Structural equilibrium underlying ligand-dependent activation of β2-adrenoreceptor

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. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS Access

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customer support SIMILAR CONTENT BEING VIEWED BY OTHERS STRUCTURES OF Β1-ADRENERGIC RECEPTOR IN COMPLEX WITH GS AND LIGANDS OF DIFFERENT EFFICACIES Article Open access 14 July 2022 BINDING

KINETICS DRIVE G PROTEIN SUBTYPE SELECTIVITY AT THE Β1-ADRENERGIC RECEPTOR Article Open access 13 February 2024 MECHANISTIC INSIGHTS INTO G-PROTEIN COUPLING WITH AN AGONIST-BOUND

G-PROTEIN-COUPLED RECEPTOR Article 12 June 2024 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. REFERENCES * Rosenbaum, D. M., Rasmussen, S. G. F. & Kobilka, B. K. The structure and function of G-protein-coupled receptors. _Nature_ 459, 356–363 (2009). CAS 

PubMed  PubMed Central  Google Scholar  * Erlandson, S. C., McMahon, C. & Kruse, A. C. Structural basis for G-protein-coupled receptor signaling. _Annu. Rev. Biophys._ 47, 1–18 (2018).

CAS  Google Scholar  * Flock, T. et al. Selectivity determinants of GPCR: G-protein binding. _Nature_ 545, 317–322 (2017). CAS  PubMed  PubMed Central  Google Scholar  * Deupi, X. &

Kobilka, B. K. Energy landscapes as a tool to integrate GPCR structure, dynamics, and function. _Physiol. (Bethesda)_ 25, 293–303 (2010). CAS  Google Scholar  * Shimada, I., Ueda, T.,

Kofuku, Y., Eddy, M. T. & Wüthrich, K. GPCR drug discovery: integrating solution NMR data with crystal and cryo-EM structures. _Nat. Rev. Drug Discov._ 18, 59–82 (2018). PubMed  PubMed

Central  Google Scholar  * Xiang, J. et al. Successful strategies to determine high-resolution structures of GPCRs. _Trends Pharmacol. Sci._ 37, 1055–1069 (2016). CAS  PubMed  Google Scholar

  * Trzaskowski, B. et al. Action of molecular switches in GPCRs: theoretical and experimental studies. _Curr. Med. Chem._ 19, 1090–1109 (2012). CAS  PubMed  PubMed Central  Google Scholar 

* Edward Zhou, X., Melcher, K. & Eric Xu, H. Structural biology of G-protein‐coupled receptor signaling complexes. _Protein Sci._ 28, 487–501 (2019). CAS  PubMed  Google Scholar  *

Warne, T. et al. The structural basis for agonist and partial agonist action on a β(1)-adrenergic receptor. _Nature_ 469, 241–244 (2011). CAS  PubMed  PubMed Central  Google Scholar  *

Lebon, G. et al. Agonist-bound adenosine A2A receptor structures reveal common features of GPCR activation. _Nature_ 474, 521–525 (2011). CAS  PubMed  PubMed Central  Google Scholar  *

Egloff, P. et al. Structure of signaling-competent neurotensin receptor 1 obtained by directed evolution in Escherichia coli. _Proc. Natl Acad. Sci. USA_ 111, E655–E662 (2014). CAS  PubMed 

Google Scholar  * Peng, Y. et al. 5-HT2C receptor structures reveal the structural basis of GPCR polypharmacology. _Cell_ 172, 719–730 (2018). CAS  PubMed  PubMed Central  Google Scholar  *

Wacker, D. et al. Structural features for functional selectivity at serotonin receptors. _Science_ 340, 615–619 (2013). CAS  PubMed  PubMed Central  Google Scholar  * White, J. F. et al.

Structure of the agonist-bound neurotensin receptor. _Nature_ 490, 508–513 (2012). CAS  PubMed  PubMed Central  Google Scholar  * Rasmussen, S. G. F. et al. Structure of a

nanobody-stabilized active state of the β(2) adrenoceptor. _Nature_ 469, 175–180 (2011). CAS  PubMed  PubMed Central  Google Scholar  * Ring, A. M. et al. Adrenaline-activated structure of

β2-adrenoceptor stabilized by an engineered nanobody. _Nature_ 502, 575–579 (2013). CAS  PubMed  PubMed Central  Google Scholar  * Rasmussen, S. G. et al. Crystal structure of the β2

adrenergic receptor-Gs protein complex. _Nature_ 477, 549–555 (2011). CAS  PubMed  PubMed Central  Google Scholar  * Heydenreich, F. M., Vuckovic, Z., Matkovic, M. & Veprintsev, D. B.

Stabilization of G protein-coupled receptors by point mutations. _Front. Pharm._ 6, 82 (2015). Google Scholar  * Kofuku, Y. et al. Efficacy of the β2-adrenergic receptor is determined by

conformational equilibrium in the transmembrane region. _Nat. Commun._ 3, 1045 (2012). PubMed  PubMed Central  Google Scholar  * Liu, J. J., Horst, R., Katritch, V., Stevens, R. C. &

Wüthrich, K. Biased signaling pathways in β2-adrenergic receptor characterized by 19F-NMR. _Science_ 1106, 1106–1111 (2012). Google Scholar  * Solt, A. S. et al. Insight into partial agonism

by observing multiple equilibria for ligand-bound and Gs-mimetic nanobody-bound β1-adrenergic receptor. _Nat. Commun._ 8, 1795 (2017). PubMed  PubMed Central  Google Scholar  * Kruse, A. C.

et al. Activation and allosteric modulation of a muscarinic acetylcholine receptor. _Nature_ 504, 101–106 (2013). CAS  PubMed  PubMed Central  Google Scholar  * Manglik, A. et al.

Structural Insights into the dynamic process of β2-adrenergic receptor signaling. _Cell_ 161, 1101–1111 (2015). CAS  PubMed  PubMed Central  Google Scholar  * Gregorio, G. G. et al.

Single-molecule analysis of ligand efficacy in β2AR-G-protein activation. _Nature_ 547, 68–73 (2017). CAS  PubMed  PubMed Central  Google Scholar  * Wingler, L. M. et al. Angiotensin analogs

with divergent bias stabilize distinct receptor conformations. _Cell_ 176, 468–478 (2019). CAS  PubMed  PubMed Central  Google Scholar  * Eddy, M. T. et al. Allosteric coupling of drug

binding and intracellular signaling in the A2A adenosine receptor. _Cell_ 172, 68–80 (2018). CAS  PubMed  Google Scholar  * Roth, C. B., Hanson, M. A. & Stevens, R. C. Stabilization of

the human β2-adrenergic receptor TM4–TM3–TM5 helix interface by mutagenesis of Glu1223.41, a critical residue in GPCR structure. _J. Mol. Biol._ 376, 1305–1319 (2008). CAS  PubMed  Google

Scholar  * Gossert, A. D. et al. A simple protocol for amino acid type selective isotope labeling in insect cells with improved yields and high reproducibility. _J. Biomol. NMR_ 51, 449–456

(2011). CAS  PubMed  Google Scholar  * Battiste, J. L. & Wagner, G. Utilization of site-directed spin labeling and high-resolution heteronuclear nuclear magnetic resonance for global

fold determination of large proteins with limited nuclear Overhauser effect data. _Biochemistry_ 39, 5355–5365 (2000). CAS  PubMed  Google Scholar  * Anthis, N. J. & Clore, G. M.

Visualizing transient dark states by NMR spectroscopy. _Q. Rev. Biophys._ 48, 35–116 (2015). CAS  PubMed  PubMed Central  Google Scholar  * King, G. J. et al. The Arabidopsis B3 domain

protein VERNALIZATION1 (VRN1) is involved in processes essential for development, with structural and mutational studies revealing its DNA-binding surface. _J. Biol. Chem._ 288, 3198–3207

(2013). CAS  PubMed  Google Scholar  * Reibarkh, M., Malia, T. J. & Wagner, G. NMR distinction of single- and multiple-mode binding of small-molecule protein ligands. _J. Am. Chem. Soc._

128, 2160–2161 (2006). CAS  PubMed  PubMed Central  Google Scholar  * Hobbs, C. A., Bobay, B. G., Thompson, R. J., Perego, M. & Cavanagh, J. NMR solution structure and DNA-binding model

of the DNA-binding domain of competence protein A. _J. Mol. Biol._ 398, 248–263 (2010). CAS  PubMed  PubMed Central  Google Scholar  * Takeuchi, K., Imai, M. & Shimada, I. Dynamic

equilibrium on DNA defines transcriptional regulation of a multidrug binding transcriptional repressor, LmrR. _Sci. Rep._ 7, 267 (2017). PubMed  PubMed Central  Google Scholar  * Minato, Y.,

Ueda, T., Machiyama, A., Iwaï, H. & Shimada, I. Dynamic domain arrangement of CheA–CheY complex regulates bacterial thermotaxis, as revealed by NMR. _Sci. Rep._ 7, 16462 (2017). PubMed

  PubMed Central  Google Scholar  * Bain, A. D. Chemical exchange in NMR. _Prog. Nucl. Magn. Reson. Spectrosc._ 43, 63–103 (2003). CAS  Google Scholar  * Waudby, C. A., Ramos, A., Cabrita,

L. D. & Christodoulou, J. Two-dimensional NMR lineshape analysis. _Sci. Rep._ 6, 24826 (2016). CAS  PubMed  PubMed Central  Google Scholar  * Hukushima, K. & Nemoto, K. Exchange

Monte Carlo method and application to spin glass simulations. _J. Phys. Soc. Jpn._ 65, 1604–1608 (1996). CAS  Google Scholar  * Nagata, K., Sugita, S. & Okada, M. Bayesian spectral

deconvolution with the exchange Monte Carlo method. _Neural Netw._ 28, 82–89 (2012). PubMed  Google Scholar  * Fernández, C. & Wider, G. TROSY in NMR studies of the structure and

function of large biological macromolecules. _Curr. Opin. Struct. Biol._ 13, 570–580 (2003). PubMed  Google Scholar  * Lapinaite, A. et al. The structure of the box C/D enzyme reveals

regulation of RNA methylation. _Nature_ 502, 519–523 (2013). CAS  PubMed  Google Scholar  * Gottstein, D., Reckel, S., Dötsch, V. & Güntert, P. Requirements on paramagnetic relaxation

enhancement data for membrane protein structure determination by NMR. _Structure_ 20, 1019–1027 (2012). CAS  PubMed  Google Scholar  * Isogai, S. et al. Backbone NMR reveals allosteric

signal transduction networks in the β1-adrenergic receptor. _Nature_ 314, 1–17 (2016). Google Scholar  * Grzesiek, S., Cordier, F., Jaravine, V. & Barfield, M. Insights into biomolecular

hydrogen bonds from hydrogen bond scalar couplings. _Prog. Nucl. Magn. Reson. Spectrosc._ 45, 275–300 (2004). CAS  Google Scholar  * Zou, Y., Weis, W. I. & Kobilka, B. K. N-terminal T4

lysozyme fusion facilitates crystallization of a G protein coupled receptor. _PLoS One_ 7, e46039 (2012). CAS  PubMed  PubMed Central  Google Scholar  * Rosenbaum, D. M. et al. GPCR

engineering yields high-resolution structural insights into 2-adrenergic receptor function. _Science_ 318, 1266–1273 (2007). CAS  PubMed  Google Scholar  * Fung, J. J. et al.

Ligand-regulated oligomerization of β(2)-adrenoceptors in a model lipid bilayer. _EMBO J._ 28, 3315–3328 (2009). CAS  PubMed  PubMed Central  Google Scholar  * Nietlispach, D. Suppression of

anti-TROSY lines in a sensitivity enhanced gradient selection TROSY scheme. _J. Biomol. NMR_ 31, 161–166 (2005). CAS  PubMed  Google Scholar  * Helmus, J. J. & Jaroniec, C. P. NMRglue:

an open source Python package for the analysis of multidimensional NMR data. _J. Biomol. NMR_ 55, 355–367 (2013). CAS  PubMed  PubMed Central  Google Scholar  * Sali, A. & Blundell, T.

L. Comparative protein modelling by satisfaction of spatial restraints. _J. Mol. Biol._ 234, 779–815 (1993). CAS  PubMed  Google Scholar  * Schwieters, C. D., Bermejo, G. A. & Clore, G.

M. Xplor-NIH for molecular structure determination from NMR and other data sources. _Protein Sci._ 27, 26–40 (2018). CAS  PubMed  Google Scholar  * Iwahara, J., Schwieters, C. D. &

Clore, G. M. Ensemble approach for NMR structure refinement against 1H paramagnetic relaxation enhancement data arising from a flexible paramagnetic group attached to a macromolecule. _J.

Am. Chem. Soc._ 126, 5879–5896 (2004). CAS  PubMed  Google Scholar  * McConnell, H. M. Reaction rates by nuclear magnetic resonance. _J. Chem. Phys._ 28, 430–431 (1958). CAS  Google Scholar

  Download references 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.).

AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Shunsuke Imai, Tomoki Yokomizo, Yutaka Kofuku, Yutaro

Shiraishi, Takumi Ueda & Ichio Shimada Authors * Shunsuke Imai View author publications You can also search for this author inPubMed Google Scholar * Tomoki Yokomizo View author

publications You can also search for this author inPubMed Google Scholar * Yutaka Kofuku View author publications You can also search for this author inPubMed Google Scholar * Yutaro

Shiraishi View author publications You can also search for this author inPubMed Google Scholar * Takumi Ueda View author publications You can also search for this author inPubMed Google

Scholar * Ichio Shimada View author publications You can also search for this author inPubMed Google Scholar 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. CORRESPONDING AUTHOR Correspondence to Ichio Shimada. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Figs.

1–13 and Supplementary Table 1 REPORTING SUMMARY RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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 Download citation * Received: 13 July

2019 * Revised: 01 December 2019 * Accepted: 17 December 2019 * Published: 20 January 2020 * Issue Date: April 2020 * DOI: https://doi.org/10.1038/s41589-019-0457-5 SHARE THIS ARTICLE Anyone

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