Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 20 (4), 419-25

Crystal Structure of Oligomeric β1-adrenergic G Protein-Coupled Receptors in Ligand-Free Basal State

Affiliations

Crystal Structure of Oligomeric β1-adrenergic G Protein-Coupled Receptors in Ligand-Free Basal State

Jianyun Huang et al. Nat Struct Mol Biol.

Abstract

G protein-coupled receptors (GPCRs) mediate transmembrane signaling. Before ligand binding, GPCRs exist in a basal state. Crystal structures of several GPCRs bound with antagonists or agonists have been solved. However, the crystal structure of the ligand-free basal state of a GPCR, the starting point of GPCR activation and function, had not yet been determined. Here we report the X-ray crystal structure of the ligand-free basal state of a GPCR in a lipid membrane-like environment. Oligomeric turkey β1-adrenergic receptors display two dimer interfaces. One interface involves the transmembrane domain (TM) 1, TM2, the C-terminal H8 and extracellular loop 1. The other interface engages residues from TM4, TM5, intracellular loop 2 and extracellular loop 2. Structural comparisons show that this ligand-free state is in an inactive conformation. This provides the structural basis of GPCR dimerization and oligomerization.

Figures

Figure 1
Figure 1
Structure of the ligand-free basal state β1-AR. a, β1-AR crystallographic packing. The dashed box indicates one crystallographic asymmetric unit. Chain A: green; chain B: magenta. b and c, Molecular surface representation of oligomers of β1-AR. Within the same layer, β1-ARs form oligomers with two dimer interfaces. The N- and C-termini are indicated. c, Top view (from the extracellular surface) of the β1-AR oligomers. The TMs are labeled as I to VII.
Figure 2
Figure 2
Dimer interface 1 of β1-AR oligomers. a and b, The surface involved in dimer interface 1 is highlighted in green (chain A) and in magenta (chain B). The helix 8 is labeled as VIII and the extracellular loop 1 as ECL1. c and d, Residues in TM1, TM2 and ECL1 are involved in the dimer formation. e, Residues in H8 are involved in the dimer formation.
Figure 3
Figure 3
Dimer interface 2 of β1-AR oligomers. a and b, The surface involved in dimer interface 2 is highlighted in green (chain A) and in magenta (chain B). The intracellular loop 2 is labeled as ICL2 and the extracellular loop 2 as ECL2. c, Residues in TM5 and ECL2 are involved in dimer formation. d, Residues in TM4 and ICL2 are involved in dimer formation. e. Disulfide crosslinking experiments with Cys mutants of β1-AR with copper phenanthroline. One representative experiment of three is shown (left panel). The dimer fraction is quantified as dimer/(monomer + dimer). Results are means and s.d. (n = 3; *, p < 0.05; **, p < 0.001, Student’s t-test) (right panel).
Figure 4
Figure 4
The ligand-free basal state of β1-AR in an inactive conformation and with a contracted ligand-binding pocket. a and b, 2Fo-Fc map (blue mesh) of the cytoplasmic ends of TM3 and TM6 showing the ionic-lock salt bridge between Arg1393.50 and Glu2856.30. The electron density is contoured at 1.0 σ level and the dashed line shows the distance between Arg1393.50 and Glu2856.30. c, Comparison of the ligand-free state of β1-AR (in cyan, molecule B) and the cyanopindolol-bound β1-AR with TM6 in the bent conformation (in magenta, PDB code 2YCX, molecule A). The ionic-lock is present in both structures. d, Comparison of the ligand-free state of β1-AR (in cyan, molecule B) and the cyanopindolol-bound β1-AR with TM6 in the straight conformation (in gold, PDB code 2YCY, molecule B). The ionic-lock is present in the ligand-free state, but not in the cyanopindolol-bound β1-AR with TM6 in the straight conformation. Structures were aligned using all seven TM segments. Parts of the helices in front are removed for clarity. e, Representative regions of 2Fo-Fc map (blue mesh) around the ligand-binding pocket of β1-AR (molecule B, cyan), showing the empty pocket. The electron density is contoured at 1.2 σ level. f, Comparison of the ligand-free state of β1-AR (in cyan, molecule B) and the antagonist cyanopindolol-bound β1-AR (in yellow, PDB code 2VT4, molecule B). g, Comparison of the ligand-free state of β1-AR (in cyan, molecule B) and the agonist isoprenaline-bound β1-AR (in magenta, PDB code 2Y03, molecule A). The ligand-binding pockets are viewed from the extracellular surface and ECL2 is hidden for clarity. The dash lines represent the key hydrogen bonds involved in ligand binding. h, Comparison of the ligand-binding pockets for the empty ligand-free state β1-AR structure (molecule B, cyan), β1-AR with the antagonist cyanopindolol-bound (molecule B, yellow), and β1-AR with the agonist isoprenaline-bound (molecule A, magenta). The ligands and ECL2 are removed for clarity. The distances between the Cα atoms of Ser211 and Asn329 are represented as dashes and labeled.
Figure 5
Figure 5
Docking of Gs onto β1-AR dimer. a and b, The complex of β2-AR and Gs (PDB code 3SN6) was aligned with molecule B of the β1-AR dimer with the TM1-TM2-H8 interface. β1-AR is in green and β2-AR is in magenta. Gs α-subunit (the Ras-like and the α-helical (AH) domains) is in yellow. Gβ subunit is in cyan. Gγ subunit is in blue. c and d, The complex of β2-AR and Gs was aligned with molecule B of the β1-AR dimer with the TM4-TM5 interface. The steric collision is indicated by dashed circles.
Figure 6
Figure 6
Comparison of the β1-AR oligomer with the μ-opioid receptor oligomer. a, β1-AR is in green and μ-opioid receptor (PDB code 4DKL) is in magenta. Top panel, side view of the oligomers. Bottom panel, top view (from the extracellular surface) of the oligomers. The alignment was performed between molecule A ofβ1-AR and one molecule in μ-opioid receptor using all seven TM segments. b, Docking of Gs ontoβ1-AR tetramer. The complex of β2-AR and Gs (PDB code 3SN6) was aligned with molecule B of the β1-AR dimer with the TM1-TM2-H8 interface.

Comment in

Similar articles

See all similar articles

Cited by 94 articles

See all "Cited by" articles

References

    1. Pierce KL, Premont RT, Lefkowitz RJ. Seven-transmembrane receptors. Nat Rev Mol Cell Biol. 2002;3:639–50. - PubMed
    1. Oldham WM, Hamm HE. Heterotrimeric G protein activation by G-protein-coupled receptors. Nat Rev Mol Cell Biol. 2008;9:60–71. - PubMed
    1. Vassart G, Costagliola S. G protein-coupled receptors: mutations and endocrine diseases. Nat Rev Endocrinol. 2011;7:362–72. - PubMed
    1. Kenakin T, Miller LJ. Seven transmembrane receptors as shapeshifting proteins: the impact of allosteric modulation and functional selectivity on new drug discovery. Pharmacol Rev. 2010;62:265–304. - PMC - PubMed
    1. Lappano R, Maggiolini M. G protein-coupled receptors: novel targets for drug discovery in cancer. Nat Rev Drug Discov. 2011;10:47–60. - PubMed

Publication types

Associated data

LinkOut - more resources

Feedback