(A) Position of the labeled residues in GHSR sequence. Red labeling indicates positions that were deleterious to GHSR expression and/or function. Green labeling indicates positions that did not …
HTRF ratio for GHSR and its mutants.
Luminescence values for the GTP turnover assay.
GHSR emission intensity.
Size-exclusion profile of the GHSR-containing nanodiscs. The nanodiscs were run on an S200 increase column (10×300) using a 25 mM HEPES, 150 mM NaCl, 0.5 mM EDTA, pH 7.5 buffer as the eluent and a …
SDS-PAGE of the GHSR-containing nanodiscs.
The emission intensity of the dy647 acceptor at 665 nm and that of the Lumi4-Tb donor at 620 nm were measured after excitation of the donor at 337 nm. A 10−7 M concentration of fluorescent ghrelin …
HTRF ratio for GHSR- and BLT-containing discs.
The left panels show free GTP for the wild-type receptor and each of the mutants used in the present work in the absence or in the presence of ligands (ligands indicated in each panel). All data is …
(A) Schematic representation of the construct used. This construct includes a TEV cleavage site and two additional glycines 11 residues after the receptor N-terminus. (B,C) Size-exclusion …
Absorbance of GHSR-containing nanodiscs before and after TEV cleavage.
Emission intensity of GHSR-containing nanodiscs before and after TEV cleavage.
(A) Structure of the fluorescent ghrelin peptide used in the ligand-binding experiments. (B) RP-HPLC chromatogram; RT = 2.16 min, UV purity (214 nm) = 99%; (C) MS-ESI(+) spectrum: Mcalculated = 3150.…
SDS-PAGE profile of the Gαqβ1γ2 trimer used in the functional assays. The G protein trimer was run on a 15% polyacrylamide-0.1% SDS gel, with Coomassie blue staining.
SDS-PAGE profile of the G protein used in the GTP-binding assays.
H parameter for the 7H4MC-labeled GHSR in the absence of ligand (apo) and in the presence of JMV3011, ghrelin, JMV3002, or SPA (substance-P analog). All ligands were used at a 10 µM concentration. …
H parameter for GHSR and its mutants.
The spectra were recorded in the absence of ligand. The labeling positions are given in a snake-like plot of GHSR (Shiimura et al., 2020) (A) (red: positions deleterious to GHSR function; green: …
7-H4MC emission intensity of the apo GHSR mutants.
The left panel shows the hydration parameter for each of the mutants in the absence of ligand. Data is from Figure 2. The right panel shows the mean difference between the apo state of each of the …
The left panels show the hydration parameter for each of the mutants in the absence or in the presence of ligands. Data are from Figure 2. The right panels show the mean difference between the apo …
All ligands were used at a 10 µM concentration (JMV3011: antagonist, ghrelin: full agonist, JMV3002: Gq-biased agonist, substance-P analog [SPA]: inverse agonist). The excitation wavelength was set …
7-H4MC emission intensity of the GHSR 5.58 mutant in the presence of ligands.
All ligands were used at a 10 µM concentration (JMV3011: antagonist, ghrelin: full agonist, JMV3002: Gq-biased agonist, substance-P analog [SPA]: inverse agonist). The excitation wavelength was set …
pa 7-H4MC emission intensity of the GHSR 6.44 mutant in the presence of ligands.
(E and J) panels show simulations where GHSR transited from inactive to active (E) or from active to inactive (J) states, respectively. The backbone of the protein is represented as a …
Red and green squares represent inactive and active experimental structures, respectively, whereas the black squares stand for structures describing intermediates. Both purple squares represent …
GHSR is represented in white ribbons. Volumetric maps in solid surface represent the water distribution with a probability of presence of 0.3. Meshes represent the most probable (probability of 0.3) …
The dark isocontours in mesh represent a probability of presence of the mutated residue of 0.3, while the light isocontours in mesh represent a probability of presence of water of 0.3. The protein …
Protein backbone is represented in cartoon; the green arrows emphasize the difference between the two models.
Protein atoms are represented as lines and cartoon, each receptor has been colored differently.
The receptor is represented as cartoon and black arrows represent the direction and amplitude of eigenvectors. (A and C) represent a side and an intracellular view of the first eigenvector …
The receptor is represented in transparent cartoon colored by residue number from red (N-terminus) to blue (C-terminus). The Cα atoms kept for the PCA are represented in spheres.
H parameter for L-(7-hydroxycoumarin-4-yl) (7H4MC)-labeled GHSR assembled into nanodiscs containing or not phosphatidylinositol-4,5-bisphosphate (PIP2) (2.5% PIP2-to-total lipids molar ratio), in …
H parameter as a function of PIP2 in the nanodiscs.
Names correspond to uniprot names of GPCR and have been placed in respect to the projection. Red names correspond to activated receptors, purple to intermediate conformations and blue names to …
The 5 MD simulations from the inactive state are represented in blue, and the 5 MD simulations from the active state are represented in orange. Running averages on top of curves are shown for clarity.
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Strain, strain background | BL21(DE3) Escherichia coli | Sigma-Aldrich | CMC0014 | Chemically competent cells |
Recombinant DNA reagent | pEvol-aaRS | doi: 10.1021/ja062666k | ||
Recombinant DNA reagent | pMSP1E3D1 | Addgene | #20066 | |
Recombinant DNA reagent | pET21a-α5-GHSR (transfected construct; Homo sapiens) | doi: 10.1074/jbc.M111.288324 | ||
Peptide, recombinant protein | Ghrelin | This work | Synthesis is described in the Materials and methods section | |
Peptide, recombinant protein | Fluorescent ghrelin | This work | Labeling is described in the Materials and methods section | |
Peptide, recombinant protein | Thrombin | Sigma | T7009 | |
Commercial assay or kit | GTPase-GloTM assay | Promega | V7681 | |
Chemical compound, drug | 7H4MC-ethylglycine | This work | Synthesis is described the Materials and methods section | |
Chemical compound, drug | Ampicillin | Sigma | A9518 | |
Chemical compound, drug | Chloramphenicol | Calbiochem | 220551 | |
Chemical compound, drug | IPTG | Sigma | I6758 | |
Chemical compound, drug | Amphipol A8-35 | Anatrace | A835 100 MG | |
Chemical compound, drug | β-DDM | Anatrace | D310 | |
Chemical compound, drug | Cholesteryl-hemisuccinate | Anatrace | CH210 | |
Chemical compound, drug | POPC | Avanti Polar Lipids | 850457C | |
Chemical compound, drug | POPG | Avanti Polar Lipids | 840457C | |
Chemical compound, drug | PIP2 | Avanti Polar Lipids | 850155P | |
Chemical compound, drug | Bio-Beads SM-2 | BIO-RAD | 1528920 | |
Chemical compound, drug | Lumi4-Tb NHS | CisBio | 62TBSPEA | |
Chemical compound, drug | DY647P1-maleimide | Dyomics | 647P1-03 | |
Chemical compound, drug | Amine reactive Tb chelate | Fisher | 11563467 | |
Chemical compound, drug | NiNTA Superflow | Qiagen | 30430 | |
Chemical compound, drug | Streptavidin-agarose | Thermofisher | 20361 | |
Chemical compound, drug | Superdex S200 increase 10×300 GL | GE Healthcare (Cytiva) | 28990944 | |
Chemical compound, drug | Source 15Q 4.6×100 PE | GE Healthcare (Cytiva) | 17518101 | |
Chemical compound, drug | ZebaSpin 40K MWCO column | Thermofisher | 87766 | |
Software, algorithm | Prism | GraphPad | Version 8.4.3 | |
Software, algorithm | VMD | doi: 10.1016/0263-7855(96)00018-5 | ||
Software, algorithm | Bio3D | doi: 10.1093/bioinformatics/btl461 | ||
Software, algorithm | Pymol | Schrodinger LLC | ||
Software, algorithm | Gromacs 2020.3 | doi: 10.5281/zenodo.3923645 |
Equilibration procedure.
List of structures used for principal component analysis.