Structural insights into the human D1 and D2 dopamine receptor signaling complexes.

The D1- and D2-dopamine receptors (D1R and D2R), which signal through Gs and Gi, respectively, represent the principal stimulatory and inhibitory dopamine receptors in the central nervous system. D1R and D2R also represent the main therapeutic targets for Parkinson's disease, schizophrenia, and many other neuropsychiatric disorders, and insight into their signaling is essential for understanding both therapeutic and side effects of dopaminergic drugs. Here, we report four cryoelectron microscopy (cryo-EM) structures of D1R-Gs and D2R-Gi signaling complexes with selective and non-selective dopamine agonists, including two currently used anti-Parkinson's disease drugs, apomorphine and bromocriptine. These structures, together with mutagenesis studies, reveal the conserved binding mode of dopamine agonists, the unique pocket topology underlying ligand selectivity, the conformational changes in receptor activation, and potential structural determinants for G protein-coupling selectivity. These results provide both a molecular understanding of dopamine signaling and multiple structural templates for drug design targeting the dopaminergic system.

Conformational transitions and activation of the adhesion receptor CD97.

Adhesion G protein-coupled receptors (aGPCRs) are evolutionarily ancient receptors involved in a variety of physiological and pathophysiological processes. Modulators of aGPCR, particularly antagonists, hold therapeutic promise for diseases like cancer and immune and neurological disorders. Hindered by the inactive state structural information, our understanding of antagonist development and aGPCR activation faces challenges. Here, we report the cryo-electron microscopy structures of human CD97, a prototypical aGPCR that plays crucial roles in immune system, in its inactive apo and G13-bound fully active states. Compared with other family GPCRs, CD97 adopts a compact inactive conformation with a constrained ligand pocket. Activation induces significant conformational changes for both extracellular and intracellular sides, creating larger cavities for Stachel sequence binding and G13 engagement. Integrated with functional and metadynamics analyses, our study provides significant mechanistic insights into the activation and signaling of aGPCRs, paving the way for future drug discovery efforts.

GPCRs steer Gi and Gs selectivity via TM5-TM6 switches as revealed by structures of serotonin receptors.

Serotonin (or 5-hydroxytryptamine, 5-HT) is an important neurotransmitter that activates 12 different G protein-coupled receptors (GPCRs) through selective coupling of Gs, Gi, or Gq proteins. The structural basis for G protein subtype selectivity by these GPCRs remains elusive. Here, we report the structures of the serotonin receptors 5-HT4, 5-HT6, and 5-HT7 with Gs, and 5-HT4 with Gi1. The structures reveal that transmembrane helices TM5 and TM6 alternate lengths as a macro-switch to determine receptor's selectivity for Gs and Gi, respectively. We find that the macro-switch by the TM5-TM6 length is shared by class A GPCR-G protein structures. Furthermore, we discover specific residues within TM5 and TM6 that function as micro-switches to form specific interactions with Gs or Gi. Together, these results present a common mechanism of Gs versus Gi protein coupling selectivity or promiscuity by class A GPCRs and extend the basis of ligand recognition at serotonin receptors.

Structures of the human dopamine D3 receptor-Gi complexes.

The dopamine system, including five dopamine receptors (D1R-D5R), plays essential roles in the central nervous system (CNS), and ligands that activate dopamine receptors have been used to treat many neuropsychiatric disorders. Here, we report two cryo-EM structures of human D3R in complex with an inhibitory G protein and bound to the D3R-selective agonists PD128907 and pramipexole, the latter of which is used to treat patients with Parkinson's disease. The structures reveal agonist binding modes distinct from the antagonist-bound D3R structure and conformational signatures for ligand-induced receptor activation. Mutagenesis and homology modeling illuminate determinants of ligand specificity across dopamine receptors and the mechanisms for Gi protein coupling. Collectively our work reveals the basis of agonist binding and ligand-induced receptor activation and provides structural templates for designing specific ligands to treat CNS diseases targeting the dopaminergic system.

Molecular Basis for Hormone Recognition and Activation of Corticotropin-Releasing Factor Receptors.

Corticotropin-releasing factor (CRF) and the three related peptides urocortins 1-3 (UCN1-UCN3) are endocrine hormones that control the stress responses by activating CRF1R and CRF2R, two members of class B G-protein-coupled receptors (GPCRs). Here, we present two cryoelectron microscopy (cryo-EM) structures of UCN1-bound CRF1R and CRF2R with the stimulatory G protein. In both structures, UCN1 adopts a single straight helix with its N terminus dipped into the receptor transmembrane bundle. Although the peptide-binding residues in CRF1R and CRF2R are different from other members of class B GPCRs, the residues involved in receptor activation and G protein coupling are conserved. In addition, both structures reveal bound cholesterol molecules to the receptor transmembrane helices. Our structures define the basis of ligand-binding specificity in the CRF receptor-hormone system, establish a common mechanism of class B GPCR activation and G protein coupling, and provide a paradigm for studying membrane protein-lipid interactions for class B GPCRs.

Structures of full-length glycoprotein hormone receptor signalling complexes.

Luteinizing hormone and chorionic gonadotropin are glycoprotein hormones that are related to follicle-stimulating hormone and thyroid-stimulating hormone1,2. Luteinizing hormone and chorionic gonadotropin are essential to human reproduction and are important therapeutic drugs3-6. They activate the same G-protein-coupled receptor, luteinizing hormone-choriogonadotropin receptor (LHCGR), by binding to the large extracellular domain3. Here we report four cryo-electron microscopy structures of LHCGR: two structures of the wild-type receptor in the inactive and active states; and two structures of the constitutively active mutated receptor. The active structures are bound to chorionic gonadotropin and the stimulatory G protein (Gs), and one of the structures also contains Org43553, an allosteric agonist7. The structures reveal a distinct 'push-and-pull' mechanism of receptor activation, in which the extracellular domain is pushed by the bound hormone and pulled by the extended hinge loop next to the transmembrane domain. A highly conserved 10-residue fragment (P10) from the hinge C-terminal loop at the interface between the extracellular domain and the transmembrane domain functions as a tethered agonist to induce conformational changes in the transmembrane domain and G-protein coupling. Org43553 binds to a pocket of the transmembrane domain and interacts directly with P10, which further stabilizes the active conformation. Together, these structures provide a common model for understanding the signalling of glycoprotein hormone receptors and a basis for drug discovery for endocrine diseases.

Structural basis of GABAB receptor-Gi protein coupling.

G-protein-coupled receptors (GPCRs) have central roles in intercellular communication1,2. Structural studies have revealed how GPCRs can activate G proteins. However, whether this mechanism is conserved among all classes of GPCR remains unknown. Here we report the structure of the class-C heterodimeric GABAB receptor, which is activated by the inhibitory transmitter GABA, in its active form complexed with Gi1 protein. We found that a single G protein interacts with the GB2 subunit of the GABAB receptor at a site that mainly involves intracellular loop 2 on the side of the transmembrane domain. This is in contrast to the G protein binding in a central cavity, as has been observed with other classes of GPCR. This binding mode results from the active form of the transmembrane domain of this GABAB receptor being different from that of other GPCRs, as it shows no outside movement of transmembrane helix 6. Our work also provides details of the inter- and intra-subunit changes that link agonist binding to G-protein activation in this heterodimeric complex.

Structures of the glucocorticoid-bound adhesion receptor GPR97-Go complex.

Adhesion G-protein-coupled receptors (GPCRs) are a major family of GPCRs, but limited knowledge of their ligand regulation or structure is available1-3. Here we report that glucocorticoid stress hormones activate adhesion G-protein-coupled receptor G3 (ADGRG3; also known as GPR97)4-6, a prototypical adhesion GPCR. The cryo-electron microscopy structures of GPR97-Go complexes bound to the anti-inflammatory drug beclomethasone or the steroid hormone cortisol revealed that glucocorticoids bind to a pocket within the transmembrane domain. The steroidal core of glucocorticoids is packed against the 'toggle switch' residue W6.53, which senses the binding of a ligand and induces activation of the receptor. Active GPR97 uses a quaternary core and HLY motif to fasten the seven-transmembrane bundle and to mediate G protein coupling. The cytoplasmic side of GPR97 has an open cavity, where all three intracellular loops interact with the Go protein, contributing to the high basal activity of GRP97. Palmitoylation at the cytosolic tail of the Go protein was found to be essential for efficient engagement with GPR97 but is not observed in other solved GPCR complex structures. Our work provides a structural basis for ligand binding to the seven-transmembrane domain of an adhesion GPCR and subsequent G protein coupling.

Structural insights into the lipid and ligand regulation of serotonin receptors.

Serotonin, or 5-hydroxytryptamine (5-HT), is an important neurotransmitter1,2 that activates the largest subtype family of G-protein-coupled receptors3. Drugs that target 5-HT1A, 5-HT1D, 5-HT1E and other 5-HT receptors are used to treat numerous disorders4. 5-HT receptors have high levels of basal activity and are subject to regulation by lipids, but the structural basis for the lipid regulation and basal activation of these receptors and the pan-agonism of 5-HT remains unclear. Here we report five structures of 5-HT receptor-G-protein complexes: 5-HT1A in the apo state, bound to 5-HT or bound to the antipsychotic drug aripiprazole; 5-HT1D bound to 5-HT; and 5-HT1E in complex with a 5-HT1E- and 5-HT1F-selective agonist, BRL-54443. Notably, the phospholipid phosphatidylinositol 4-phosphate is present at the G-protein-5-HT1A interface, and is able to increase 5-HT1A-mediated G-protein activity. The receptor transmembrane domain is surrounded by cholesterol molecules-particularly in the case of 5-HT1A, in which cholesterol molecules are directly involved in shaping the ligand-binding pocket that determines the specificity for aripiprazol. Within the ligand-binding pocket of apo-5-HT1A are structured water molecules that mimic 5-HT to activate the receptor. Together, our results address a long-standing question of how lipids and water molecules regulate G-protein-coupled receptors, reveal how 5-HT acts as a pan-agonist, and identify the determinants of drug recognition in 5-HT receptors.

Structural basis of GPBAR activation and bile acid recognition.

The G-protein-coupled bile acid receptor (GPBAR) conveys the cross-membrane signalling of a vast variety of bile acids and is a signalling hub in the liver-bile acid-microbiota-metabolism axis1-3. Here we report the cryo-electron microscopy structures of GPBAR-Gs complexes stabilized by either the high-affinity P3954 or the semisynthesized bile acid derivative INT-7771,3 at 3 Å resolution. These structures revealed a large oval pocket that contains several polar groups positioned to accommodate the amphipathic cholic core of bile acids, a fingerprint of key residues to recognize diverse bile acids in the orthosteric site, a putative second bile acid-binding site with allosteric properties and structural features that contribute to bias properties. Moreover, GPBAR undertakes an atypical mode of activation and G protein coupling that features a different set of key residues connecting the ligand-binding pocket to the Gs-coupling site, and a specific interaction motif that is localized in intracellular loop 3. Overall, our study not only reveals unique structural features of GPBAR that are involved in bile acid recognition and allosteric effects, but also suggests the presence of distinct connecting mechanisms between the ligand-binding pocket and the G-protein-binding site in the G-protein-coupled receptor superfamily.

Structural basis and molecular mechanism of biased GPBAR signaling in regulating NSCLC cell growth via YAP activity.

The G protein-coupled bile acid receptor (GPBAR) is the membrane receptor for bile acids and a driving force of the liver-bile acid-microbiota-organ axis to regulate metabolism and other pathophysiological processes. Although GPBAR is an important therapeutic target for a spectrum of metabolic and neurodegenerative diseases, its activation has also been found to be linked to carcinogenesis, leading to potential side effects. Here, via functional screening, we found that two specific GPBAR agonists, R399 and INT-777, demonstrated strikingly different regulatory effects on the growth and apoptosis of non-small cell lung cancer (NSCLC) cells both in vitro and in vivo. Further mechanistic investigation showed that R399-induced GPBAR activation displayed an obvious bias for ß-arrestin 1 signaling, thus promoting YAP signaling activation to stimulate cell proliferation. Conversely, INT-777 preferentially activated GPBAR-Gs signaling, thus inactivating YAP to inhibit cell proliferation and induce apoptosis. Phosphorylation of GPBAR by GRK2 at S310/S321/S323/S324 sites contributed to R399-induced GPBAR-ß-arrestin 1 association. The cryoelectron microscopy (cryo-EM) structure of the R399-bound GPBAR-Gs complex enabled us to identify key interaction residues and pivotal conformational changes in GPBAR responsible for the arrestin signaling bias and cancer cell proliferation. In summary, we demonstrate that different agonists can regulate distinct functions of cell growth and apoptosis through biased GPBAR signaling and control of YAP activity in a NSCLC cell model. The delineated mechanism and structural basis may facilitate the rational design of GPBAR-targeting drugs with both metabolic and anticancer benefits.

Identification and mechanism of G protein-biased ligands for chemokine receptor CCR1.

Biased signaling of G protein-coupled receptors describes an ability of different ligands that preferentially activate an alternative downstream signaling pathway. In this work, we identified and characterized different N-terminal truncations of endogenous chemokine CCL15 as balanced or biased agonists targeting CCR1, and presented three cryogenic-electron microscopy structures of the CCR1-Gi complex in the ligand-free form or bound to different CCL15 truncations with a resolution of 2.6-2.9 Å, illustrating the structural basis of natural biased signaling that initiates an inflammation response. Complemented with pharmacological and computational studies, these structures revealed it was the conformational change of Tyr291 (Y2917.43) in CCR1 that triggered its polar network rearrangement in the orthosteric binding pocket and allosterically regulated the activation of ß-arrestin signaling. Our structure of CCL15-bound CCR1 also exhibited a critical site for ligand binding distinct from many other chemokine-receptor complexes, providing new insights into the mode of chemokine recognition.

Constitutive signal bias mediated by the human GHRHR splice variant 1.

Alternative splicing of G protein-coupled receptors has been observed, but their functions are largely unknown. Here, we report that a splice variant (SV1) of the human growth hormone-releasing hormone receptor (GHRHR) is capable of transducing biased signal. Differing only at the receptor N terminus, GHRHR predominantly activates Gs while SV1 selectively couples to ß-arrestins. Based on the cryogenic electron microscopy structures of SV1 in the apo state or GHRH-bound state in complex with the Gs protein, molecular dynamics simulations reveal that the N termini of GHRHR and SV1 differentiate the downstream signaling pathways, Gs versus ß-arrestins. As suggested by mutagenesis and functional studies, it appears that GHRH-elicited signal bias toward ß-arrestin recruitment is constitutively mediated by SV1. The level of SV1 expression in prostate cancer cells is also positively correlated with ERK1/2 phosphorylation but negatively correlated with cAMP response. Our findings imply that constitutive signal bias may be a mechanism that ensures cancer cell proliferation.

RbpA and σB association regulates polyphosphate levels to modulate mycobacterial isoniazid-tolerance.

To facilitate survival under drug stresses, a small population of Mycobacterium tuberculosis can tolerate bactericidal concentrations of drugs without genetic mutations. These drug-tolerant mycobacteria can be induced by environmental stresses and contribute to recalcitrant infections. However, mechanisms underlying the development of drug-tolerant mycobacteria remain obscure. Herein, we characterized a regulatory pathway which is important for the tolerance to isoniazid (INH) in Mycobacterium smegmatis. We found that the RNA polymerase binding protein RbpA associates with the stress response sigma factor σB , to activate the transcription of ppk1, the gene encoding polyphosphate kinase. Subsequently, intracellular levels of inorganic polyphosphate increase to promote INH-tolerant mycobacteria. Interestingly, σB and ppk1 expression varied proportionately in mycobacterial populations and positively correlated with tolerance to INH in individual mycobacteria. Moreover, sigB and ppk1 transcription are both induced upon nutrient depletion, a condition that stimulates the formation of INH-tolerant mycobacteria. Over-expression of ppk1 in rbpA knockdown or sigB deleted strains successfully restored the number of INH-tolerant mycobacteria under both normal growth and nutrient starved conditions. These data suggest that RbpA and σB regulate ppk1 expression to control drug tolerance both during the logarithmic growth phase and under the nutrition starved conditions.

Association of ω with the C-Terminal Region of the ß' Subunit Is Essential for Assembly of RNA Polymerase in Mycobacterium tuberculosis.

The ω subunit is the smallest subunit of bacterial RNA polymerase (RNAP). Although homologs of ω are essential in both eukaryotes and archaea, this subunit has been known to be dispensable for RNAP in Escherichia coli and in other bacteria. In this study, we characterized an indispensable role of the ω subunit in Mycobacterium tuberculosis Unlike the well-studied E. coli RNAP, the M. tuberculosis RNAP core enzyme cannot be functionally assembled in the absence of the ω subunit. Importantly, substitution of M. tuberculosis ω with ω subunits from E. coli or Thermus thermophilus cannot restore the assembly of M. tuberculosis RNAP. Furthermore, by replacing different regions in M. tuberculosis ω with the corresponding regions from E. coli ω, we found a nonconserved loop region in M. tuberculosis ω essential for its function in RNAP assembly. From RNAP structures, we noticed that the location of the C-terminal region of the ß' subunit (ß'CTD) in M. tuberculosis RNAP but not in E. coli or T. thermophilus RNAP is close to the ω loop region. Deletion of this ß'CTD in M. tuberculosis RNAP destabilized the binding of M. tuberculosis ω on RNAP and compromised M. tuberculosis core assembly, suggesting that these two regions may function together to play a role in ω-dependent RNAP assembly in M. tuberculosis Sequence alignment of the ω loop and the ß'CTD regions suggests that the essential role of ω is probably restricted to mycobacteria. Together, our study characterized an essential role of M. tuberculosis ω and highlighted the importance of the ω loop region in M. tuberculosis RNAP assembly.IMPORTANCE DNA-dependent RNA polymerase (RNAP), which consists of a multisubunit core enzyme (α2ßß'ω) and a dissociable σ subunit, is the only enzyme in charge of transcription in bacteria. As the smallest subunit, the roles of ω remain the least well studied. In Escherichia coli and some other bacteria, the ω subunit is known to be nonessential for RNAP. In this study, we revealed an essential role of the ω subunit for RNAP assembly in the human pathogen Mycobacterium tuberculosis, and a mycobacterium-specific ω loop that plays a role in this function was also characterized. Our study provides fresh insights for further characterizing the roles of bacterial ω subunit.

Characterization of a Minimal Type of Promoter Containing the -10 Element and a Guanine at the -14 or -13 Position in Mycobacteria.

Three key promoter elements, i.e., -10, -35, and T-15G-14N, are recognized by the σ subunit of RNA polymerase. Among them, promoters with the -10 element and either -35 or T-15G-14N are known to initiate transcription efficiently, but recent systematic analyses have identified a large group of promoters in Mycobacterium tuberculosis that contain only a -10 consensus. How these promoters initiate transcription remains poorly understood. Here, we show that promoters containing the -10 element and an upstream G located at the -14 or -13 position can successfully initiate transcription in mycobacteria. Importantly, this new type of promoter is active in the absence of other promoter consensuses, suggesting that it is a minimal promoter type. Mutation of the upstream G in promoters decreased the efficiencies of their binding with RNA polymerase and their abilities to initiate transcription in both in vitro and in vivo analyses. A glutamic acid in σ region 3.0 is essential for recognizing G-14 and G-13 and is conserved in both principal and principal-like σ factors in mycobacteria, indicating that recognition of this minimal type of promoter might be a common mechanism for transcription initiation. Consistently, more than 70% of the identified promoters in M. tuberculosis contained G-14 or G-13 upstream of the conserved -10 element, and thousands of promoters in representative mycobacterial species have been predicted using the -10 consensus and G-14 or G-13 Altogether, our study presents a universal mechanism for transcription initiation from a minimal promoter in mycobacteria, which might also be applicable to other bacteria.IMPORTANCE In contrast to the detailed information for recognizing classic promoters in the model organism Escherichia coli, very little is known about how transcription is initiated in the human pathogen Mycobacterium tuberculosis In this study, we characterized a new type of promoter in mycobacteria that requires only a -10 consensus and an upstream G-14 or G-13 Residues important for recognizing the -10 element and the upstream G are conserved in σA and σB from mycobacterial species. According to such features, thousands of promoters in mycobacteria can be predicted using the -10 consensus and G-14 or G-13, which suggests that transcription from this new type of promoter might be widespread. Our findings provide insightful information for characterizing promoters in mycobacteria.

σ(E) -dependent activation of RbpA controls transcription of the furA-katG operon in response to oxidative stress in mycobacteria.

Mycobacterium tuberculosis adopts various strategies to cope with oxidative stress during infection. Transcriptional regulators, including σ factors, make important contributions to this stress response, but how these proteins cooperate with each other is largely unknown. In this study, the role of RbpA and its cooperation with σ factors in response to oxidative stress are investigated. Knock down expression of rbpA in Mycobacterium smegmatis attenuated bacterial survival in the presence of H2 O2 . Additionally, transcription of the rbpA gene was induced by H2 O2 in a σ(E) -dependent manner. After induction, RbpA interacts with the principal sigma factor, σ(A) , to control the transcription of furA-katG operon, which encodes an H2 O2 scavenging enzyme. Moreover, this regulation is responsible for the role of σ(E) in oxidative response because bacterial survival was attenuated and transcription of the furA-katG operon was down-regulated with H2 O2 treatment in sigE deletion mutant (ΔsigE), and over-expression of RbpA in ΔsigE strain restored all of these phenotypes. Taken together, our study first illustrated a mechanism for σ(E) in response to oxidative stress through regulation of rbpA transcription. This study was also the first to demonstrate that RbpA is required for the full response to oxidative stress by cooperating with the principal σ(A) .

Structural basis of tethered agonism and G protein coupling of protease-activated receptors.

Protease-activated receptors (PARs) are a unique group within the G protein-coupled receptor superfamily, orchestrating cellular responses to extracellular proteases via enzymatic cleavage, which triggers intracellular signaling pathways. Protease-activated receptor 1 (PAR1) is a key member of this family and is recognized as a critical pharmacological target for managing thrombotic disorders. In this study, we present cryo-electron microscopy structures of PAR1 in its activated state, induced by its natural tethered agonist (TA), in complex with two distinct downstream proteins, the Gq and Gi heterotrimers, respectively. The TA peptide is positioned within a surface pocket, prompting PAR1 activation through notable conformational shifts. Contrary to the typical receptor activation that involves the outward movement of transmembrane helix 6 (TM6), PAR1 activation is characterized by the simultaneous downward shift of TM6 and TM7, coupled with the rotation of a group of aromatic residues. This results in the displacement of an intracellular anion, creating space for downstream G protein binding. Our findings delineate the TA recognition pattern and highlight a distinct role of the second extracellular loop in forming ß-sheets with TA within the PAR family, a feature not observed in other TA-activated receptors. Moreover, the nuanced differences in the interactions between intracellular loops 2/3 and the Gα subunit of different G proteins are crucial for determining the specificity of G protein coupling. These insights contribute to our understanding of the ligand binding and activation mechanisms of PARs, illuminating the basis for PAR1's versatility in G protein coupling.

Molecular Determinant Underlying Selective Coupling of Primary G-Protein by Class A GPCRs.

G-protein-coupled receptors (GPCRs) transmit downstream signals predominantly via G-protein pathways. However, the conformational basis of selective coupling of primary G-protein remains elusive. Histamine receptors H2R and H3R couple with Gs- or Gi-proteins respectively. Here, three cryo-EM structures of H2R-Gs and H3R-Gi complexes are presented at a global resolution of 2.6-2.7 Å. These structures reveal the unique binding pose for endogenous histamine in H3R, wherein the amino group interacts with E2065.46 of H3R instead of the conserved D1143.32 of other aminergic receptors. Furthermore, comparative analysis of the H2R-Gs and H3R-Gi complexes reveals that the structural geometry of TM5/TM6 determines the primary G-protein selectivity in histamine receptors. Machine learning (ML)-based structuromic profiling and functional analysis of class A GPCR-G-protein complexes illustrate that TM5 length, TM5 tilt, and TM6 outward movement are key determinants of the Gs and Gi/o selectivity among the whole Class A family. Collectively, the findings uncover the common structural geometry within class A GPCRs that determines the primary Gs- and Gi/o-coupling selectivity.

Structural genomics of the human dopamine receptor system.

The dopaminergic system, including five dopamine receptors (D1R to D5R), plays essential roles in the central nervous system (CNS); and ligands that activate dopamine receptors have been used to treat many neuropsychiatric disorders, including Parkinson's Disease (PD) and schizophrenia. Here, we report cryo-EM structures of all five subtypes of human dopamine receptors in complex with G protein and bound to the pan-agonist, rotigotine, which is used to treat PD and restless legs syndrome. The structures reveal the basis of rotigotine recognition in different dopamine receptors. Structural analysis together with functional assays illuminate determinants of ligand polypharmacology and selectivity. The structures also uncover the mechanisms of dopamine receptor activation, unique structural features among the five receptor subtypes, and the basis of G protein coupling specificity. Our work provides a comprehensive set of structural templates for the rational design of specific ligands to treat CNS diseases targeting the dopaminergic system.