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, 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.
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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.
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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.
The authors declare no competing interests.
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
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.
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.
a–d, 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.
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.
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.
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.
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. e–h, 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).
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.
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. d–f, 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.
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.
This file contains Supplementary Figs. 1-3, Supplementary Tables 1-3, and Supplementary references.
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.
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.
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.
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.
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Park, J., Fu, Z., Frangaj, A. et al. Structure of human GABAB receptor in an inactive state. Nature 584, 304–309 (2020). https://doi.org/10.1038/s41586-020-2452-0
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