Selective activation of the -opioid receptor (DOP) has great potential for the treatment of chronic pain, benefitting from ancillary anxiolytic and antidepressant-like effects. Moreover, DOP agonists show reduced adverse effects as compared to -opioid receptor (MOP) agonists that are in the spotlight of the current "opioid crisis." Here, we report the first crystal structures of the DOP in an activated state, in complex with two relevant and structurally diverse agonists: the potent opioid agonist peptide KGCHM07 and the small-molecule agonist DPI-287 at 2.8 and 3.3 Å resolution, respectively. Our study identifies key determinants for agonist recognition, receptor activation, and DOP selectivity, revealing crucial differences between both agonist scaffolds. Our findings provide the first investigation into atomic-scale agonist binding at the DOP, supported by site-directed mutagenesis and pharmacological characterization. These structures will underpin the future structure-based development of DOP agonists for an improved pain treatment with fewer adverse effects. RESULTS Activation-related changes in the DOPBoth agonist-bound structures are in an activated state. Unless otherwise indicated, we will use the higher-resolution BRIL-DOP-KGCHM07 structure for comparison with previously published inactive-state antagonist-bound DOP structures [Protein Data Bank (PDB) 4N6H and 4RWD] (16, 17) and with active-state structures of the MOP (PDB 5C1M and 6DDF) (18,20) and KOP (PDB 6B73) (19). First, the agonist-bound DOP structures display large outward movements of the intracellular parts of helices V (4.5 Å) and VI (9.4 to 11.2 Å), and a 3.9 Å inward movement of helix VII ( Fig. 2A), which is a common feature of the active conformational states of GPCRs (21). The shift of helix VII at the level of residue N314 7.49 [superscripts according to the Ballesteros and Weinstein numbering (22)] (Fig. 3A), which leads to a collapse of the allosteric sodium-binding pocket in active-state GPCR structures (23), is even more pronounced in the determined DOP structures as compared to the active MOP and KOP (Fig. 3B and fig. S1). However, this greater shift of N314 7.49 in the DOP might be affected by three mutations in the sodium-binding pocket (N90 2.45 S, D95 2.50 G, N131 3.35 S) that were introduced during construct design. The activation-related outward movement of helix VI at the level of residue F270 6.44 is greater in the agonist-bound DOP than in the MOP and KOP. On the contrary, the very tips of helix VI (at position 6.28 as a reference) are more tilted by 4 to 6 Å in the active-state MOP and KOP ( fig. S1), likely due to the bound G protein or nanobody, respectively, pushing helix VI tips further outward (24). Elucidating the active -opioid receptor crystal structure with peptide and smallmolecule agonists. Sci. Adv. 5, eaax9115 (2019).

Background:The N111G and N111W mutations make the AT 1 receptor constitutively active and inactivable, respectively. Results: The orientation and interactions of D74 2.50 are influenced by the residue at position 111 3.35 . Conclusion: H-bond formation between D742.50 and N46 1.50 is critical for AT 1 receptor activation. Significance: This novel molecular switch could be involved in the GPCR activation mechanism as it involves highly conserved residues D 2.50 and N 1.50 .

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Background: The N111G and D74N mutations bias the AT 1 receptor for the G q/11 and ␤-arrestin pathways, respectively. Results: Structural rearrangements of theAT 1 receptor are induced by the N111G mutation and AngII. Conclusion: Activation of the G q/11 and ␤-arrestin pathways is associated with a decreased and increased stability, respectively, of the ground state of the receptor. Significance: Distinct conformations of AT 1 receptor are associated with distinct pathways.

The octapeptide angiotensin II (AngII) exerts a variety of cardiovascular effects through the activation of the AngII type 1 receptor (AT 1 ), a G protein-coupled receptor. The AT 1 receptor engages and activates several signaling pathways, including heterotrimeric G proteins G q and G 12 , as well as the extracellular signal-regulated kinases (ERK) 1/2 pathway. Additionally, following stimulation, barrestin is recruited to the AT 1 receptor, leading to receptor desensitization. It is increasingly recognized that specific ligands selectively bind and favor the activation of some signaling pathways over others, a concept termed ligand bias or functional selectivity. A better understanding of the molecular basis of functional selectivity may lead to the development of better therapeutics with fewer adverse effects. In the present study, we developed assays allowing the measurement of six different signaling modalities of the AT 1 receptor. Using a series of AngII peptide analogs that were modified in positions 1, 4, and 8, we sought to better characterize the molecular determinants of AngII that underlie functional selectivity of the AT 1 receptor in human embryonic kidney 293 cells. The results reveal that position 1 of AngII does not confer functional selectivity, whereas position 4 confers a bias toward ERK signaling over G q signaling, and position 8 confers a bias toward barrestin recruitment over ERK activation and G q signaling. Interestingly, the analogs modified in position 8 were also partial agonists of the protein kinase C (PKC)-dependent ERK pathway via atypical PKC isoforms PKCz and PKCi.