Function-related conformational dynamics of G protein–coupled receptors revealed by NMR

  • Takumi Ueda
  • Yutaka Kofuku
  • Junya Okude
  • Shunsuke Imai
  • Yutaro Shiraishi
  • Ichio ShimadaEmail author


G protein–coupled receptors (GPCRs) function as receptors for various neurotransmitters, hormones, cytokines, and metabolites. GPCR ligands impart differing degrees of signaling in the G protein and arrestin pathways, in phenomena called biased signaling, and each ligand for a given GPCR has a characteristic level of ability to activate or deactivate its target, which is referred to as its efficacy. The ligand efficacies and biased signaling of GPCRs remarkably affect the therapeutic properties of the ligands. However, these features of GPCRs can only be partially understood from the crystallography data, although numerous GPCR structures have been solved. NMR analyses have revealed that GPCRs have multiple interconverting substates, exchanging on various timescales, and that the exchange rates are related to the ligand efficacies and biased signaling. In addition, NMR analyses of GPCRs in the lipid bilayer environment of rHDLs revealed that the exchange rates are modulated by the lipid bilayer environment, highlighting the importance of the function-related dynamics in the lipid bilayer. In this review, we will describe several solution NMR studies that have clarified the conformational dynamics related to the ligand efficacy and biased signaling of GPCRs.


Nuclear magnetic resonance Membrane protein Adrenergic receptor Opioid receptor Nanodiscs 



This work is supported by The Ministry of Education, Culture, Sports, Science and Technology (MEXT)/Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers JP17H06097, JP18H04540, JP16H01531, JP17H04999, JP16H01353, JP15K18843, JP15J12409, and JP17J1142, and the development of core technologies for innovative drug development based upon IT and the development of innovative drug discovery technologies for middle-sized molecules, from the Japan Agency for Medical Research and Development, AMED (I.S.).

Compliance with ethical standards

Conflict of interest

Takumi Ueda declares that he has no conflict of interest. Yutaka Kofuku declares that he has no conflict of interest. Junya Okude declares that he has no conflict of interest. Shunsuke Imai declares that he has no conflict of interest. Yutaro Shiraishi declares that he has no conflict of interest. Ichio Shimada declares that he has no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.


  1. Ballesteros JA, Weinstein H (1995) Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G-protein coupled receptors. Methods Neurosci 25:366–428CrossRefGoogle Scholar
  2. Bayburt TH, Grinkova YV, Sligar SG (2002) Self-assembly of discoidal phospholipid bilayer nanoparticles with membrane scaffold proteins. Nano Lett 2:853–856CrossRefGoogle Scholar
  3. Bhabha G, Lee J, Ekiert DC, Gam J, Wilson IA, Dyson HJ, Benkovic SJ, Wright PE (2011) A dynamic knockout reveals that conformational fuctuations influence the chemical step of enzyme catalysis. Science 332:234–238CrossRefPubMedCentralGoogle Scholar
  4. Bohn LM, Lefkowitz RJ, Gainetdinov RR, Peppel K, Caron MG, Lin FT (1999) Enhanced morphine analgesia in mice lacking β-arrestin 2. Science 286:2495–2498CrossRefGoogle Scholar
  5. Brüschweiler S, Yang Q, Run C, Chou JJ (2015) Substrate-modulated ADP/ATP-transporter dynamics revealed by NMR relaxation dispersion. Nat Struct Mol Biol 22:636–641CrossRefPubMedCentralGoogle Scholar
  6. Bünemann M, Bücheler MM, Philipp M, Lohse MJ, Hein L (2001) Activation and deactivation kinetics of alpha 2A- and alpha 2C-adrenergic receptor-activated G protein-activated inwardly rectifying K+ channel currents. J Biol Chem 276:47512–47517CrossRefGoogle Scholar
  7. Butterfoss GL, DeRose EF, Gabel SA, Perera L, Krahn JM, Mueller GA, Zheng X, London RE (2010) Conformational dependence of 13C shielding and coupling constants for methionine methyl groups. J Biomol NMR 48:31–47CrossRefPubMedCentralGoogle Scholar
  8. Chen XT, Pitis P, Liu G, Yuan C, Gotchev D, Cowan CL, Rominger DH, Koblish M, Dewire SM, Crombie AL, Violin JD, Yamashita DS (2013) Structure-activity relationships and discovery of a G protein biased μ opioid receptor ligand, [(3-methoxythiophen-2-yl)methyl]({2-[(9R)-9-(pyridin-2-yl)-6-oxaspiro-[4.5]decan- 9-yl]ethyl})amine (TRV130), for the treatment of acute severe pain. J Med Chem 56:8019–8031CrossRefGoogle Scholar
  9. Cherezov V, Rosenbaum DM, Hanson MA, Rasmussen SG, Thian FS, Kobilka TS, Choi HJ, Kuhn P, Weis WI, Kobilka BK, Stevens RC (2007) High-resolution crystal structure of an engineered human β2-adrenergic G protein-coupled receptor. Science 318:1258–1265CrossRefPubMedCentralGoogle Scholar
  10. Fenalti G, Giguere PM, Katritch V, Huang XP, Thompson AA, Cherezov V, Roth BL, Stevens RC (2014) Molecular control of δ-opioid receptor signalling. Nature 506:191–196CrossRefPubMedCentralGoogle Scholar
  11. Gregorio GG, Masureel M, Hilger D, Terry DS, Juette M, Zhao H, Zhou Z, Perez-Aguilar JM, Hauge M, Mathiasen S, Javitch JA, Weinstein H, Kobilka BK, Blanchard SC (2017) Single-molecule analysis of ligand efficacy in β2AR-G-protein activation. Nature 547:68–73CrossRefPubMedCentralGoogle Scholar
  12. Hanania NA, Dickey BF, Bond RA (2010) Clinical implications of the intrinsic efficacy of β-adrenoceptor drugs in asthma: full, partial and inverse agonism. Curr Opin Pulm Med 16:1–5CrossRefPubMedCentralGoogle Scholar
  13. Hauser AS, Attwood MM, Rask-Andersen M, Schiöth HB, Gloriam DE (2017) Trends in GPCR drug discovery: new agents, targets and indications. Nat Rev Drug Discov 16:829–842CrossRefGoogle Scholar
  14. Huang W, Manglik A, Venkatakrishnan AJ, Laeremans T, Feinberg EN, Sanborn AL, Kato HE, Livingston KE, Thorsen TS, Kling RC, Granier S, Gmeiner P, Husbands SM, Traynor JR, Weis WI, Steyaert J, Dror RO, Kobilka BK (2015) Structural insights into μ-opioid receptor activation. Nature 524:315–321CrossRefPubMedCentralGoogle Scholar
  15. Ikeya T, Ban D, Lee D, Ito Y, Kato K, Griesinger C (2018) Solution NMR views of dynamical ordering of biomacromolecules. Biochim Biophys Acta 1862:287–306CrossRefGoogle Scholar
  16. Imai S, Osawa M, Mita K, Toyonaga S, Machiyama A, Ueda T, Takeuchi K, Oiki S, Shimada I (2012) Functional equilibrium of the KcsA structure revealed by NMR. J Biol Chem 287:39634–39641CrossRefPubMedCentralGoogle Scholar
  17. Kahsai AW, Xiao K, Rajagopal S, Ahn S, Shukla AK, Sun J, Oas TG, Lefkowitz RJ (2011) Multiple ligand-specific conformations of the β2-adrenergic receptor. Nat Chem Biol 7:692–700CrossRefPubMedCentralGoogle Scholar
  18. Kerns SJ, Agafonov RV, Cho YJ, Pontiggia F, Otten R, Pachov DV, Kutter S, Phung LA, Murphy PN, Thai V, Alber T, Hagan MF, Kern D (2015) The energy landscape of adenylate kinase during catalysis. Nat Struct Mol Biol 22:124–131CrossRefPubMedCentralGoogle Scholar
  19. Koehl A, Hu H, Maeda S, Zhang Y, Qu Q, Paggi JM, Latorraca NR, Hilger D, Dawson R, Matile H, Schertler GFX, Granier S, Weis WI, Dror RO, Manglik A, Skiniotis G, Kobilka BK (2018) Structure of the μ-opioid receptor-G. Nature 558:547–552CrossRefPubMedCentralGoogle Scholar
  20. Kofuku Y, Ueda T, Okude J, Shiraishi Y, Kondo K, Maeda M, Tsujishita H, Shimada I (2012) Efficacy of the β2-adrenergic receptor is determined by conformational equilibrium in the transmembrane region. Nat Commun 3:1045CrossRefPubMedCentralGoogle Scholar
  21. Kofuku Y, Ueda T, Okude J, Shiraishi Y, Kondo K, Mizumura T, Suzuki S, Shimada I (2014) Functional dynamics of deuterated β2−adrenergic receptor in lipid bilayers revealed by NMR spectroscopy. Angew Chem Int Ed 53:13376–13379CrossRefGoogle Scholar
  22. Kofuku Y, Yokomizo T, Imai S, Shiraishi Y, Natsume M, Itoh H, Inoue M, Nakata K, Igarashi S, Yamaguchi H, Mizukoshi T, Suzuki EI, Ueda T, Shimada I (2018) Deuteration and selective labeling of alanine methyl groups of β 2-adrenergic receptor expressed in a baculovirus-insect cell expression system. J Biomol NMR 71(3):185–192CrossRefGoogle Scholar
  23. Lange OF, Lakomek NA, Farès C, Schröder GF, Walter KF, Becker S, Meiler J, Grubmüller H, Griesinger C, de Groot BL (2008) Recognition dynamics up to microseconds revealed from an RDC-derived ubiquitin ensemble in solution. Science 320:1471–1475CrossRefGoogle Scholar
  24. Liu JJ, Horst R, Katritch V, Stevens RC, Wüthrich K (2012) Biased signaling pathways in β2-adrenergic receptor characterized by 19F-NMR. Science 335:1106–1110CrossRefPubMedCentralGoogle Scholar
  25. Majumdar S, Devi LA (2018) Strategy for making safer opioids bolstered. Nature 553:286–288CrossRefGoogle Scholar
  26. Manglik A, Kruse AC, Kobilka TS, Thian FS, Mathiesen JM, Sunahara RK, Pardo L, Weis WI, Kobilka BK, Granier S (2012) Crystal structure of the μ-opioid receptor bound to a morphinan antagonist. Nature 485:321–326CrossRefPubMedCentralGoogle Scholar
  27. Manglik A, Kim TH, Masureel M, Altenbach C, Yang Z, Hilger D, Lerch MT, Kobilka TS, Thian FS, Hubbell WL, Prosser RS, Kobilka BK (2015) Structural insights into the dynamic process of β2-adrenergic receptor signaling. Cell 161:1101–1111CrossRefPubMedCentralGoogle Scholar
  28. Minato Y, Suzuki S, Hara T, Kofuku Y, Kasuya G, Fujiwara Y, Igarashi S, Suzuki E, Nureki O, Hattori M, Ueda T, Shimada I (2016) Conductance of P2X4 purinergic receptor is determined by conformational equilibrium in the transmembrane region. Proc Natl Acad Sci U S A 113:4741–4746CrossRefPubMedCentralGoogle Scholar
  29. Mittermaier A, Kay L (2009) Observing biological dynamics at atomic resolution using NMR. Trends Biochem Sci 34:601–611CrossRefGoogle Scholar
  30. Morrison EA, DeKoster GT, Dutta S, Vafabakhsh R, Clarkson MW, Bahl A, Kern D, Ha T, Henzler-Wildman KA (2011) Antiparallel EmrE exports drugs by exchanging between asymmetric structures. Nature 481:45–50CrossRefPubMedCentralGoogle Scholar
  31. Nikolaev VO, Bünemann M, Hein L, Hannawacker A, Lohse MJ (2004) Novel single chain cAMP sensors for receptor-induced signal propagation. J Biol Chem 279:37215–37218CrossRefGoogle Scholar
  32. Nishida N, Osawa M, Takeuchi K, Imai S, Stampoulis P, Kofuku Y, Ueda T, Shimada I (2014) Functional dynamics of cell surface membrane proteins. J Magn Reson 241:86–96CrossRefGoogle Scholar
  33. Okude J, Ueda T, Kofuku Y, Sato M, Nobuyama N, Kondo K, Shiraishi Y, Mizumura T, Onishi K, Natsume M, Maeda M, Tsujishita H, Kuranaga T, Inoue M, Shimada I (2015) Identification of a conformational equilibrium that determines the efficacy and functional selectivity of the μ-opioid receptor. Angew Chem Int Ed Engl 54:15771–15776CrossRefPubMedCentralGoogle Scholar
  34. Osawa M, Takeuchi K, Ueda T, Nishida N, Shimada I (2012) Functional dynamics of proteins revealed by solution NMR. Curr Opin Struct Biol 22:660–669CrossRefGoogle Scholar
  35. Pándy-Szekeres G, Munk C, Tsonkov TM, Mordalski S, Harpsøe K, Hauser AS, Bojarski AJ, Gloriam DE (2018) GPCRdb in 2018: adding GPCR structure models and ligands. Nucleic Acids Res 46:D440–D446CrossRefGoogle Scholar
  36. Perkins SJ, Wüthrich K (1979) Ring current effects in the conformation dependent NMR chemical shifts of aliphatic protons in the basic pancreatic trypsin inhibitor. Biochim Biophys Acta 576:409–423CrossRefGoogle Scholar
  37. Rasmussen SG, DeVree BT, Zou Y, Kruse AC, Chung KY, Kobilka TS, Thian FS, Chae PS, Pardon E, Calinski D, Mathiesen JM, Shah ST, Lyons JA, Caffrey M, Gellman SH, Steyaert J, Skiniotis G, Weis WI, Sunahara RK, Kobilka BK (2011) Crystal structure of the β2 adrenergic receptor-Gs protein complex. Nature 477:549–555CrossRefPubMedCentralGoogle Scholar
  38. Saucerman JJ, Brunton LL, Michailova AP, McCulloch AD (2003) Modeling beta-adrenergic control of cardiac myocyte contractility in silico. J Biol Chem 278:47997–48003CrossRefGoogle Scholar
  39. Shimada I, Ueda T, Kofuku Y, Eddy MT, Wüthrich K (2018) GPCR drug discovery: integrating solution NMR data with crystal and cryo-EM structures. Nat Rev Drug DiscovGoogle Scholar
  40. Smith JS, Lefkowitz RJ, Rajagopal S (2018) Biased signalling: from simple switches to allosteric microprocessors. Nat Rev Drug DiscovGoogle Scholar
  41. Staus DP, Strachan RT, Manglik A, Pani B, Kahsai AW, Kim TH, Wingler LM, Ahn S, Chatterjee A, Masoudi A, Kruse AC, Pardon E, Steyaert J, Weis WI, Prosser RS, Kobilka BK, Costa T, Lefkowitz RJ (2016) Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation. Nature 535:448–452CrossRefPubMedCentralGoogle Scholar
  42. Swift JL, Godin AG, Doré K, Freland L, Bouchard N, Nimmo C, Sergeev M, De Koninck Y, Wiseman PW, Beaulieu JM (2011) Quantification of receptor tyrosine kinase transactivation through direct dimerization and surface density measurements in single cells. Proc Natl Acad Sci U S A 108:7016–7021CrossRefPubMedCentralGoogle Scholar
  43. Urban JD, Clarke WP, von Zastrow M, Nichols DE, Kobilka B, Weinstein H, Javitch JA, Roth BL, Christopoulos A, Sexton PM, Miller KJ, Spedding M, Mailman RB (2007) Functional selectivity and classical concepts of quantitative pharmacology. J Pharmacol Exp Ther 320:1–13CrossRefGoogle Scholar
  44. West GM, Chien EY, Katritch V, Gatchalian J, Chalmers MJ, Stevens RC, Griffin PR (2011) Ligand-dependent perturbation of the conformational ensemble for the GPCR β2 adrenergic receptor revealed by HDX. Structure 19:1424–1432CrossRefPubMedCentralGoogle Scholar

Copyright information

© International Union for Pure and Applied Biophysics (IUPAB) and Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Takumi Ueda
    • 1
    • 2
  • Yutaka Kofuku
    • 1
  • Junya Okude
    • 1
  • Shunsuke Imai
    • 1
  • Yutaro Shiraishi
    • 1
  • Ichio Shimada
    • 1
    Email author
  1. 1.Graduate School of Pharmaceutical SciencesThe University of TokyoTokyoJapan
  2. 2.Precursory Research for Embryonic Science and TechnologyJapan Science and Technology AgencyKawaguchiJapan

Personalised recommendations