell plate was spun at 190 × g for 2 min and conditioned medium (CM; 80 μl per well) was transferred to an empty 96-well plate (“CM plate”). Alkaline phosphatase reaction solution (10 mM p-nitrophenylphosphate, 120 mM Tris-HCl, pH 9.5, 40 mM NaCl, and 10 mM MgCl₂) was dispensed into the cell plates and CM plates (80 µl). The absorbance of the plates at 405 nm was measured using a microplate reader (SpectraMax 340 PC384, Molecular Devices) before and after incubation for 1 or 2 h at room temperature. Ligand-induced AP-TGFα release was obtained by calculating AP activity in conditioned media and subtracting a vehicle-treated spontaneous AP-TGFα signal³². Using Prism 8 software (GraphPad Prism), the AP-TGFα release signals were fitted with a four-parameter sigmoidal concentration–response curve, from which EC₅₀ or IC₅₀ and E_max values were obtained. Negative logarithmic values of EC₅₀ (pEC₅₀) were used to calculate the mean and SEM.

The equilibrium dissociation constant (K_B) was calculated for each experiment performed in parallel from the IC₅₀ values (for spiperone and risperidone), an EC₅₀ value (for dopamine), a Hill slope (K, for dopamine), and the tested concentration of dopamine (A; 1 µM), as follows⁶⁶:

$$K_B=\frac{{\mathrm{IC}}_{50}}{1+{\left(\frac{A}{{\mathrm{EC}}_{50}}\right)}^K}$$ 1

The resulting K_B values were logarithmically transformed and their negative values (pK_B) were used to calculate the difference between the pK_B values (∆pK_B) for a mutant (MT) and the wild-type (WT) receptor, derived from  parallelly conducted experiments, as follows:

$$\mathrm{\Delta }p{K}_B=p{K}_B\left(\mathrm{MT}\right)-p{K}_B\left(\mathrm{WT}\right)$$ 2

Mean and SEM values of the pK_B and the ∆pK_B values were calculated.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Source data

Source Data ^{(22.5KB, xlsx)}

Author contributions

D.I. designed constructs, expressed, purified, and crystallized the receptor. D.I. and T.U. generated the antibody. D.I., T.F., and C.M. prepared microcrystals. D.I., T.F., Y.Y., T.T., A.Y., and E.N. collected data at SACLA. T.N. processed the data. K.T. contributed to the beamline operation at SACLA. Y.S. and N.N. prepared mutants. A.I. and F.M.N.K. designed, performed, and analyzed the functional assay under J.A.’s supervision. H.A. and K.T.K. performed the binding assay. T.S. designed the constructs and solved and refined the structure as well as supervised the project. D.I, S.I., and T.S. wrote the manuscript. S.I. advised T.S. All authors discussed the results and commented on the manuscript.

Data availability

The coding sequence of human D₂R is available in UniProt with accession code P14416. The protein coordinate and atomic structure factor have been deposited in the Protein Data Bank (PDB) with accession code 7DFP. The raw diffraction images have been deposited to CXIDB (https://cxidb.org/) with accession code 110. Other data are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

Competing interests

The authors declare no competing interests.

Supplementary information

Supplementary information is available for this paper at 10.1038/s41467-020-20221-0.