Concerted conformational dynamics and water movements in the ghrelin G protein-coupled receptor

  1. Maxime Louet
  2. Marina Casiraghi
  3. Marjorie Damian
  4. Mauricio GS Costa
  5. Pedro Renault
  6. Antoniel AS Gomes
  7. Paulo R Batista
  8. Céline M'Kadmi
  9. Sophie Mary
  10. Sonia Cantel
  11. Severine Denoyelle
  12. Khoubaib Ben Haj Salah
  13. David Perahia
  14. Paulo M Bisch
  15. Jean-Alain Fehrentz
  16. Laurent J Catoire
  17. Nicolas Floquet
  18. Jean-Louis Banères  Is a corresponding author
  1. IBMM, Univ Montpellier, CNRS, ENSCM, France
  2. Laboratoire de Biologie Physico-Chimique des Protéines Membranaires, UMR 7099, CNRS, Université de Paris, Institut de Biologie Physico-Chimique (FRC 550), France
  3. Laboratoire de Biologie et Pharmacologie Appliquées, UMR 8113 CNRS, Ecole Normale Supérieure Paris-Saclay, France
  4. Programa de Computação Científica, Fundação Oswaldo Cruz, Brazil
  5. Laboratório de Física Biológica, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil
7 figures, 1 table and 3 additional files

Figures

Figure 1 with 6 supplements
Growth hormone secretagogue receptor (GHSR) labeling.

(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 …

Figure 1—figure supplement 1
The growth hormone secretagogue receptor (GHSR)-containing nanodiscs.

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 …

Figure 1—figure supplement 1—source data 1

SDS-PAGE of the GHSR-containing nanodiscs.

https://cdn.elifesciences.org/articles/63201/elife-63201-fig1-figsupp1-data1-v1.xlsx
Figure 1—figure supplement 2
Control for the ligand-binding properties of the isolated growth hormone secretagogue receptor (GHSR) in 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 …

Figure 1—figure supplement 2—source data 1

HTRF ratio for GHSR- and BLT-containing discs.

https://cdn.elifesciences.org/articles/63201/elife-63201-fig1-figsupp2-data1-v1.xlsx
Figure 1—figure supplement 3
Statistical analysis of the GTP turnover assay.

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 …

Figure 1—figure supplement 4
N-terminal labeling of growth hormone secretagogue receptor (GHSR).

(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 …

Figure 1—figure supplement 4—source data 1

Absorbance of GHSR-containing nanodiscs before and after TEV cleavage.

https://cdn.elifesciences.org/articles/63201/elife-63201-fig1-figsupp4-data1-v1.xlsx
Figure 1—figure supplement 4—source data 2

Emission intensity of GHSR-containing nanodiscs before and after TEV cleavage.

https://cdn.elifesciences.org/articles/63201/elife-63201-fig1-figsupp4-data2-v1.xlsx
Figure 1—figure supplement 5
Fluorescent ghrelin characterization.

(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.…

Figure 1—figure supplement 6
The G protein trimer.

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.

Figure 1—figure supplement 6—source data 1

SDS-PAGE profile of the G protein used in the GTP-binding assays.

https://cdn.elifesciences.org/articles/63201/elife-63201-fig1-figsupp6-data1-v1.xlsx
Figure 2 with 6 supplements
Local hydration of growth hormone secretagogue receptor (GHSR) as a function of ligands.

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. …

Figure 2—figure supplement 1
Emission spectra of L-(7-hydroxycoumarin-4-yl) (7H4MC)-ethylglycine incorporated into isolated growth hormone secretagogue receptor (GHSR).

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: …

Figure 2—figure supplement 1—source data 1

7-H4MC emission intensity of the apo GHSR mutants.

https://cdn.elifesciences.org/articles/63201/elife-63201-fig2-figsupp1-data1-v1.xlsx
Figure 2—figure supplement 2
Statistical analysis of the data in Figure 2.

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 …

Figure 2—figure supplement 3
Structure of the ligands used throughout this work.
Figure 2—figure supplement 4
Statistical analysis of the data in Figure 2.

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 …

Figure 2—figure supplement 5
Emission spectra of L-(7-hydroxycoumarin-4-yl) (7H4MC)-ethylglycine incorporated at position 5.58 of growth hormone secretagogue receptor (GHSR) in the presence of pharmacologically distinct ligands or phosphatidylinositol-4,5-bisphosphate (PIP2).

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 …

Figure 2—figure supplement 5—source data 1

7-H4MC emission intensity of the GHSR 5.58 mutant in the presence of ligands.

https://cdn.elifesciences.org/articles/63201/elife-63201-fig2-figsupp5-data1-v1.xlsx
Figure 2—figure supplement 6
Emission spectra of L-(7-hydroxycoumarin-4-yl) (7H4MC)-ethylglycine incorporated at position 6.44 of growth hormone secretagogue receptor (GHSR) in the presence of pharmacologically distinct ligands or phosphatidylinositol-4,5-bisphosphate (PIP2) in the nanodiscs.

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 …

Figure 2—figure supplement 6—source data 1

pa 7-H4MC emission intensity of the GHSR 6.44 mutant in the presence of ligands.

https://cdn.elifesciences.org/articles/63201/elife-63201-fig2-figsupp6-data1-v1.xlsx
Figure 3 with 1 supplement
Water distribution in growth hormone secretagogue receptor (GHSR) as observed along the five independent 5 µs molecular dynamics (MD) simulations starting from either the inactive (A to E) or the active (F to J) states of the receptor.

(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 …

Figure 3—figure supplement 1
Projection of structures and molecular dynamics (MD) conformers on the first two eigenvectors obtained from the principal component analysis (PCA) of experimental structures.

Red and green squares represent inactive and active experimental structures, respectively, whereas the black squares stand for structures describing intermediates. Both purple squares represent …

Figure 4 with 5 supplements
Amino acid positions and hydration patterns of inactive growth hormone secretagogue receptor (GHSR) (A) and active GHSR (B) explored by molecular dynamics (MD) simulations.

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) …

Figure 4—figure supplement 1
Intersections between water distributions and predicted L-(7-hydroxycoumarin-4-yl) (7H4MC) orientations in the inactive growth hormone secretagogue receptor (GHSR) (A and B) and the active GHSR (C and D) at positions 268 (A and C) and 315 (B and D).

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 …

Figure 4—figure supplement 2
Structural differences between both starting conformers: inactive (blue) and active (orange).

Protein backbone is represented in cartoon; the green arrows emphasize the difference between the two models.

Figure 4—figure supplement 3
Orientation of F2726.44 in all experimental-inactive (A) and experimental-active (B) structures.

Protein atoms are represented as lines and cartoon, each receptor has been colored differently.

Figure 4—figure supplement 4
Motions encoded by the first two eigenvectors resulting from the principal component analysis (PCA) of the conserved Cα coordinates from all experimental structures.

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 …

Figure 4—figure supplement 5
Residue selection for principal component analysis (PCA).

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.

Impact of lipids on the local hydration of growth hormone secretagogue receptor (GHSR).

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 …

Figure 5—source data 1

H parameter as a function of PIP2 in the nanodiscs.

https://cdn.elifesciences.org/articles/63201/elife-63201-fig5-data1-v1.xlsx
Author response image 1
Projections along the first-two eigenvectors inferred from a Principal Component Analysis (PCA) of all experimental structures.

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 …

Author response image 2
Root Mean Square Deviation (RMSD) of all MD simulations with initial structures as references.

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.

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain backgroundBL21(DE3)
Escherichia coli
Sigma-AldrichCMC0014Chemically competent cells
Recombinant DNA reagentpEvol-aaRSdoi: 10.1021/ja062666k
Recombinant DNA reagentpMSP1E3D1Addgene#20066
Recombinant DNA reagentpET21a-α5-GHSR (transfected construct; Homo sapiens)doi: 10.1074/jbc.M111.288324
Peptide, recombinant proteinGhrelinThis workSynthesis is described in the Materials and methods section
Peptide, recombinant proteinFluorescent ghrelinThis workLabeling is described in the Materials and methods section
Peptide, recombinant proteinThrombinSigmaT7009
Commercial assay or kitGTPase-GloTM assayPromegaV7681
Chemical compound, drug7H4MC-ethylglycineThis workSynthesis is described the Materials and methods section
Chemical compound, drugAmpicillinSigmaA9518
Chemical compound, drugChloramphenicolCalbiochem220551
Chemical compound, drugIPTGSigmaI6758
Chemical compound, drugAmphipol A8-35AnatraceA835 100 MG
Chemical compound, drugβ-DDMAnatraceD310
Chemical compound, drugCholesteryl-hemisuccinateAnatraceCH210
Chemical compound, drugPOPCAvanti Polar Lipids850457C
Chemical compound, drugPOPGAvanti Polar Lipids840457C
Chemical compound, drugPIP2Avanti Polar Lipids850155P
Chemical compound, drugBio-Beads SM-2BIO-RAD1528920
Chemical compound, drugLumi4-Tb NHSCisBio62TBSPEA
Chemical compound, drugDY647P1-maleimideDyomics647P1-03
Chemical compound, drugAmine reactive Tb chelateFisher11563467
Chemical compound, drugNiNTA SuperflowQiagen30430
Chemical compound, drugStreptavidin-agaroseThermofisher20361
Chemical compound, drugSuperdex S200 increase 10×300 GLGE Healthcare (Cytiva)28990944
Chemical compound, drugSource 15Q 4.6×100 PEGE Healthcare (Cytiva)17518101
Chemical compound, drugZebaSpin 40K MWCO columnThermofisher87766
Software, algorithmPrismGraphPadVersion 8.4.3
Software, algorithmVMDdoi: 10.1016/0263-7855(96)00018-5
Software, algorithmBio3Ddoi: 10.1093/bioinformatics/btl461
Software, algorithmPymolSchrodinger LLC
Software, algorithmGromacs 2020.3doi: 10.5281/zenodo.3923645

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