Structure of human GABAB receptor in an inactive state

Abstract

The human GABAB receptor—a member of the class C family of G-protein-coupled receptors (GPCRs)—mediates inhibitory neurotransmission and has been implicated in epilepsy, pain and addiction1. A unique GPCR that is known to require heterodimerization for function2,3,4,5,6, the GABAB receptor has two subunits, GABAB1 and GABAB2, that are structurally homologous but perform distinct and complementary functions. GABAB1 recognizes orthosteric ligands7,8, while GABAB2 couples with G proteins9,10,11,12,13,14. Each subunit is characterized by an extracellular Venus flytrap (VFT) module, a descending peptide linker, a seven-helix transmembrane domain and a cytoplasmic tail15. Although the VFT heterodimer structure has been resolved16, the structure of the full-length receptor and its transmembrane signalling mechanism remain unknown. Here we present a near full-length structure of the GABAB receptor at atomic resolution, captured in an inactive state by cryo-electron microscopy. Our structure reveals several ligands that preassociate with the receptor, including two large endogenous phospholipids that are embedded within the transmembrane domains to maintain receptor integrity and modulate receptor function. We also identify a previously unknown heterodimer interface between transmembrane helices 3 and 5 of both subunits, which serves as a signature of the inactive conformation. A unique ‘intersubunit latch’ within this transmembrane interface maintains the inactive state, and its disruption leads to constitutive receptor activity.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Cryo-EM structure of human GABAB receptor.
Fig. 2: Transmembrane heterodimer interface of the GABAB receptor.
Fig. 3: Ca2+ binding in GABAB1b.
Fig. 4: Identification of endogenous phospholipid ligands of the GABAB receptor.

Data availability

All data are included in this paper and its Supplementary Information. Cryo-EM density maps of the GABAB receptor have been deposited in the Electron Microscopy Data Bank (https://www.ebi.ac.uk/pdbe/emdb/) under accession code EMD-21685. Atomic coordinates for the GABAB receptor structure have been deposited in the RCSB Protein Data Bank under accession code 6WIV. Raw cryo-EM images have been deposited in the Electron Microscopy Public Image Archive (https://www.ebi.ac.uk/pdbe/emdb/empiar/) under accession code EMPIAR-10410.

References

  1. 1.

    Bettler, B., Kaupmann, K., Mosbacher, J. & Gassmann, M. Molecular structure and physiological functions of GABAB receptors. Physiol. Rev. 84, 835–867 (2004).

    CAS  PubMed  Google Scholar 

  2. 2.

    Jones, K. A. et al. GABAB receptors function as a heteromeric assembly of the subunits GABABR1 and GABABR2. Nature 396, 674–679 (1998).

    ADS  CAS  PubMed  Google Scholar 

  3. 3.

    Kaupmann, K. et al. GABAB-receptor subtypes assemble into functional heteromeric complexes. Nature 396, 683–687 (1998).

    ADS  CAS  PubMed  Google Scholar 

  4. 4.

    White, J. H. et al. Heterodimerization is required for the formation of a functional GABAB receptor. Nature 396, 679–682 (1998).

    ADS  CAS  PubMed  Google Scholar 

  5. 5.

    Kuner, R. et al. Role of heteromer formation in GABAB receptor function. Science 283, 74–77 (1999).

    ADS  CAS  PubMed  Google Scholar 

  6. 6.

    Ng, G. Y. et al. Identification of a GABAB receptor subunit, gb2, required for functional GABAB receptor activity. J. Biol. Chem. 274, 7607–7610 (1999).

    CAS  PubMed  Google Scholar 

  7. 7.

    Kaupmann, K. et al. Expression cloning of GABAB receptors uncovers similarity to metabotropic glutamate receptors. Nature 386, 239–246 (1997).

    ADS  CAS  PubMed  Google Scholar 

  8. 8.

    Malitschek, B. et al. The N-terminal domain of γ-aminobutyric acidB receptors is sufficient to specify agonist and antagonist binding. Mol. Pharmacol. 56, 448–454 (1999).

    CAS  PubMed  Google Scholar 

  9. 9.

    Galvez, T. et al. Allosteric interactions between GB1 and GB2 subunits are required for optimal GABAB receptor function. EMBO J. 20, 2152–2159 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Margeta-Mitrovic, M., Jan, Y. N. & Jan, L. Y. Function of GB1 and GB2 subunits in G protein coupling of GABAB receptors. Proc. Natl Acad. Sci. USA 98, 14649–14654 (2001).

    ADS  CAS  PubMed  Google Scholar 

  11. 11.

    Robbins, M. J. et al. GABAB2 is essential for G-protein coupling of the GABAB receptor heterodimer. J. Neurosci. 21, 8043–8052 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Duthey, B. et al. A single subunit (GB2) is required for G-protein activation by the heterodimeric GABAB receptor. J. Biol. Chem. 277, 3236–3241 (2002).

    CAS  PubMed  Google Scholar 

  13. 13.

    Havlickova, M. et al. The intracellular loops of the GB2 subunit are crucial for G-protein coupling of the heteromeric γ-aminobutyrate B receptor. Mol. Pharmacol. 62, 343–350 (2002).

    CAS  PubMed  Google Scholar 

  14. 14.

    Monnier, C. et al. Trans-activation between 7TM domains: implication in heterodimeric GABAB receptor activation. EMBO J. 30, 32–42 (2011).

    ADS  CAS  PubMed  Google Scholar 

  15. 15.

    Pin, J. P. & Bettler, B. Organization and functions of mGlu and GABAB receptor complexes. Nature 540, 60–68 (2016).

    ADS  CAS  PubMed  Google Scholar 

  16. 16.

    Geng, Y., Bush, M., Mosyak, L., Wang, F. & Fan, Q. R. Structural mechanism of ligand activation in human GABAB receptor. Nature 504, 254–259 (2013).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Burmakina, S., Geng, Y., Chen, Y. & Fan, Q. R. Heterodimeric coiled-coil interactions of human GABAB receptor. Proc. Natl Acad. Sci. USA 111, 6958–6963 (2014).

    ADS  CAS  PubMed  Google Scholar 

  18. 18.

    Conklin, B. R., Farfel, Z., Lustig, K. D., Julius, D. & Bourne, H. R. Substitution of three amino acids switches receptor specificity of Gqα to that of Giα. Nature 363, 274–276 (1993).

    ADS  CAS  PubMed  Google Scholar 

  19. 19.

    Xue, L. et al. Rearrangement of the transmembrane domain interfaces associated with the activation of a GPCR hetero-oligomer. Nat. Commun. 10, 2765 (2019).

    ADS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Koehl, A. et al. Structural insights into the activation of metabotropic glutamate receptors. Nature 566, 79–84 (2019); correction 567, E10 (2019).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Geng, Y. et al. Structural mechanism of ligand activation in human calcium-sensing receptor. eLife 5, e13662 (2016).

    PubMed  PubMed Central  Google Scholar 

  22. 22.

    Wise, A. et al. Calcium sensing properties of the GABAB receptor. Neuropharmacology 38, 1647–1656 (1999).

    CAS  PubMed  Google Scholar 

  23. 23.

    Galvez, T. et al. Ca2+ requirement for high-affinity gamma-aminobutyric acid (GABA) binding at GABAB receptors: involvement of serine 269 of the GABABR1 subunit. Mol. Pharmacol. 57, 419–426 (2000).

    CAS  PubMed  Google Scholar 

  24. 24.

    Hanson, M. A. et al. Crystal structure of a lipid G protein-coupled receptor. Science 335, 851–855 (2012).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Rasmussen, S. G. et al. Crystal structure of the β2 adrenergic receptor-Gs protein complex. Nature 477, 549–555 (2011).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Thal, D. M., Glukhova, A., Sexton, P. M. & Christopoulos, A. Structural insights into G-protein-coupled receptor allostery. Nature 559, 45–53 (2018).

    ADS  CAS  PubMed  Google Scholar 

  27. 27.

    Palczewski, K. et al. Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289, 739–745 (2000).

    ADS  CAS  PubMed  Google Scholar 

  28. 28.

    Doré, A. S. et al. Structure of class C GPCR metabotropic glutamate receptor 5 transmembrane domain. Nature 511, 557–562 (2014).

    ADS  PubMed  Google Scholar 

  29. 29.

    Wu, H. et al. Structure of a class C GPCR metabotropic glutamate receptor 1 bound to an allosteric modulator. Science 344, 58–64 (2014).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Goehring, A. et al. Screening and large-scale expression of membrane proteins in mammalian cells for structural studies. Nat. Protocols 9, 2574–2585 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Margeta-Mitrovic, M., Jan, Y. N. & Jan, L. Y. A trafficking checkpoint controls GABAB receptor heterodimerization. Neuron 27, 97–106 (2000).

    CAS  PubMed  Google Scholar 

  32. 32.

    Pagano, A. et al. C-terminal interaction is essential for surface trafficking but not for heteromeric assembly of GABAB receptors. J. Neurosci. 21, 1189–1202 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Reeves, P. J., Callewaert, N., Contreras, R. & Khorana, H. G. Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. Proc. Natl Acad. Sci. USA 99, 13419–13424 (2002).

    ADS  CAS  PubMed  Google Scholar 

  34. 34.

    Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Zhang, K. Gctf: real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    CAS  Google Scholar 

  38. 38.

    Scheres, S. H. & Chen, S. Prevention of overfitting in cryo-EM structure determination. Nat. Methods 9, 853–854 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Tan, Y. Z. et al. Addressing preferred specimen orientation in single-particle cryo-EM through tilting. Nat. Methods 14, 793–796 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Punjani, A. & Fleet, D. J. 3D variability analysis: directly resolving continuous flexibility and discrete heterogeneity from single particle cryo-EM images. bioRxiv https://doi.org/10.1101/2020.04.08.032466 (2020).

  42. 42.

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Barad, B. A. et al. EMRinger: side chain-directed model and map validation for 3D cryo-electron microscopy. Nat. Methods 12, 943–946 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Novotny, M., Madsen, D. & Kleywegt, G. J. Evaluation of protein fold comparison servers. Proteins 54, 260–270 (2004).

    CAS  PubMed  Google Scholar 

  47. 47.

    Goddard, T. D. et al. UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018).

    CAS  Google Scholar 

  48. 48.

    Morin, A. et al. Collaboration gets the most out of software. eLife 2, e01456 (2013).

    PubMed  PubMed Central  Google Scholar 

  49. 49.

    Geng, Y. et al. Structure and functional interaction of the extracellular domain of human GABAB receptor GBR2. Nat. Neurosci. 15, 970–978 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Quick, M. & Javitch, J. A. Monitoring the function of membrane transport proteins in detergent-solubilized form. Proc. Natl Acad. Sci. USA 104, 3603–3608 (2007).

    ADS  CAS  PubMed  Google Scholar 

  51. 51.

    Gupta, K. et al. Identifying key membrane protein lipid interactions using mass spectrometry. Nat. Protocols 13, 1106–1120 (2018).

    CAS  PubMed  Google Scholar 

  52. 52.

    Mafu, S. et al. Biosynthesis of the microtubule-destabilizing diterpene pseudolaric acid B from golden larch involves an unusual diterpene synthase. Proc. Natl Acad. Sci. USA 114, 974–979 (2017).

    CAS  PubMed  Google Scholar 

  53. 53.

    Kind, T. et al. LipidBlast in silico tandem mass spectrometry database for lipid identification. Nat. Methods 10, 755–758 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Mukherjee, R. S., McBride, E. W., Beinborn, M., Dunlap, K. & Kopin, A. S. Point mutations in either subunit of the GABAB receptor confer constitutive activity to the heterodimer. Mol. Pharmacol. 70, 1406–1413 (2006).

    CAS  PubMed  Google Scholar 

  55. 55.

    Chen, K. L. B., Amarasiriwardena, C. J. & Christiani, D. C. Determination of total arsenic concentrations in nails by inductively coupled plasma mass spectrometry. Biol. Trace Elem. Res. 67, 109–125 (1999).

    CAS  PubMed  Google Scholar 

  56. 56.

    Pruszkowski, E., Neubauer, K. & Thomas, R. An overview of clinical applications by inductively coupled plasma mass spectrometry. Atomic Spectroscopy 19, 111–115 (1998).

    CAS  Google Scholar 

  57. 57.

    Hollenstein, K. et al. Structure of class B GPCR corticotropin-releasing factor receptor 1. Nature 499, 438–443 (2013).

    ADS  CAS  PubMed  Google Scholar 

  58. 58.

    Wang, C. et al. Structure of the human smoothened receptor bound to an antitumour agent. Nature 497, 338–343 (2013).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank R. Henderson for early-stage cryo-EM investigation and critical reading of the manuscript; C. S. Zuker for advice and financial support; C. Karan and R. Realubit for assistance with the EnVision plate reader at Columbia Genome Center; A. Sobolevsky, K. Saotome and E. Cao for BacMam vectors; Y. H. Wong for a Gαqi5 chimera plasmid; and B. K. Kobilka and B. Skiniotis for advice. Titan Krios data collection was performed at the Simons Electron Microscopy Center, directed by B. O. Carragher and C. Potter, supported by grants from the Simons Foundation (SF349247), NYSTAR and the National Institutes of Health (NIH; GM103310). This work was supported by NIH grants R01GM088454 (to Q.R.F.), R01GM125801 (to Q.R.F., P.A.S. and M.Q.), R01GM107462 (to W.A.H.), P41GM116799 (to W.A.H.), and U2C ES030158 for lipid identification (to O.F.). Q.R.F. was an Irma Hirschl Career Scientist, Pew Scholar, McKnight Scholar and Schaefer Scholar.

Author information

Affiliations

Authors

Contributions

J.P., J.L., Q.R.F. and K.M.R. cultured cells and purified protein; Z.F. and A.F. prepared cryo-EM grids; Z.F., A.F., Q.R.F. and O.B.C. collected cryo-EM data; Z.F. and A.F. performed initial image processing; O.B.C. processed cryo-EM data to high resolution; L.M. and Q.R.F. built and refined models; Q.R.F., L.M., A.F., J.P. and Z.F. analysed structures; T.S. and O.F. identified phospholipids and GABA by mass spectrometry; V.N.S. and J.G. conducted elemental analysis; Q.R.F, J.L., J.P., A.F., J.T., X.L. and J.P.W. performed mutagenesis and cell-based functional assays; M.Q. performed and analysed radioligand binding studies; B.C, Y.G., H.Z. and Y.K. generated expression plasmids and carried out early protein purification trials; R.G., W.J.R., E.T.E., R.K.H. and Z.Y. assisted with cryo-EM data collection; S.C., Z.L., W.J.R. and E.T.E. performed initial cryo-EM characterization; R.K.S. measured molecular mass; B.K. screened detergents; W.A.H. contributed to structural analysis; Q.R.F., P.A.S. and J.A.J. supervised functional analyses; J.F., O.B.C. and H.Y. supervised cryo-EM studies; Q.R.F. and A.F. wrote the paper; T.S., V.N.S., M.Q., Z.F. and R.K.S. contributed Methods sections; all authors contributed to revision of the manuscript; Q.R.F., A.F., J.P., O.B.C., T.S., Z.F., J.L. and M.Q. prepared figures; Q.R.F. conceived and supervised the project.

Corresponding authors

Correspondence to Oliver B. Clarke or Joachim Frank or Qing R. Fan.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks Ryan Hibbs, Bernhard Bettler and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Purification and functional analysis of the human GABAB receptor.

a, Superose 6 size-exclusion chromatography profile of detergent-purified GABAB1b(1–802)–GABAB2(1–819) complex. b, SDS PAGE gel of the size-exclusion peak fraction from a under reducing conditions. For gel source data, see Supplementary Fig. 2. c, Dose-dependent [3H]GABA binding to purified GABAB1b(1–802)–GABAB2(1–819) complex, reaching maximum molar ratios of GABA-to-receptor binding at 0.98 ± 0.03 mol mol−1. Each data point represents the mean of triplicate measurements from a typical experiment. The experiment was repeated four times with similar results. Data were subjected to nonlinear regression fitting, and the dissociation constant (Kd = 1.3 ± 0.16 µM) is reported as means ± s.e.m. of the fit. d, Functional analysis comparison of full-length and truncated WT GABAB receptor. Shown is the dose-dependent baclofen-stimulated receptor response in cells transiently expressing Gαqi5 (abbreviated as Gqi) with full-length GABAB heterodimer or the C-terminally truncated GABAB1b(1–802)–GABAB2(1–819) complex. Cells transfected with Gαqi5 alone were used as a negative control. Relative agonist-stimulated activity was measured by IP1 accumulation, and is expressed as a percentage of maximum wild-type activity induced by baclofen relative to the activity of Gαqi5 alone. Data points represent averages ± s.e.m. of multiple experiments (n), each with quadruplicate measurements. Cell surface expression level was 106% for the GABAB1b(1–802)–GABAB2(1–819) complex in comparison with the full-length WT/WT heterodimer.

Extended Data Fig. 2 Cryo-EM imaging of human GABAB receptor.

a, Workflow of cryo-EM data processing. b, A representative motion-corrected cryo-electron micrograph of the GABAB receptor. c, Reference-free 2D class averages, highlighting clear density for transmembrane helices. d, Global density map, coloured according to local resolution, in full and clipped views perpendicular to the plane of the membrane. e, Global FSC curve (purple) corrected by high-resolution noise substitution. The overall resolution as determined by an FSC cut-off value of 0.143 (blue line) is 3.3 Å. f, 3D FSC curves, measuring directional resolution anisotropy. Plots show the global half-map FSC (red solid line; left-hand y-axis), together with the spread of directional resolution values within ± 1 standard deviation of the mean (area encompassed by green dashed lines), and a histogram (turquoise; right-hand y-axis) of 100 such directional resolution values sampled evenly over the 3D FSC threshold value of 0.143 (blue line). The sphericity value reported by 3D FSC is 0.958 out of 1. g, h, Separate FSC curves for the locally refined reconstructions of ECDs g) and transmembrane domains (h). The blue lines mark the resolution corresponding to an FSC value of 0.143 (ECD, 3.1 Å; transmembrane region, 3.4 Å). The adjacent diagrams show the masks (translucent surface) used for each local refinement.

Extended Data Fig. 3 Structural model of the GABAB receptor fit within the cryo-EM map.

ad, Cryo-EM density maps and refined models for the LB1 interface helices (H–B and H–C) in the extracellular VFT (a), the linker between the VFT and transmembrane domains (b), all seven transmembrane helices of the GABAB1b subunit (c), the ten modelled transmembrane cholesterols (d) and all seven transmembrane helices of the GABAB2 subunit (e). The density map is a composite of the locally refined reconstructions for the ECD and transmembrane domains. The N- and C-terminal residues of each helix are labelled.

Extended Data Fig. 4 Architecture of the GABAB receptor.

a, Structure of the GABAB receptor in four views related by 90° rotations about an axis perpendicular to the membrane. GABAB1b (blue) and GABAB2 (green) are rendered as ribbons; Ca2+ is shown as a green sphere; phospholipids (PE 38:5 and PC 38:2) are presented as yellow space-filling models; the observed N-linked glycans (NAGs) and cholesterols (CLRs) are shown respectively as grey and pink ball-and-stick models. Transmembrane helices 1–7, along with N and C termini, are marked for each subunit. b, Cryo-EM density map of the GABAB receptor, in the same orientations and colour scheme as in a. The map is composed of local reconstructions for the ECD and transmembrane domain, which were independently refined to 3.1 Å and 3.4 Å, respectively. c, Linker and transmembrane domain of the GABAB receptor viewed from the extracellular and intracellular sides.

Extended Data Fig. 5 Heterodimer conformation and interface features of the GABAB receptor.

a, b, Cryo-EM structure of near full-length GABAB receptor (cyan) superimposed with the crystal structure of its extracellular VFT module in the inactive state (PDB code 4MQE; purple) (a) or active state (PDB code 4MS3; red) (b). The middle panel shows the heterodimeric receptor structures superimposed based on the LB1 domain of the GABAB1b subunit. The two side panels show superpositions of individual GABAB1b and GABAB2 subunits on the basis of their respective LB1 domains. In b, the green line denotes the axis of rotation that relates the LB2 domains of near full-length and VFT structures of GABAB1b (rotation χ = 28°; screw translation τχ = 0.6 Å), or near full-length and VFT structures of GABAB2 (rotation χ = 7°; screw translation τχ = 0.3 Å). c, d, Extracellular LB2 domains viewed from the C-terminal end. Superposition of near full-length (cyan) and VFT structures (c, inactive state, purple; d, active state, red) based on the LB1 domain of the GABAB1b subunit. Within each heterodimeric complex, the C termini of the LB2 domains in the GABAB1b and GABAB2 subunits are shown as spheres, and the distance between the two C termini is marked by dotted lines. e, Cryo-EM structure of full-length mGlu5 in the inactive (PDB code 6N52) and active (PDB code 6N51) conformations20. f, Molecular surface of the GABAB1b–GABAB2 complex, showing the plane of the heterodimer interface for the extracellular and transmembrane domains. Structural elements involved in heterodimer formation are highlighted in cartoon format (ectodomain, H–B and H–C helices; transmembrane domain, TM5 and TM3 helices). g, The GABAB transmembrane domain viewed from the extracellular side, comparing the locations of core (I, II, IIIa, IIIb) versus peripheral cholesterol-mediated heterodimer contacts from different layers. Heterodimer contacts mediated by two cholesterols (CLR6 and CLR3) are shown at the bottom.

Extended Data Fig. 6 Extracellular ligand binding in GABAB1b.

a, b, Molecular surface (a) and ribbon representation (b) of the GABAB1b subunit, showing the location of the Ca2+-binding site and an unmodelled density at the interdomain crevice of VFT. c, d, Functional analysis of the impact of endogenous Ca2+. Basal activity (c) and dose-dependent baclofen-stimulated receptor response (d) in cells transiently expressing the Gαqi5 subunit (abbreviated as Gqi) with different combinations of WT and mutant GABAB-receptor subunits (GABAB1b E309K, abbreviated as E309K; GABAB1b E423R, abbreviated as E423R). IP1 accumulation by the WT/WT heterodimer was measured in the presence and absence of 2.5 mM EGTA. Cells transfected with Gαqi5 alone were used as negative controls. Relative activity in both graphs is expressed as a percentage of the maximum wild-type activity induced by baclofen relative to the activity of Gαqi5 alone. Data points represent averages ± s.e.m. of multiple experiments (n), each with quadruplicate measurements. **P = 0.0016, ***P = 0.0002, ****P < 0.0001; one-way ANOVA with Bonferroni’s post hoc test was used to calculate statistical differences in basal activity (c). Cell surface expression level was 107% for E309K/WT and 87% for E423R/WT mutants in comparison with the WT/WT heterodimer. e, Fitting of GABA into the extra density (contoured at 7.0σ) at the orthosteric ligand-binding site and its potential interaction with GABAB1b. f, Concentration of endogenous GABA in the supernatant and lysate of HEK 293 GnTI cells after recombinant expression of the GABAB receptor, as well as cell culture medium and lysis buffer controls, as detected by mass spectrometry.

Extended Data Fig. 7 Endogenous phospholipid-binding sites of the GABAB receptor.

a, b, Ribbon representation of the GABAB1b (a) and GABAB2 (b) transmembrane domains, highlighting the cryo-EM density for phospholipids contoured at 4.0σ. Phospholipids are rendered in ball-and-stick representation. c, d, Electrostatic potential surface of the lipid-binding pocket in GABAB1b (c) and GABAB2 (d). The phospholipids are shown as spheres. Charged residues that directly contact the phosphate group of each lipid are marked. eh, Comparison of phospholipids bound to GABAB subunits with ligands bound to the class A GPCRs rhodopsin27 (PDB code 1F88) and S1P1 receptor24 (PDB code 3V2Y) (e), the class B GPCR corticotropin-releasing factor receptor 1 (CRF1; ref. 57; PDB code 4K5Y) (f), the class C GPCRs mGlu1 (ref. 29; PDB code 4OR2) and mGlu5 (ref. 28; PDB code 4OO9) (g), and the class F GPCR Smoothened58 (PDB code 4JKV) (h). In each panel, the Cα trace of the GABAB1b linker and transmembrane domain is shown in two orthogonal views in grey, and the superimposed GABAB ligands PE 38:5 and PC 38:2 are shown as blue and green stick models, respectively. Various GPCRs were overlapped onto the transmembrane domain of GABAB1b to bring their bound ligands into superposition. i, j, Schematic diagrams of the specific contacts between GABAB1b and PE 38:5 (i), and between GABAB2 and PC 38:2 (j). Selected contacts between residues and phospholipids are highlighted; hydrogen bonds, red dotted lines; hydrophobic contacts, black wiggled lines; polar interactions, green curved lines; pi-stacking interactions, orange box wave. Red zigzags indicate contacts that are mediated by main-chain atoms. Lipid-interacting residues that are conserved in the two subunits are highlighted in bold and include: (1) head group (GABAB1b, His 643 and Arg 5493.32; GABAB2, His 647 and Arg 5563.32); (2) 20-carbon fatty acyl chain (GABAB1b, Phe 5573.40, Tyr 6575.44 and Ala 7036.54; GABAB2, Tyr 5643.40, Tyr 6615.44 and Ala 7076.54); (3) 18-carbon fatty acyl chain (GABAB1b, Ile 7247.36; GABAB2 Ile7287.36).

Extended Data Fig. 8 Endogenous phospholipid interactions with GABAB receptor.

a, c, e, Orthogonal views of a potential access channel in GABAB1b, GABAB2 and S1P1 in molecular surface representation, along with phospholipids PE 38:5, PC 38:2 or the sphingolipid mimic ML056, in space-filling representation. Side views (left) show an opening between helices TM5 and TM6 in GABAB1b (a) and GABAB2 (c), and between TM1 and TM7 in S1P1 (e), while top views (right) highlight the blocked entrance to the lipid-binding pocket from the extracellular side. In all cases, ECL1 and ECL3 (orange), ECL2 (pale brown), the linker of GABAB-receptor subunits (pink) and the N-terminal helix of S1P1 (pink) are distinguished by colour. b, d, f, The same information as in a, c, e but with ribbon models for GABAB1b (b), GABAB2 (d), and S1P1 (ref. 24) (PDB code 3V2Y) (f). Lipids are in stick format. g, h, Functional effect of a GABAB2 lipid-binding site mutation. Basal activity (g) and dose-dependent baclofen-stimulated receptor response (h) in cells transiently expressing Gαqi5 (Gqi) with WT GABAB receptor or WT GABAB1b and mutant GABAB2 R714A (R714A) heterodimer. Cells transfected with Gαqi5 alone were used as negative controls. Relative activity in both graphs was measured by IP1 accumulation, and expressed as a percentage of the maximum wild-type activity induced by baclofen relative to the activity of Gαqi5 alone. Data points represent averages ± s.e.m. of multiple experiments (n), each with quadruplicate measurements. **P = 0.0016; one-way ANOVA with Bonferroni’s post hoc test was used to calculate statistical differences in basal activity (g). Cell surface expression level was 77% for the WT/R714A mutant in comparison with the WT/WT heterodimer.

Extended Data Fig. 9 Comparison of the GABAB transmembrane domain with other GPCRs.

a, Superposition of GABAB1b and GABAB2 subunits based on their VFT modules. The short purple line denotes the axis of rotation that relates the linker and transmembrane domains of GABAB1b and GABAB2 (rotation χ = 23°; screw translation τχ = 0.01 Å). b, Superposition of the linker and transmembrane domains of the GABAB1b and GABAB2 subunits. c, Superposition of the linker and transmembrane domains of each GABAB subunit with the class C GPCR mGlu5 (ref. 20; PDB code 6N52) in three different views, with arrows revealing inward extracellular shifts in TM5 and TM7, as well as an inward intracellular shift in TM3, in mGlu5 compared with either GABAB subunit. df, Superposition of the transmembrane helices of each GABAB subunit with the class A GPCR rhodopsin27 (PDB code 1F88) (d), the class B GPCR CRF1 (ref. 57; PDB code 4K5Y) (e), and the class F GPCR Smoothened58 (PDB code 4JKV) (f). Arrows indicate large shifts in transmembrane helix positions between GABAB subunits and other GPCRs, such as outward extracellular movements in class A (TM2 and TM6), class B (TM1, TM2 and TM7), and class F (TM2, TM4 and TM5). There are also inward intracellular shifts in TM7 in all comparisons, and outward intracellular shifts in TM5 in class A, B and F.

Extended Data Fig. 10 Conserved motifs in GABAB, rhodopsin and mGlu receptors.

a, b, The ‘ionic lock’ and FxPKxx motifs in the GABAB1b (a) and GABAB2 (b) subunits. The ‘ionic locks’ consist of Asp 684 of ICL3 and Lys 5673.50 in GABAB1b, and Asp 688 of ICL3 and Lys 5743.50 in GABAB2. The FxPKxx motifs include the conserved Lys 7397.51 of GABAB1b and Lys 7437.51 of GABAB2, which interact with the ‘ionic locks’ through Asn2.39 (GABAB1b N5132.39; GABAB2 N5202.39) and a serine (GABAB1b S508; GABAB2 S515) in ICL1. c, The ‘ionic lock’ and NPxxY motifs in class A rhodopsin27. d, e, The ‘ionic lock’ and FxPKxY motifs in the class C receptors mGlu1 (ref. 29; d) and mGlu5 (ref. 28; e), which are in close proximity as in GABAB subunits. Key residues of the motifs are displayed as stick models. Hydrogen bonds are indicated by black dotted lines. Interactions between participating residues of the ‘ionic lock’ are denoted by red dotted lines with distances labelled. Distances between the Cα atoms of the ‘ionic lock’ residues are marked by brown dotted lines.

Supplementary information

41586_2020_2452_MOESM3_ESM.mp4

Global conformational variability analysis of GABAB receptor linkers. Motion between the ECD and transmembrane domains as revealed by 3D variability analysis of the entire receptor. The ECD and transmembrane domains flex back and forth about the linker region.

41586_2020_2452_MOESM4_ESM.mp4

Global conformational variability analysis of GABAB receptor membrane-proximal domains. Motion between the membrane-proximal LB2 domains of VFT as revealed by 3D variability analysis of the entire receptor. The LB2 domains of VFT approach and withdraw, as do their associated linkers.

41586_2020_2452_MOESM5_ESM.mp4

Local conformational variability analysis of GABAB receptor membrane-proximal domains. Motion between the membrane-proximal LB2 domains of VFT as revealed by 3D variability analysis of the ECD. The LB2 domains of VFT approach and withdraw, as do their associated linkers.

41586_2020_2452_MOESM6_ESM.mp4

Local conformational variability analysis of GABAB receptor transmembrane domains. Motion between the transmembrane domains as revealed by 3D variability analysis of the transmembrane region. The extracellular ends of the transmembrane domains approach and withdraw.

Supplementary Information

This file contains Supplementary Figs. 1-3, Supplementary Tables 1-3, and Supplementary references.

Reporting Summary

Supplementary Video 1|

Global conformational variability analysis of GABAB receptor linkers. Motion between the ECD and transmembrane domains as revealed by 3D variability analysis of the entire receptor. The ECD and transmembrane domains flex back and forth about the linker region.

Supplementary Video 2|

Global conformational variability analysis of GABAB receptor membrane-proximal domains. Motion between the membrane-proximal LB2 domains of VFT as revealed by 3D variability analysis of the entire receptor. The LB2 domains of VFT approach and withdraw, as do their associated linkers.

Supplementary Video 3|

Local conformational variability analysis of GABAB receptor membrane-proximal domains. Motion between the membrane-proximal LB2 domains of VFT as revealed by 3D variability analysis of the ECD. The LB2 domains of VFT approach and withdraw, as do their associated linkers.

Supplementary Video 4|

Local conformational variability analysis of GABAB receptor transmembrane domains. Motion between the transmembrane domains as revealed by 3D variability analysis of the transmembrane region. The extracellular ends of the transmembrane domains approach and withdraw.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Park, J., Fu, Z., Frangaj, A. et al. Structure of human GABAB receptor in an inactive state. Nature (2020). https://doi.org/10.1038/s41586-020-2452-0

Download citation

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.