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Structural equilibrium underlying ligand-dependent activation of β2-adrenoreceptor

Nature Chemical Biology volume 16, pages 430–439 (2020)Cite this article

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Abstract

G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins mediating cellular signals in response to extracellular stimuli. Although three-dimensional structures showcase snapshots that can be sampled in the process and nuclear magnetic resonance detects conformational equilibria, the mechanism by which agonist-activated GPCRs interact with various effectors remains elusive. Here, we used paramagnetic nuclear magnetic resonance for leucine amide resonances to visualize the structure of β2-adrenoreceptor in the full agonist-bound state, without thermostabilizing mutations abolishing its activity. The structure exhibited a unique orientation of the intracellular half of the transmembrane helix 6, forming a cluster of G-protein-interacting residues. Furthermore, analyses of efficacy-dependent chemical shifts of the residues near the pivotal PIF microswitch identified an equilibrium among three conformations, including one responsible for the varied signal level in each ligand-bound state. Together, these results provide a structural basis for the dynamic activation of GPCRs and shed light on GPCR-mediated signal transduction.

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Fig. 1: 1H-15N TROSY spectra of [2,3,3–2H,15N]-Leu-labeled β2AR-Δ.
Fig. 2: PRE analyses in the formoterol-bound state.
Fig. 3: PRE structural model of the agonist-activated β2AR.
Fig. 4: Ligand-dependent spectral changes.
Fig. 5: Model of β2AR activation regulated by ligand-dependent equilibrium shift.

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Data availability

Atomic coordinates for β2AR-Δ in the fully activated state have been deposited in the PDB under accession code 6KR8. The NMR data and restraints used in the structure calculations have been deposited in the Biological Magnetic Resonance Data Bank under accession number 36284. The other data that support the findings of this study are available from the corresponding author upon reasonable request.

Code availability

All code used in this study is available from the corresponding author upon reasonable request.

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Acknowledgements

This work is supported by The Ministry of Education, Culture, Sports, Science and Technology and the Japan Society for the Promotion of Science KAKENHI grant number JP17H06097 and by the development of innovative drug discovery technologies for middle-sized molecules from the Japan Agency for Medical Research and Development (to I.S.).

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Authors and Affiliations

  1. Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan

    Shunsuke Imai, Tomoki Yokomizo, Yutaka Kofuku, Yutaro Shiraishi, Takumi Ueda & Ichio Shimada

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  1. Shunsuke Imai
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Contributions

S.I. designed the study, constructed β2AR-Δ and its variants, purified proteins, conducted GTP turnover assays with T.Y., acquired NMR spectra, analyzed the PRE data and calculated the PRE structure and wrote the manuscript. Y.K. established the purification protocol of β2AR at the early stage of the project and constructed the plasmid for the expression of cystathionine-γ-synthase. Y.S. prepared the virus stock for the coexpression of the Gs heterotrimer and cultured the cells by using the virus stock. T.U. performed the exchange Monte Carlo calculation. I.S. designed the study, analyzed the data and wrote the manuscript.

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Correspondence to Ichio Shimada.

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Imai, S., Yokomizo, T., Kofuku, Y. et al. Structural equilibrium underlying ligand-dependent activation of β2-adrenoreceptor. Nat Chem Biol 16, 430–439 (2020). https://doi.org/10.1038/s41589-019-0457-5

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