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Energetic analysis of the rhodopsin–G-protein complex links the α5 helix to GDP release

Nature Structural & Molecular Biology volume 21, pages 56–63 (2014)Cite this article

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Abstract

We present a model of interaction of Gi protein with the activated receptor (R*) rhodopsin, which pinpoints energetic contributions to activation and reconciles the β2 adrenergic receptor–Gs crystal structure with new and previously published experimental data. In silico analysis demonstrated energetic changes when the Gα C-terminal helix (α5) interacts with the R* cytoplasmic pocket, thus leading to displacement of the helical domain and GDP release. The model features a less dramatic domain opening compared with the crystal structure. The α5 helix undergoes a 63° rotation, accompanied by a 5.7-Å translation, that reorganizes interfaces between α5 and α1 helices and between α5 and β6-α5. Changes in the β6-α5 loop displace αG. All of these movements lead to opening of the GDP-binding pocket. The model creates a roadmap for experimental studies of receptor-mediated G-protein activation.

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Figure 1: Overall structure of β2AR–Gs complex, our model of the R*–Gi complex and the unbound Gi heterotrimer.
Figure 2: Placement of helical domain and rotation of α5 as observed by EPR measurements.
Figure 3: Agreement of unified model with available experimental data.
Figure 4: Rosetta energetic analysis.
Figure 5: Rosetta energetic analysis of the interface between α5 and Gαi GTPase.
Figure 6: Agreement of unified model with new structural data.
Figure 7: Validation of the model energetic predictions.

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Acknowledgements

Work in J.M.'s laboratory is supported through the US National Institutes of Health (NIH) (R01 GM080403, R01 MH090192 and R01 GM099842) and US National Science Foundation (Career 0742762). NIH National Research Service Award (MH086222) provided additional support (N.S.A.). Work in the laboratory of H.E.H. is supported through the NIH (EY006062 to H.E.H.). NIH provided additional support (U54 GM084757 to R.A.S.). The authors would like to thank S. Deluca of Vanderbilt University for implementing the Rosetta per-residue interface energy tool and H. Mchaourab of Vanderbilt University for his support with the DEER measurements (support to NIH S10 RR027091).

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Author notes

  1. Nathan S Alexander and Anita M Preininger: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, Vanderbilt University, Nashville, Tennessee, USA

    Nathan S Alexander & Jens Meiler

  2. Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, USA

    Anita M Preininger, Ali I Kaya, Heidi E Hamm & Jens Meiler

  3. Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee, USA

    Richard A Stein

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Contributions

N.S.A., A.M.P., A.I.K., H.E.H. and J.M. designed the experiments. N.S.A., A.M.P., A.I.K. and R.A.S. collected data. All authors contributed analysis. N.S.A., A.M.P., H.E.H. and J.M. wrote the manuscript with input from A.I.K. and R.A.S.

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Correspondence to Heidi E Hamm or Jens Meiler.

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Integrated supplementary information

Supplementary Figure 1 Alignment of Gαi and Gαs sequences used for construction of the initial comparative model of the R*–Gαi complex.

Alignment is from1. α-Helices are labeled in red. β-strands are labeled in blue. All secondary structure elements are labeled according to convention in the manuscript. Residue ranges critical for the energetic analysis are framed and named according to convention in the manuscript.

Supplementary Figure 2 Receptor comparative model construction.

(a) Alignment of B2- adrenergic receptor with metarhodopsin as performed by MUSTANG2. Amino acids groups for the energetic analysis are outlined in bold and named according to sequence location. Strands are highlighted in blue. Helices are highlighted in red; dark red is used to separate consecutive helices. Residues with green text were rebuilt using the Rosetta loop building protocol. (b) Superimposition of the receptor comparative model (orange/green) overlayed on the template structure B2-adrenergic receptor (grey). Residues rebuilt using the Rosetta loop building protocol are colored in green.

Supplementary Figure 3 Residues of the model construction basis.

(a) Residues rebuilt using the Rosetta loop building protocol are shown in magenta coloring. (b) Residues with experimental data are shown in magenta coloring. Regions not rebuilt using the Rosetta loop building protocol are structured as from the template 3sn6, with the exception of the helical domain (green) which undergoes rigid body movements. In (b) DEER distances measured are shown as red lines. (c) Distances measured in Gα by DEER. (d) In black is the model before relaxation, which is compared to the colored model after relaxation. The rmsd between them is 4.1 Å, excluding the helical domain residues 57-180. This shows that the relaxation protocol is a small perturbation protocol.

Supplementary Figure 4 DEER distributions compared to model and experimental structures.

(a) Comparison of the experimental distance distribution as observed in EPR DEER measurements (blue) with the predicted distribution computed from the ensemble model of receptor unbound Gαi (red). (b) Comparison of the experimental distance distribution as observed in EPR DEER measurements (blue) with the predicted distribution computed from the ensemble model of the R*-Gi complex (red). In green we show the distance distribution of our previous model which reproduces average distance accurately but not the distance distribution3. In magenta we display the distance distribution based on the crystal structure4.

Supplementary Figure 5 Comparison of receptor structures.

(a) Comparison of the activated rhodopsin structure (3DQB, grey) with the β2AR (cyan). The ligand does not significantly perturb the structure. (b) Comparison of model receptor (salmon) with activated rhodopsin structure (3DQB, black).

Supplementary Figure 6 Characterization experiments of double mutants.

(a) Binding of doubly spin-labeled mutant G proteins to rhodopsin in disc membranes. (b) Basal (grey) and receptor (black) catalyzed nucleotide exchange rates for the doubly spin-labeled mutant α-subunit. Bars represent the mean of a minimum of three independent experiments, and error bars show standard error of the mean.

Supplementary Figure 7 DEER echo decays and the corresponding distance distribution fits.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Tables 1–9 and Supplementary Note (PDF 2490 kb)

Supplementary Data Set 1

Ten model structures representing the receptor-unbound model of Gαiβγ (ZIP 2955 kb)

Supplementary Data Set 2

Nine model structures representing the R*–Gi complex (ZIP 1920 kb)

Modeled interaction of activated rhodopsin (R*) with Gi.

The receptor starts in the non-activated conformation (red) and then moves into the activated state (orange). Upon receptor activation, the α5 C-terminal helix of Gαi (yellow, blue) binds to R*. The helical domain (green) opens away Gαi-GTPase (grey) and GDP is released (spheres). Gβ is shown in brown; Gγ shown in black. (MOV 297 kb)

Agreement of unified model with single-particle EM class averages, relating to Figure 3d. (MOV 1301 kb)

Energetics of helical domain–Gαi interface in free Gαi, relating to Figure 4a. (MOV 1645 kb)

Energetics of the GDP-Gαi interface in free Gαi, relating to Figure 4b. (MOV 1428 kb)

Energetics of R*–Gαi interface in the R*–Gαi complex, relating to Figure 4c. (MOV 788 kb)

Energetics of the interface between α5–Gαi-GTPase in the basal state, relating to Figure 5a. (MOV 1246 kb)

Energetics of the interface between α5–Gαi-GTPase in the R*–Gαi complex, relating to Figure 5b. (MOV 867 kb)

Rotation of the α5 C-terminal helix of Gαi upon receptor binding.

As α5 binds to activated rhodopsin (R*), α5 undergoes a 63° rotation Gαi-GTPase. Asparagine 341 is shown as sticks for reference. (MOV 653 kb)

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Alexander, N., Preininger, A., Kaya, A. et al. Energetic analysis of the rhodopsin–G-protein complex links the α5 helix to GDP release. Nat Struct Mol Biol 21, 56–63 (2014). https://doi.org/10.1038/nsmb.2705

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