Cryo-EM structure of the rhodopsin-Gαi-βγ complex reveals binding of the rhodopsin C-terminal tail to the gβ subunit
- Paul Scherrer Institute, Switzerland
- University of Basel, Switzerland
- ETH Zurich, Switzerland
- Hoffmann-La Roche Ltd, Switzerland
Figures
Figure 1 with 8 supplements
Cryo-EM structure of the rhodopsin-Gi-Fab16 complex.
(A) GPCR signaling complexes. (B) EM density map of the complex (rhodopsin – blue, Gαi – green, Gβ – yellow, Gγ – magenta, Fab16 – white). (C) Atomic model of the complex (same color code as B). The …
Figure 1—figure supplement 1
Purification of the rhodopsin-Gi and rhodopsin-Gi-Fab16 complexes.
(A) SDS-PAGE analysis of individual proteins and main peak fraction of corresponding size-exclusion chromatography (SEC). (B) SEC profiles of rhodopsin-Gαi and rhodopsin-Gi-Fab16 complexes used for …
Figure 1—figure supplement 2
Cryo-EM maps of rhodopsin-Gi complexes with and without Fab16.
(A) Small dataset of rhodopsin-Gi (without Fab16) obtained using a Falcon III detector. (B) Small dataset of rhodopsin-Gi-Fab16 obtained using a Falcon III detector. (C) Gold-standard FSC curves …
Figure 1—figure supplement 3
Image processing of the rhodopsin-Gi-Fab16 complex acquired with K2.
(A) A representative micrograph. (B) Selected 2D class averages. (C) Image-processing workflow of the data processing. (D) Local resolution of the 3D reconstruction from (C). (E) Fourier Shell …
Figure 1—figure supplement 4
Image processing details.
(A) Angular distribution of the final set of particles (115’000 particles). Removal of particles that contribute to the over-represented views did not improve neither worsen map quality.(B) Spatial …
Figure 1—figure supplement 5
3D classification reveals the flexibility of the AH domain of Gαi.
(A) Density map of one 3D class obtained during classification in RELION. The AH domain of the Gα subunit, highlighted in red, becomes visible only at high threshold when visualizing the density map …
Figure 1—figure supplement 6
Details of the cryo-EM density map of rhodopsin-Gi-Fab16 with a fitted atomic model.
(A) The segmented density map shows individual regions of rhodopsin (transmembrane helices (TM), intracellular and extracellular loops (ICL, ECL), helix 8 (H8) and the C-terminal tail (C-tail), …
Figure 1—figure supplement 7
Comparison of the bovine rhodopsin-Gi complex to the other GPCR-G protein complexes.
(A) The complex structures are aligned to the Cα atoms of the rhodopsin-Gi complex (receptor – blue; Gα – green; Gβ – yellow; Gγ – magenta; peptide ligand – salmon). (B) Binding interface between …
Figure 1—figure supplement 8
Details of the source organism of the Gα, Gβ, and Gγ proteins used to obtain GPCR G-protein complexes for structure determination.
https://doi.org/10.7554/eLife.46041.010
Figure 2 with 4 supplements
The C-terminal tail of rhodopsin.
(A) The EM map is contoured at two different levels to show the continuity of the density. The weakening at the end of H8 may arise from impaired interactions of the receptor with the detergent …
Figure 2—figure supplement 1
Electrostatic potential.
(A) Three-dimensional structure of the rhodopsin (blue) - Gα (green) – Gβγ (yellow and magenta) complex. The alpha carbons of the interacting residues in each component are displayed as spheres. (B) …
Figure 2—figure supplement 2
Sequence conservation in Gα and Gβγ.
(A) Sequence logo depicting the sequence conservation in the α3 helix of the Ras domain in different G protein subtypes. The size of the letters represents frequency within a sequence alignment of …
Figure 2—figure supplement 3
Flexible fitting of the C-tail in the electron density.
Structural model of rhodopsin displaying the electron density at a 10σ cuf-off around H8 and the C-tail (left), and snapshots of this region during the MDFF simulation (right).
Figure 2—figure supplement 4
Structure of the C-tail in rhodopsin and M1R.
(A) Three-dimensional structure of the rhodopsin (blue) - Gαi (green) – Gβ (yellow) complex. (B) Three-dimensional structure of the M1 muscarinic acetylcholine receptor (blue) – Gα11 (green) – Gβ …
Figure 3 with 5 supplements
Binding of the Gα α5 helix in GPCR-G protein complexes.
(A) Overview of a subset of GPCR-G protein complexes used for this analysis. For a complete list of the complexes used, see Figure 3—figure supplement 2. The complexes are shown from the cytoplasmic …
Figure 3—figure supplement 1
Residue-residue contact list between GPCRs and Gα H5.
11–26 within 4 Å.
Figure 3—figure supplement 2
Residue-residue contacts between GPCRs and Gα H5.
11–26.
Figure 3—figure supplement 3
Contacts observed between ICL2/ICL3 and the Gα subunit.
(A) Intracellular loop (ICL) 2 and 3 in the EM density map (B) Schematic overview of the contacts between the intracellular loops of the receptor and Gα. (C) Sequence of ICL2 in the available …
Figure 3—figure supplement 4
Residue-residue contacts between ICL2/3 and Gα.
Contacts at the regions of ICL2 and 3 are identified using a 4 Å cut-off. Residue-residue contacts are listed for the regions of ICL2 (top panel) and ICL3 (bottom panel). The residues of Gα are …
Figure 3—figure supplement 5
Region near ICL2 in available structures.
View of the existing GPCR-G protein complex structures centered at the ICL2 region. The structures are aligned to the Cα atoms of rhodopsin. The regions of the G protein involved in contacting ICL2 …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifier | Addition information |
|---|---|---|---|---|
| Strain, strain background (E. coli) | BL21(DE3) | Sigma-Aldrich | SA: CMC0014 | |
| Cell line (H. sapiens) | HEK293 GnTI- | Reeves laboratory (gift) | Under MTA with MIT, Cambridge, MA, US. | PMID: 12370423 |
| Cell line (M. musculus) | Hybridoma | Hoffmann-La Roche (by collaboration) | Under MTA with Hoffmann-La Roche, Basel, Switzerland. | Maeda et al., 2018 |
| Biological sample (B. taurus) | Bovine retinae | WL Lawson Company (USA) | https://wllawsoncompany.com/ | |
| Chemical compound, drug | Blue Sepharose 6 Fast Flow | GE Healthcare | GEH: 17094801 | |
| Chemical compound, drug | Chelating Sepharose Fast Flow immobilized metal affinity chromatography resin | GE Healthcare | GEH: 17057501 | |
| Chemical compound, drug | HiTrap Protein G Sepharose HP column | GE Healthcare | GEH: 17040401 | |
| Chemical compound, drug | Immobilized papain agarose resin | Thermo Scientific | TS: 20341 | |
| Chemical compound, drug | Protein A Sepharose | GE Healthcare | GEH: 17078001 | |
| Chemical compound, drug | CNBr-activated sepharose 4B | GE Healthcare | GEH: 17043001 | |
| Chemical compound, drug | Ultra-low IgG fetal bovine serum | Gibco | Gibco: 16250078 | |
| Chemical compound, drug | Interleukin-6 recombinant mouse protein | Invitrogen | Invitrogen: PMC0064 | |
| Chemical compound, drug | cOmplete EDTA-free protease inhibitor cocktail | Sigma-Aldrich | SA: 1873580001 | |
| Chemical compound, drug | Dodecyl β-maltoside | Anatrace | Anatrace: D310 | |
| Chemical compound, drug | Lauryl-maltoside neopentyl glycol | Anatrace | Anatrace: NG310 | |
| Chemical compound, reagent | 1D4 antibody | Cell Essentials Inc | http://www.cell-essentials.com/ | |
| Chemical compound, drug | 1D4 peptide | Peptide 2.0 | https://www.peptide2.com/ | |
| Chemical compound, drug | 9-cis retinal | Sigma-Aldrich | SA: R5754 | |
| Chemical compound, drug | Apyrase | New England Biolabs | NEB: M0398 | |
| Software, algorithm | XDS | http://xds.mpimf-heidelberg.mpg.de | PMID: 20124692; RRID: SCR_015652 | |
| Software, algorithm | PHENIX | https://www.phenix-online.org | Adams et al., 2010; RRID: SCR_014224 | |
| Software, algorithm | UCSF Chimera | https://www.cgl.ucsf.edu/chimera | Pettersen et al., 2004; RRID: SCR_004097 | |
| Software, algorithm | PyMOL | Schrödinger LLC | https://pymol.org | RRID:SCR_000305 |
| Software, algorithm | FOCUS | https://focus.c-cina.unibas.ch/about.php | Biyani et al., 2017 | |
| Software, algorithm | MotionCor2 | http://msg.ucsf.edu/em/software/motioncor2.html | Zheng et al., 2017; RRID: SCR_016499 | |
| Software, algorithm | cryoSPARC | Structura Biotechnology Inc. | https://cryosparc.com | PMID: 28165473; RRID: SCR_016501 |
| Software, algorithm | RELION 2, 3 | http://www2.mrc-lmb.cam.ac.uk/relion | RRID:SCR_016274 | |
| Software, algorithm | NAMD | http://www.ks.uiuc.edu/Research/namd | RRID:SCR_014894 | |
| Software, algorithm | Coot | https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot | Emsley and Cowtan, 2004; RRID: SCR_014222 |
Additional files
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Supplementary file 1
Supplementary Tables.
Supplementary Table 1. Cryo-EM data collection and refinement statistics. Supplementary Table 2. Crystallographic data and structural refinement of Fab16.
- https://doi.org/10.7554/eLife.46041.022
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Transparent reporting form
- https://doi.org/10.7554/eLife.46041.023
