Mechanism of dopamine binding and allosteric modulation of the human D1 dopamine receptor

Abstract

Dear Editor, Dopamine acts as an essential neurotransmitter whose signaling is conducted through five G protein-coupled receptors (GPCRs), dopamine D1 to D5 receptors (DRD1–DRD5). The D1like receptors, comprising DRD1 and DRD5, primarily couple to the Gs family of G proteins to activate adenylyl cyclase and induce cAMP production. DRD1 is the most abundantly expressed dopamine receptor in the CNS. It is the central receptor mediating excitatory dopamine signaling in multiple dopaminergic pathways. Dysregulation of DRD1 signaling has been directly linked to Parkinson’s disease (PD), schizophrenia, and drug abuse. Due to its fundamental functions in human diseases, DRD1 has long been the subject of intensive drug development efforts toward the treatment of neuropsychiatric diseases. A majority of DRD1 agonists, including the SKF compounds, targets the orthosteric pocket of DRD1, but none has passed clinical trials for neuropsychiatric symptoms to date. GPCR positive allosteric modulators (PAMs) have been proposed to provide unique advantages over orthosteric agonists including greater receptor subtype selectivity, saturable therapeutic effects and the ability to maintain spatial and temporal patterns of endogenous dopamine signaling, which collectively may lead to reduced side effects. Multiple groups have reported that DRD1 PAMs, such as LY3154207, CID2886111, and DETQ, stimulate DRD1 signaling. With ongoing clinical investigation, DRD1 PAMs may offer new therapeutic opportunities for PD. Despite significant efforts, the structural basis of DRD1 ligand binding and allosteric regulation properties remains poorly understood, which has significantly impeded the discovery of potential DRD1-selective drugs with minimal side effects. Here, we report two structures of DRD1–Gs complexes activated by the endogenous ligand, dopamine, and a synthetic agonist, SKF81297, both in the presence of LY3154207, respectively (Fig. 1a, b). We used an engineered miniGs (miniGαs_DN) to assemble DRD1–Gs signaling complexes (Supplementary information, Fig. S1). To obtain stable DRD1–Gs complexes for structural studies, we coexpressed wild-type (WT) human DRD1, miniGαs_DN, rat Gβ1 and bovine Gγ2 in Sf9 insect cells. The complexes were prepared as described in Supplementary information and purified to homogeneity for single-particle cryo-EM studies (Supplementary information, Fig. S2). Two different DRD1 PAMs, CID2886111 and LY3154207, which bind to different sites on DRD1 as shown by prior studies, were added to further stabilize the dopaminebound DRD1–Gs complex. The structures of DRD1–Gs complexed with dopamine/LY3154207 and SKF81297/LY3154207 were determined at a global resolution of 3.2 Å and 3.0 Å, respectively (Fig. 1a, b; Supplementary information, Fig. S3 and Table S1). The relatively high-resolution maps allowed us to unambiguously model most portions of DRD1 from S21 to Y348, the Gs heterotrimer, the orthosteric agonists, and the nanobody Nb35 (Supplementary information, Figs. S4 and S5a). In addition, in the SKF81297-bound DRD1 structure, clear density for LY3154207 was observed above intracellular loop 2 (ICL2) (Fig. 1b; Supplementary information, Figs. S4 and S5a), allowing us to define the binding pose of LY3154207 and the allosteric site. In the dopamine-bound DRD1 structure, the binding pose of LY3154207 can be defined (Fig. 1b; Supplementary information, Figs. S4 and S5a), but no density was observed for CID2886111. The overall structures of LY3154207-bound DRD1 with dopamine and SKF81297 are quite similar, with a root mean square deviation (RMSD) value of 0.6 Å for the main chain Cα atoms. However, the orthosteric binding pocket (OBP) of SKF81297 is narrower compared to that of dopamine (Fig. 1c; Supplementary information, Fig. S5b). In both structures, DRD1 adopts a canonical seven-helical transmembrane domain (TMD), the ligand-binding pockets are located at the extracellular part of the TMD and the Gprotein coupling interface is located at the cytoplasmic side (Fig. 1a, b). In the dopamine-bound DRD1 structure, dopamine occupies the OBP composed of residues from TM3, TM5–7 and capped by extracellular loop 2 (ECL2) (Fig. 1d; Supplementary information, Fig. S5a). The primary amine group forms direct ionic contacts with the carboxylate group of D103 (superscript based on Ballesteros-Weinstein numbering rules of GPCRs), which is highly conserved in aminergic GPCRs. Such interaction is further enhanced by hydrogen bond interactions among D103, S107, and W321 (Fig. 1d). The catechol moiety forms hydrogen bond interactions with S198 and S202 from TM5 and N292 from TM6 (Fig. 1d). These findings agree well with the mutational results from previous studies reporting that S198 and S202 are pivotal for dopamine binding. In addition to the polar interaction network, hydrophobic residues I104, L190, W285, F288, F289 and V317 form extensive hydrophobic interactions with dopamine to further stabilize the dopamine binding (Fig. 1d). For SKF81297, although it shares the same catechol moiety as dopamine and its benzazepine ring overlaps well with the phenylethylamine moiety of dopamine, the conformation of the catechol group of SKF81297 is slightly different from that of dopamine (Fig. 1d; Supplementary information, Fig. S5c). As a result, the catechol moiety of SKF81297 forms hydrogen bonds with S198 but not S202 (Supplementary information, Fig. S5c). The extra benzene group of SKF81297 occupies a small extended binding pocket (EBP) at the extracellular vestibule formed by residues V100, L190, S188 and F313 (Supplementary information, Fig. S5c), which contributes to its higher affinity to DRD1 than dopamine. Interestingly, the side chain of D187 points towards polar residues K81 and D314 in the SKF81297-bound DRD1 but not in the dopamine-bound DRD1, forming a potential polar interaction network (Supplementary information, Fig. S5d). The clustering of the side chains of these three polar residues leads to a narrower ligand-binding pocket for SKF81297 than that for dopamine. To validate the structural findings in dopamine-binding pockets in DRD1, we mutated residues near the pockets and analyzed the expression levels and cAMP accumulation of these DRD1 mutants