Structure of endothelin ETB receptor-Gi complex in a conformation stabilized by unique NPxxL motif

Abstract

Endothelin type B receptor (ET_BR) plays a crucial role in regulating blood pressure and humoral homeostasis, making it an important therapeutic target for related diseases. ET_BR activation by the endogenous peptide hormones endothelin (ET)-1-3 stimulates several signaling pathways, including G_s, G_i/o, G_q/11, G_12/13, and β-arrestin. Although the conserved NPxxY motif in transmembrane helix 7 (TM7) is important during GPCR activation, ET_BR possesses the lesser known NPxxL motif. In this study, we present the cryo-EM structure of the ET_BR-G_i complex, complemented by MD simulations and functional studies. These investigations reveal an unusual movement of TM7 to the intracellular side during ET_BR activation and the essential roles of the diverse NPxxL motif in stabilizing the active conformation of ET_BR and organizing the assembly of the binding pocket for the α5 helix of G_i protein. These findings enhance our understanding of the interactions between GPCRs and G proteins, thereby advancing the development of therapeutic strategies.

Fig. 1. Cryo-EM structure of the ET-1–ET_BR–DNG_i complex.

a Cryo-EM density map of the ET-1–ET_BR–DNG_i–scFv16 complex. Green: ET_BR, salmon: ET-1, magenta: DNGα_i Ras-like domain, blue: Gβ, orange: Gγ, and gray: scFv16. The inset shows the ET-1 model with the corresponding density at a contour level of 4.0 σ. b Molecular model of the ET-1–ET_BR–DNG_i complex in the same view and color scheme as in a. c Comparison of the G_i-stabilized active state of ET-1–ET_BR (green), partially active state of ET-1–ET_BR (blue), and bosentan-bound inactive ET_BR (red). Black arrows represent helical movements from inactive to active state of ET_BR.

Fig. 2. G_i-coupled ET_BR is in an active conformation.

a Superposition of the G_i-bound ET_BR structure (green) with the partially active-state crystal structure of ET-1-bound ET_BR (blue) and the inactive-state crystal structure of the antagonist bosentan-bound ET_BR (magenta). b–e Close-up views of conserved motifs involved in receptor activation. Arrows indicate the repositioning of side chains from the inactive to active state. f, g Concentration–response curves for ET-1-induced G_i signaling activity in the NanoBiT G-protein dissociation assay of ET_BR–wild-type (WT) and mutant receptors. Symbols and error bars represent mean and standard error of the mean (SEM), respectively, from three independent experiments, each performed in duplicate or triplicate. Signaling of reduced amounts of WT ET_BR (% of plasmid DNA transfected) for G_i is shown in gray. Data for these figures and expression levels of WT and mutant receptors measured by [¹²⁵I]ET-1 binding are shown in Supplementary Fig. 8 and Table 1a, b.

Fig. 3. Hydrophobic interactions between ET_BR and NK₁R in the active state.

Hydrophobic interactions around R^3.50 and L/Y^7.53 of ET_BR (a, b) and NK₁R (c, d), respectively. a The downward motion of TM7 of ET_BR is stabilized by N382^7.49 and L386^7.53 in the NPxxL motif through a series of hydrophobic interactions with I140^2.43, L195^3.46, etc. The density around all rendered residues at a contour level of 5.0 σ is shown as a mesh. b The large hydrophobic side chains of L348^H5.20 and L353^H5.25 of Gα_i penetrate deeply into the hydrophobic pocket formed by TM3, TM5, TM6, and TM7 of ET_BR. I343^H5.15 and I344^H5.16 form additional interactions with ICL2. The density around the rendered residues of the α5 helix of Gα_i is shown as a mesh at a contour level of 5.0 σ. c The downward motion of TM7 of NK₁R is stabilized by E78^2.50, N301^7.49, and Y305^7.53 in NPxxY through a series of hydrogen-bond interactions as well as hydrophobic interactions with L71^2.43, V126^3.46, etc. d The large hydrophobic side chains of L353^H5.20 and L358^H5.25 of Gα_q penetrate deeply into the hydrophobic pocket formed by TM3, TM5, TM6, and TM7 of NK₁R. Identical residues among G_i, G_o, and G_s are denoted by “*” before the amino acid label, but a conserved residue (L348^H5.15 of Gα_q) in d was omitted because it does not contact the receptor. The NPxxL motif leads to the formation of a larger cavity than NPxxY (indicated by a dashed oval).

Fig. 4. Interface between ET_BR and Gα_i.

a Close-up view of the interaction between ET_BR and the α5 helix of Gα_i. Hydrogen bonds are indicated by black dotted lines. b Schematic representation of direct contacts between ET_BR and the α5 helix of Gα_i. Hydrogen-bonded and hydrophobic contacts are indicated by dashed and solid lines, respectively. Receptor residues involved in hydrogen bonding are numbered according to Ballesteros–Weinstein numbering, and Gα_i residues involved in hydrogen bonding are numbered according to CGN numbering. Gα_i and conserved Gα_q and Gα_s residues are in magenta, homologous residues of Gα_q and Gα_s are in orange, and others are shown in yellow.

Fig. 5. Validation of the interface residues of the ET_BR–G_i complex in the NanoBiT G_i-protein dissociation assay.

Symbols and error bars represent mean and standard error of the mean (SEM), respectively, from three independent experiments, each performed in duplicate or triplicate. a–c The replaced interface residues of ET_BR were examined. Data for these figures and the expression levels of WT and mutant receptors are shown in Supplementary Table 1c–e. d The replaced interface residues of Gα_i were examined. Mutant G_i show luminescence counts comparable with those of WT. Data for this figure are shown in Supplementary Table 1f.

Fig. 6. Intermolecular and intramolecular interactions observed in MD simulations.

a Hydrogen-bond interactions in each run are represented by red lines. b Water densities in the cavity formed by transmembrane regions TM3, TM6, and TM7 in run 1 are superposed on the initial structure. Time evolution of distances between R199^3.50 and N382^7.49 (c) and between L195^3.46 and L386^7.53 (d) are shown. Distances were calculated as the minimum distance between all possible pairs of heavy atoms of two residues.