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The dynamics of agonist-β2-adrenergic receptor activation induced by binding of GDP-bound Gs protein

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Abstract

There is considerable uncertainty about the mechanism by which the β2-adrenergic receptor (β2AR) is activated. Here we use molecular metadynamics computations to predict the mechanism by which an agonist induces the activation of the β2AR and its cognate Gs protein. We found that binding agonist alone to the inactive β2AR does not break the ionic lock and hence does not drive the β2AR towards the activated conformation. However, we found that attaching the inactive Gs protein to the agonist-bound inactive β2AR (containing the ionic lock) leads to partial insertion of Gαs-α5 into the core of β2AR, which breaks the ionic lock, leading to activation of the Gs protein coupled to β2AR. Upon activation, the Gαs protein undergoes a remarkable opening of the GDP binding pocket, making the GDP available for exchange or release. Concomitantly, Gαs-α5 undergoes a remarkable expansion in the β2AR cytoplasmic region after the ionic lock is broken, inducing TM6 to displace outward by ~5 Å from TM3.

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Fig. 1: Ligand-first activation mechanism for β2AR and its cognate Gs protein.
Fig. 2: Inactive state of β2AR.
Fig. 3: Agonists do not stabilize the active conformation of β2AR.
Fig. 4: The inactive Gs-protein-bound GDP attaches to the agonist-bound β2AR with its closed cytoplasmic region.
Fig. 5: Activation of Gs protein-bound GDP triggers activation of BI67107-bound β2AR.
Fig. 6: Comparison of our predicted active state of BI167107–β2AR–Gs (GDP) with crystal structures.

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Data availability

All data generated or analysed during this study are included in this published Article (and its Supplementary Information files). We deposited (https://figshare.com/articles/dataset/free_energy_files/22217674) the metadynamics protocol and associate free-energy results along with the initial and final protein complex structures. Source data are provided with this paper.

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GROMACS (https://www.gromacs.org/) and Plumed (https://www.plumed.org/) are all available as open source.

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Acknowledgements

A.M., S.-K.K. and W.A.G. acknowledge support from the NIH (R01HL155532 and R35HL150807).

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Authors and Affiliations

  1. Materials and Process Simulation Center, Caltech, Pasadena, CA, USA

    Amirhossein Mafi, Soo-Kyung Kim & William A. Goddard III

  2. California Institute of Technology, Pasadena, CA, USA

    Amirhossein Mafi, Soo-Kyung Kim & William A. Goddard III

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  1. Amirhossein Mafi
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  3. William A. Goddard III
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Contributions

W.A.G. and A.M. designed the project. A.M. carried out all calculations. A.M. and S.-K.K. prepared all figures and tables and the Supplementary Information. A.M. wrote the manuscript with W.A.G. and S.-K.K.

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Correspondence to William A. Goddard III.

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Extended data

Extended Data Fig. 1 Agonists alone do not activate β2AR.

(a) The optimized inverse agonist (ICI118551)-bound β2AR, featuring the ionic lock in the cytoplasmic region. (b) ICI118551-bound β2AR: MetaMD free energy of the distance between TM3 [the center of mass of Cα for residues 129-137] and TM6 [the center of mass of Cα for residues 266-277]. (c) inverse agonist: ICI118551: MetaMD free energy of the strength for the ionic lock between R1313.50(CZ)- E2686.30(CD). The weighted averages and the standard deviations were calculated for the reported ΔGif within the converged period. The metaMD free energies were reweighted31 for estimation of the error. We used block averaging (n = 951 blocks) to report the standard deviation. (d) The free energy of breaking the ionic lock in the presence of (inverse agonist and agonists) and in the absence of ligands. Comparison of our optimized ICI118551 (inverse-agonist)-bound β2AR with (e) epinephrine-bound β2AR and (f) BI167107-bound β2AR. All RMSDs were calculated for the backbone atoms on the TM domain of β2AR.

Source data

Extended Data Fig. 2 Agonist binding is the main driver for activation of the β2AR-Gs protein complex.

The optimized (a) unliganded-β2AR (apo); (c) carazolol-bound -β2AR (inverse agonist); and (e) ICI118551-bound -β2AR (inverse agonist) coupled to inactive Gs protein-bound GDP. (b), (d) & (f) MetaMD free energy versus the distance between the α-helical (AH) [the center of mass of Cαs for the residues 69-204] and Ras-like [the center of mass of Cαs for the residues 223-241, 250-285, and 294-358] subdomains. The weighted averages and the standard deviations were calculated for the reported ΔGif within the converged period. The metaMD free energies were reweighted31 for estimating the error. We used block averaging (n = 951 blocks) to report the standard deviation for each data point.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–20, Tables 1–5 and Videos 1–3.

Supplementary Video 1

MetaMD simulation of β2AR-bound BI167107 structure, starting from the activated structure (PDB 3SN6), but Gs protein was eliminated.

Supplementary Video 2

MetaMD simulation of β2AR-bound BI167107-GS-GDP structure from the intracellular view, showing a remarkable expansion in the cytoplasmic cavity of β2AR.

Supplementary Video 3

MetaMD simulation of β2AR-bound BI167107-GS-GDP structure, showing a remarkable GS protein opening, making GDP water exposed.

Source data

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Extended Data Fig./Table 1

Statistical source data.

Source Data Extended Data Fig./Table 2

Statistical source data.

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Mafi, A., Kim, SK. & Goddard, W.A. The dynamics of agonist-β2-adrenergic receptor activation induced by binding of GDP-bound Gs protein. Nat. Chem. 15, 1127–1137 (2023). https://doi.org/10.1038/s41557-023-01238-6

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