Ligand recognition and allosteric regulation of DRD1-Gs signaling complexes.

Dopamine receptors, including D1- and D2-like receptors, are important therapeutic targets in a variety of neurological syndromes, as well as cardiovascular and kidney diseases. Here, we present five cryoelectron microscopy (cryo-EM) structures of the dopamine D1 receptor (DRD1) coupled to Gs heterotrimer in complex with three catechol-based agonists, a non-catechol agonist, and a positive allosteric modulator for endogenous dopamine. These structures revealed that a polar interaction network is essential for catecholamine-like agonist recognition, whereas specific motifs in the extended binding pocket were responsible for discriminating D1- from D2-like receptors. Moreover, allosteric binding at a distinct inner surface pocket improved the activity of DRD1 by stabilizing endogenous dopamine interaction at the orthosteric site. DRD1-Gs interface revealed key features that serve as determinants for G protein coupling. Together, our study provides a structural understanding of the ligand recognition, allosteric regulation, and G protein coupling mechanisms of DRD1.

Tethered peptide activation mechanism of the adhesion GPCRs ADGRG2 and ADGRG4.

Adhesion G protein-coupled receptors (aGPCRs) constitute an evolutionarily ancient family of receptors that often undergo autoproteolysis to produce α and ß subunits1-3. A tethered agonism mediated by the 'Stachel sequence' of the ß subunit has been proposed to have central roles in aGPCR activation4-6. Here we present three cryo-electron microscopy structures of aGPCRs coupled to the Gs heterotrimer. Two of these aGPCRs are activated by tethered Stachel sequences-the ADGRG2-ß-Gs complex and the ADGRG4-ß-Gs complex (in which ß indicates the ß subunit of the aGPCR)-and the other is the full-length ADGRG2 in complex with the exogenous ADGRG2 Stachel-sequence-derived peptide agonist IP15 (ADGRG2(FL)-IP15-Gs). The Stachel sequences of both ADGRG2-ß and ADGRG4-ß assume a U shape and insert deeply into the seven-transmembrane bundles. Constituting the FXφφφXφ motif (in which φ represents a hydrophobic residue), five residues of ADGRG2-ß or ADGRG4-ß extend like fingers to mediate binding to the seven-transmembrane domain and activation of the receptor. The structure of the ADGRG2(FL)-IP15-Gs complex reveals the structural basis for the improved binding affinity of IP15 compared with VPM-p15 and indicates that rational design of peptidic agonists could be achieved by exploiting aGPCR-ß structures. By converting the 'finger residues' to acidic residues, we develop a method to generate peptidic antagonists towards several aGPCRs. Collectively, our study provides structural and biochemical insights into the tethered activation mechanism of aGPCRs.

Structural basis of ligand recognition and activation of the histamine receptor family.

Histamine is a biogenic amine that is critical in various physiological and pathophysiological processes, including but not limited to allergic reactions, wakefulness, gastric acid secretion and neurotransmission. Here, we determine 9 cryo-electron microscopy (cryo-EM) structures of the 4 histamine receptors in complex with four different G protein subtypes, with endogenous or synthetic agonists bound. Inside the ligand pocket, we identify key motifs for the recognition of histamine, the distinct binding orientations of histamine and three subpockets that facilitate the design of specific ligands. In addition, we also identify key residues responsible for the selectivity of immethridine. Moreover, we reveal distinct structural features as determinants of Gq vs. Gs or Gs vs. Gi coupling differences among the histamine receptors. Our study provides a structural framework for understanding the ligand recognition and G protein coupling of all 4 histamine receptors, which may facilitate the rational design of ligands targeting these receptors.