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

Structure determination

We determined the X-ray crystal structure of turkey β₁-AR without ligands in the presence of synthetic lipids (Fig. 1, Table 1, and Supplementary Fig. 1, 2). Several complete data sets from individual crystals were recorded and they gave similar structures. Only one representative data set from a single crystal was described here (Table 1). Diffraction of the crystal was highly anisotropic, and the structure was solved and refined at 3.5 Å resolution. The overall quality of the electron density map was high. The structure of the ligand-free state of β₁-AR was determined by molecular replacement using the partial agonist (salbutamol)-bound β₁-AR as a search model¹⁸. Crystal packing indicated a membrane-like environment and was largely impacted by lipids in one direction (Fig. 1, Supplementary Fig. 2a and b). β₁-ARs parallely packed as oligomers with two distinct dimer interfaces (Fig. 1a–c). Each crystallographic asymmetric unit had twoβ₁-AR molecules (Supplementary Fig. 2c). These two β₁-AR molecules had similar configurations; the root mean square deviation was ~0.02 Å for all equivalent Cα atoms (Supplementary Fig. 2d). Although most of the experiments (including screening for crystallization conditions) described in this paper were done with the construct β₁-AR(H0), the presented ligand-free structure was from the construct β₁-AR(m23) since it gave a slightly higher resolution (Supplementary Fig. 1).β₁-AR(H0) is with deletions of amino acid sequences of 3–32, of 249–283 and of 366–483 and with point mutations of Cys116Leu and Cys358Ala (Supplementary Figure 1).β₁-AR(m23) is a thermostabilized β₁-AR mutant that was used in previous crystal structural studies with antagonists or agonists ^6,7.

Receptor oligomerization: the TM1-TM2-H8 dimer interface

Within the same lipid bilayer, oligomers of β₁-ARs were parallely packed with two alternating dimer interfaces (Figs. 1b, 1c, 2 and 3). This oligomeric architecture was remarkably similar to the proposed models for oligomeric GPCRs based on a large body of functional data^26–34. GPCRs can exist and function as dimers or oligomers ^26–29,35. Dimerization and oligomerization modulate various GPCR functions such as cell surface targeting, cooperativity, activation, G-protein coupling, signaling and internalization^28,29,36. In previous crystallographic studies with β₁-ARs bound with antagonists or agonists, β₁-ARs were observed as anti-parallel dimers ^10,18. The lack of oligomeric arrangement in those studies might be due to the exclusion of phospholipids in the crystallization conditions. Here, in one dimer interface (dimer interface 1), the interaction was mainly through TM1 as well as some residues from the C-terminal helical domain H8, TM2, and the extracellular loop 1 (Figs. 1b, 1c, and 2). The total buried contact surface (from both protomers) was ~1700 Ų (Fig. 2a and b). Interacting residues were mainly from TM1 (including Gln38, Gln39, Ala42, Leu46, Ala49, Leu50, Val52, Leu53, and Leu54) (Fig. 2c). Residues from other parts of the receptor also contributed to this dimer interface, including residues from TM2 (Pro96, Ala99, Thr100 and Val103) (Fig. 2c), the extracellular loop 1 (Thr106, Leu108, and Trp109) (Fig. 2c and d), and the C-terminal H8 (Arg351, Lys354, Arg355 and Leu356) (Fig. 2e). In addition to these hydrophobic and van der Waals interactions, Ser45 in one TM1 formed a hydrogen bond with Ser45 from another TM1 (Fig. 2c). Glu41 in TM1 from one monomer formed a salt bridge with Arg104 in TM2 from the second monomer (Fig. 2c). This dimer interface is similar to the one observed in the dimer of rhodopsin in the active state which uses TM1 and H8 as an interface^12,13.

Receptor oligomerization: the TM4-TM5-ICL2 dimer interface

In the second dimer interface, the interacting regions involved residues from TM4, TM5, the intracellular loop 2 (ICL2) and the extracellular loop 2 (Figs. 1b, 1c, and 3). The total buried surface (from both receptors) was ~900 Ų (Fig. 3a and b). Residues from both TM4 (including Leu171) and TM5 (including Arg205, Ala206, Ala210, Ile218 and Arg229) contributed to the hydrophobic interaction (Fig. 3c and d). The intracellular loop 2 also played a critical role in this dimer interaction (including residues Tyr140, Leu141, Thr144, Ser145, Phe147, Arg148, Ser151, and Leu152) (Fig. 3d). Two residues (Trp181 and Arg183) from extracellular loop 2 also participated in this interaction (Fig. 3c). Previous studies with human β₁-ARs using the bioluminescence resonance energy transfer method had shown that human β₁-ARs formed dimers, and that TM4 was involved in human β₁-AR dimerization ^37,38.

Disulfide trapping of β₁-AR dimers

Next we used disulphide-trapping experiments to biochemically test some representative residues identified from our structural studies for their involvement in β₁-AR dimerization in cells. Cysteine replacement of an appropriately disposed pair of residues at the dimer interfaces is expected to generate a disulphide bridge ^{30,33,39–41}. Based on our structural model, we selected a few residues from the two interfaces and mutated these residues into cysteines on the background of β₁-AR(H0). In dimer interface 1, Lys354 from one protomer interacted with Lys354 from the second protomer (Fig. 2e). In dimer interface 2, Arg148 from one protomer interacted with Arg148 from the other protomer (Fig. 3d). As a negative control, we also mutated Phe112 to Cys. Phe112 is on the extracellular side of TM3 and was not involved in the dimer interfaces based on our crystal structure. We transfected these mutants individually into CHO cells and stable cell lines were selected. After exposing the membrane preparations to the hyperoxidizing environment of Cu-phenanthroline (CuP), dimer formation was assessed by Western blot analysis of detergent-solubilized protein samples with β₁-AR antibodies (Fig. 3e). Wild-type β₁-AR (no cysteine residues in the two dimer interfaces) and Phe112Cys mutant showed only monomers with or without CuP treatment (Fig. 3e). Mutants Arg148Cys and Lys354Cys showed increased formation of dimers after CuP treatment (Fig. 3e). Arg148Cys mutants formed some dimers without CuP treatment, likely the result of air oxidation as shown for some Cys mutants of serotonin 5HT2c receptors ⁴¹. These data confirm that the two dimer interfaces identified in our structure are used under physiological conditions. Hence, our crystal structure provides a structural context for the dimerization and oligomerization of GPCRs, and the co-existence of the two types of dimers within the oligomers.

Ligand-free basal state of β₁-AR in an inactive conformation

One of the characteristics of the inactive state of class A GPCRs is the presence of the ionic-lock salt bridge between the highly conserved D(E)R^3.50Y motif in TM3 and an E/D^6.30 residue in TM6 (Ballesteros-Weinstein numbering system is in superscripts)^7,14,16,42. This ionic-lock salt bridge between Arg139^3.50 and Glu285^6.30 was present in the ligand-free state of β₁-AR (Fig. 4a and b). Hence, our data are consistent with the ligand-free basal state β₁-AR being in an inactive state.

In the first report of the crystal structure of β₁-AR bound with the antagonist cyanopindolol, the ionic-lock was absent¹⁰. In a subsequent report of the crystal structures of β₁-AR with cyanopindolol, the ionic-lock was present in some structures, but absent in others⁴³. In the structure of cyanopindolol-bound β₁-AR with the ionic-lock, the cytoplasmic end of TM6 (the G protein-interacting region) was in a bent conformation (Fig. 4c)⁴³. In the cyanopindolol-bound β₁-AR without the ionic lock, the cytoplasmic end of TM6 was in a straight conformation (Fig. 4d)⁴³. Thus, it was proposed that the presence of the ionic-lock was associated with the bent conformation of the cytoplasmic end of TM6 ⁴³. However, in the ligand-free basal state structure of β₁-AR described here, the ionic-lock existed concomitantly with the straight conformation of TM6 (Fig. 4c and d).

The basal state with a contracted ligand-binding pocket

Based on the comparisons of the crystal structures of several GPCRs in inactive and active states, it has been proposed that, while the overall GPCR structures did not change significantly, an outward movement of the cytoplasmic end of TM6 (to a lesser degree TM5 as well) relative to the receptor helix bundle core is a hallmark of the active state^{13,17,22–24}. The ligand-free basal state of β₁-AR did not display this characteristic outward movement of TM6 and TM5, consistent with its inactive conformation. Furthermore, agonist binding to β₁-AR induces the contraction of the ligand-binding pocket by ~1 Å (as measured between the Cα atoms of Ser211 and Asn329)¹⁸. The ligand-binding pocket in the ligand-free state of β₁-AR was empty (Fig. 4e and Supplementary Fig. 3). Moreover, the ligand-binding pocket of the ligand-free state of β₁-AR was narrower than those of the antagonist-bound and similar to the agonist-bound structures of β₁-AR (Fig. 4f–h). Thus, the contraction of the ligand-binding pocket may not be an essential feature of the binding of full agonists to β₁-AR.

The ligand-free basal state of GPCRs

Before ligand binding, GPCRs are in a basal state. Since agonists or inverse agonists could shift the ligand-free state to an activated state or an inactive state, respectively, the ligand-free state is likely conformationally flexible. This may partly explain the difficulty in crystallizing the ligand-free GPCRs. On the other hand, many GPCRs including β₁-AR have a low basal activity in the absence of ligands, suggesting that, in the ligand-free state, a large fraction of the receptor population is in the inactive state. For the ligand-free GPCRs, while opsin is in an active state, the ligand-free β₁-AR is in an inactive state. These differences might reflect the different crystallization conditions such as the presence of membrane-like environment in the β₁-AR structure, or the different crystal packings leading to different stabilized conformations. The crystal structures only provide a snapshot of the lowest energy conformations that these receptors could adopt under the specific crystallization conditions.

It might also be argued that the ligand-free β₁-AR observed here in the inactive state was stabilized by the use of thermostabilizing mutations. However, that is unlikely since these thermostabilizing mutations, although making β₁-AR proteins more stable at higher temperatures, do not stabilize β₁-AR(m23) in an inactive conformation. As recently reported, this mutated β₁-AR(m23) is still a functional receptor capable of binding agonists and antagonists and activating intracellular agonist responses (Supplementary Fig. 4) ⁴⁴. Furthermore, most crystal structures of this thermostabilized β₁-AR(m23) with agonists or antagonists displayed intermediate conformations without the ionic-lock and without the outward movement of the cytoplasmic end of TM6 ^10,18,43. The structure presented here was determined from β₁-AR proteins purified with alprenolol affinity purification (eluted with cyanopindolol) as the second purification step. Although the protein samples were dialyzed with buffers without cyanopindolol and the crystallization condition was at ~pH 4 which reduced antagonist binding to β₁-ARs (Supplementary Fig. 4), we could not completely exclude the possibility of a very low occupancy of cyanopindolol in the presented structure. However, use of β₁-AR proteins purified with two-rounds of nickel affinity purifications (without the alprenolol affinity purification step) resulted in similar structures although at lower resolutions.

It is known that some GPCRs display varying levels of constitutive activity (i.e. the basal activity in the absence of any ligands) which are critical for their physiological functions. Structural determinations of the ligand-free states of these GPCRs should provide molecular insights into the activation processes of GPCRs, the basal activity, and development of agents for therapeutic applications since the ligand-free state is the starting state and offers a point of comparison.

Dimer interfaces and G-protein interaction

In our crystal structure of β₁-AR oligomers, there are two dimer interfaces: one involves TM1-TM2-H8 and the other engages TM4-TM5-ICL2 (Fig. 1). Among the published crystal structures of GPCRs, there are four other GPCRs showing parallel dimers. In rhodopsin and κ-opioid receptor, the dimer interface involves residues from TM1-TM2-H8 ^12,13,45,46 (Supplementary Fig. 5a and b). This dimer interface is similar to dimer interface 1 in our β₁-AR structure. In the CXCR4 structure, there is a dimer interface involving residues from TM5 and TM6 ¹⁵ (Supplementary Fig. 5c). However, dimer interface 2 of β₁-ARs involves TM4 and TM5. Compared to β₁-AR, one monomer of CXCR4 rotates ~40° towards another monomer (Supplementary Fig. 5c). In a recent crystal structure of oligomeric μ-opioid receptor, two dimer interfaces were observed: one involves TM1-TM2-H8, and the other involves TM5-TM6 ⁴⁷. Hence, the TM1-TM2-H8 interface is rather conserved in various GPCRs. On the other hand, the TM5 interface sometime functions with TM4 and other times with TM6. Remarkably, the crystal structures of GPCRs so far only displayed these two types of dimer interfaces which are in agreement with a large body of experimental data, indicating that these two dimer interfaces are likely physiological relevant.

In addition to the TMs, intracellular regions contribute significantly to the dimer interfaces. There are four residues from H8 involved in the TM1-TM2-H8 dimer interface. Eight residues from ICL2 contribute to the TM4-TM5-ILC2 dimer interface. ICL2 is critical for interacting with G proteins based on the structural model of the complex of β₂-AR and G_s ²⁴. A Gs trimer could be docked onto a β₁-AR dimer formed through the TM1-TM2-H8 dimer interface (Fig. 5a and b). On the other hand, it was not possible to dock a Gs trimer onto the β₁-AR dimer formed via the TM4-TM5-ICL2 dimer interface without steric collisions (Fig. 5c and d). Participation of ICL2 in this dimer interface may prevent G protein coupling to the dimer formed through TM4-TM5-ICL2 interface, or G protein binding may disrupt this dimer interface. Therefore, we propose that, if the signaling unit is a pentamer (two GPCRs and one trimeric G protein), the GPCR dimer interface in this signaling unit is TM1-TM2-H8 (Fig. 5a and b). In this model, only one β₁-AR contacts with the G protein trimer, and the other β₁-AR is “spared” or could function through trans-protomer allosteric regulation (see below).

GPCR oligomerization and receptor activation

In our crystal structure, β₁-ARs form oligomers in a membrane-like environment. Although the physiological functions are not clear, studies using various approaches have indicated that GPCRs could form oligomers in cells ^{28,29,34,48,49}. While our manuscript was under review, a recent crystal structure of μ-opioid receptor also showed oligomers ⁴⁷. The β₁-AR oligomers show some similarities and some differences from the oligomers of μ-opioid receptors (Fig. 6a). Both oligomers have two dimer interfaces and share the same TM1-TM2-H8 dimer interface (Fig. 6a). However, in the second dimer interface, TM5 works together with TM4 in β₁-ARs while TM5 acts together with TM6 in μ-opioid receptors (Fig. 6a). Furthermore, while β₁-AR oligomers form a linear array in one direction, μ-opioid receptors are in a sine wave arrangement (Fig. 6a).

A docking exercise revealed that, with the oligomeric arrangement, trimeric G proteins could not be fitted in without steric hindrance (Supplementary Fig. 6). Even though this docking was speculative, it suggests that either G protein binding may disrupt the oligomers or change the oligomeric arrangement, or the oligomeric architecture would prevent the dramatic sideway-rotation and upward-translation of the helical-domain of the Gα_s subunit, relative to the Ras-like domain of the Gα_s subunit, as observed in the crystal structure of the complex of β₂-AR and Gs ²⁴. Indeed, the extent of membrane-driven oligomerization of a GPCR (such as D2 receptors and 5HT2c receptors) in the inverse agonist-bound state may be larger than in the agonist-bound state ^40,41. Moreover, inverse agonists stabilize β₂-AR oligomers, while Gs reduced the extent of oligomerization of β₂-ARs ⁵⁰. Hence oligomerization of GPCRs is sensitive to ligand binding. That is to say, agonist binding may disrupt the oligomerization of GPCRs into dimers and/or tetramers.

It is possible to dock two trimeric G proteins into a β₁-AR tetramer (Fig. 6b). Whether this model is physiologically relevant requires further experimental testing. In this model, the two protomers via the TM4-TM5-ICL2 dimer interface were “spared” (not interacting with G proteins) (Fig. 6b). An asymmetric functioning for GPCR dimers has been proposed ^34,51. Previously a dimer interface involving TM1 has been shown to be insensitive to ligand binding and the receptor activation state as shown for dopamine D2 receptors and serotonin 5HT2c receptors ^31,41. The similarity of dimer interface 1 (involving TM1) in the inactive β₁-AR and the active rhodopsin is consistent with the notion that this dimer interface involving TM1 does not undergo significant conformational changes from inactive to active states of GPCRs (Supplementary Fig. 5a). On the other hand, the dimer interface involving TM4 makes structural rearrangement during the GPCR activation process, at least in the cases of dopamine D2 receptors and serotonin 5HT2c receptors ^40,41. These imply a possible role for the TM4-TM5 dimer interface in GPCR transactivation even though the two promoters do not directly interact with G proteins in the proposed model (Fig. 6b). Based on the structures of active GPCRs, the intracellular end of TM5 moves away from the TM bundle core ²⁴. Therefore it is possible that, upon the agonist binding, the configuration change at the TM4-TM5 dimer interface is part of the receptor activation process.

β₁-AR constructs and purification of β₁-AR proteins

A cDNA plasmid for the turkey β₁-AR was obtained from Dr. E. Ross (Dallas, Texas)⁵². For pre-crystallization screening of β₁-AR constructs for structural studies, we used the fluorescence-detection size-exclusion chromatography (FSEC) method for integral membrane proteins developed by Dr. E. Gouaux and colleagues⁵³. In this screening method, the target proteins are covalently fused to GFP. The resultant fusion proteins are monitored first for expression level and pattern in whole cells by epifluorescence microscopy. After solubilization of whole cells or crude membranes, the resulting unpurified protein is analyzed by fluorescence-detection size-exclusion chromatography. A monodisperse and folded protein would generally yield a single symmetrical Gaussian peak, while a polydisperse, unstable, or unfolded protein would typically yield multiple asymmetric peaks⁵³. We PCR-subcloned different β₁-AR constructs into the pCGFP vector (from Drs. O. Boudker and E. Gouaux) and transfected them into HEK 293 cells. Based on previous studies with many different GPCRs, we focused on deletions on the N-terminus, the C-terminus, and the intracellular loop 3 region of β₁-AR. Two days after transfection, the subcellular localizations of the β₁-AR receptors were checked by fluorescence microscopy. All tested constructs expressed proteins at the plasma membrane. Membrane preparations were solubilized in a buffer containing the nonionic detergent n-dodecyl-β-D-maltoside (DDM), and the resulting supernatant was analyzed by fluorescence-detection size-exclusion chromatography. Among the ~40 β₁-AR constructs, several β₁-AR constructs displayed a nearly symmetric fluorescence peak with an apparent molecular weight of a monomer of β₁-AR in DDM (the protein-detergent complex) (Supplementary Fig. 7a). We purified the recombinant β₁-AR proteins from High5 insect cells. The stability of these β₁-AR proteins in different detergents was tested at 18°C. Most of the studies were with a β₁-AR construct [β₁-AR(H0)] with deletions of amino acid sequences of 3–32, of 249–283 and of 366–483 and with point mutations of Cys116Leu and Cys358Ala (Supplementary Figure 1). β₁-AR(H0) generated similar cAMP responses as wild-type β₁-AR when expressed in β₁-AR^−/−/β₂-AR^−/− MEF cells ⁵⁴ (Supplementary Fig. 7b).

β₁-AR mutants (with a C-terminal His₆ tag) were subcloned into a baculoviral expression vector pVL1393. Recombinant baculoviruses were picked and amplified. High5 insect cells were grown suspension in High5 Express Medium (Invitrogen) at 27°C with shaking at 110 rpm. Cells were infected at a multiplicity of infection of 5–10. After shaking for one hour, an equal volume of fresh medium was added. Cells were harvested by centrifugation 48 h after infection. Infected cells from cultures were harvested by centrifugation and the resulting pellet was resuspended in 20 mM Tris–HCl pH 8, 1 mM EDTA. Cells were flash frozen in liquid nitrogen and stored at −80°C. Cells were broken by sonication. After centrifugation at 2,000 rpm for 10 min, the supernatant was collected and centrifuged for 1 h at 45,000 rpm at 4°C in a Beckman Ti45 rotor. Membrane pellets were resuspended in the same volume of buffer and the centrifugation was repeated. The final pellet was resuspended in a buffer with a reduced EDTA concentration (0.2 mM) at 10–20 mg protein/ml and frozen in liquid nitrogen and stored at −80°C. Membranes containing 2 g of total proteins were thawed and diluted to 10 mg/ml protein in ice-cold 20 mM Tris–HCl pH 8 with 0.35 M NaCl, 2% DDM and protease inhibitors, and then stirred at 4°C for 1 hour. After centrifugation for 1 h at 45,000 rpm in a Ti45 rotor (4°C), solubilized membrane proteins were mixed with Ni-NTA beads pre-equilibrated with buffer A (20 mM Tris–HCl pH 8, 0.35 M NaCl, 10 mM imidazole, protease inhibitors and 0.025% DDM). The mixture was rolled at 4°C for 6 hours. The protein-loaded resin was washed with buffer B (20 mM Tris–HCl (pH 8.0), 500 mM NaCl, 0.025% DDM), and the bound protein was eluted by using 3 X bed volume buffer C (20 mM Tris–HCl (pH 8.0), 50 mM NaCl, 250 mM imidazole, 0.025% DDM). In some preparations, β₁-AR proteins were purified again with a second-round Ni-NTA affinity purification and used for crystallization. In other preparations, β₁-AR proteins were purified by alprenolol affinity purification. For alprenolol affinity purification, after dilute the protein sample with four volumes of buffer D (Buffer C without imidazole), the sample was incubated with alprenolol-Affi-Gel beads overnight at 4°C. Alprenolol-NH₂ was synthesized at Cornell’s chemistry core facility following a published protocol⁵⁵. Alprenolol was crosslinked to Aff-Gel-15. After washing the alprenolol beads with buffer D, β₁-AR was eluted with buffer D containing 50 μM cyanopindolol, and then dialyzed, concentrated and changed buffer to 10 mM Tris pH 7.7, 50 mM NaCl, 0.02% DDM and 0.1 mM EDTA with centricon (100 KDa cutoff)(Millipore)^56,57. SDS-PAGE showed that β₁-AR protein was >90% pure. The yield was ~ 2 mg of purified β₁-AR proteins from 1L of insect cells.

Crystallization

β1-AR(H0) proteins were initially used for screening crystallization conditions. β₁-AR at a final concentration of ~8 mg/ml in 20 mM Tris-HCl pH 7.7, 50 mM NaCl, 0.1 mM EDTA, 0.02% DDM, 0.1 mg/ml lipid (3:1:1:1 = POPC:POPE:POPG: Cholesterol) was incubated on ice for 1 hour prior to set up the tray. Crystals were obtained in several crystallization conditions. To further make sure that there were no ligands in the final crystals, we selected conditions with low pH which decreased the binding of ligands from β1-AR (ref.58) (Supplementary Fig. 4). Crystallization was performed by the vapour diffusion hanging drop method at 18°C. 1 μl β₁-AR protein sample was mixed with 1 μl crystallization buffer (0.1–0.3 M (NH₄)₂SO₄, 0.02 M NaAc, pH 3.6~4, 26~30% PEG200). With β₁-AR(H0) construct, the screened crystals yielded diffraction to ~8 Å. We then introduced the six point mutations (Arg68Ser, Met90Val, Tyr227Ala, Ala282Leu, Phe327Ala and Phe338Met) and generated a construct same as the thermostabilized β₁-AR(m23) (Supplementary Figure 1)¹⁸. Thus the ligand-free structure described here was for β₁-AR(m23) and this thermostabilized β₁-AR mutant was used in previous crystal structural studies with antagonists or agonists ^10,18. This mutant β₁-AR is able to adopt different conformations, to bind antagonists, partial agonists and agonists, as well as to activate G proteins and increase cAMP levels in cells in response to agonists^10.18,44. Crystals were formed within one week and were directly frozen in liquid nitrogen. Crystals were screened and diffraction data were collected at the National Synchrotron Light Source (beamlines X6A and X25) of the Brookhaven National Laboratory or the Advanced Photon Source beamline NE-CAT 24E at Argonne National Laboratory.

Data collection, structure determination and refinement

Diffraction data presented in this paper were collected at 100 K using synchrotron radiation (λ = 1.1000 Å) at the beamline X25, National Synchrotron Light Source (Brookhaven, USA), using a PILATUS 6M CCD detector. The crystal diffracted to ~ 3.3 Å and diffraction data were indexed and integrated with XDS, followed by merging and scaling with XSCALE⁵⁹. The crystal belongs to space group C2 and the corresponding data-collection statistics are shown in Table 1. Analysis of the final data set by the UCLA diffraction anisotropy server indicated that the diffraction was highly anisotropic, strong in two directions while weak in the third direction along the reciprocal space axis c* ⁶⁰. As guided by an <F>/<σF> cutoff of 3.0 along each reciprocal space axis, reflections were anisotropically truncated to 3.3 × 3.3 × 4.3 Å along a*, b*, c* and sharpened by application of a negative isotropic B factor of − 54.13 before use in refinement.

The structure of the ligand-free state β₁-AR was solved by molecular replacement with PHASER of the CCP4 suite using a monomer of the salbutamol-bound β₁AR-m23 structure (PDB ID Code: 2Y04, chain A) as the search model⁶¹. A total of two copies of the monomer were observed per asymmetric unit (ASU). Model refinements were performed with REFMAC5 and PHENIX followed by employing the program COOT for iterative cycles of rebuilding based on sigma-A weighted 2F_o-F_c and F_o-F_c maps, as well as non-crystallographic symmetry (NCS) averaged and unaveraged maps^62–64. During refinement, reflections within the resolution range 30-3.5 Å were selected and tight 2-fold NCS restraints were applied to chains A and B, with a notable reduction in R_free with good geometry. Refinement statistics were also presented in Table 1, and the stereochemical quality of the refined structure was validated using MolProbity⁶⁵. The Ramachandran plot distribution for residues in the structure was 95.6% in the favored region and 4.4% in allowed region. The high R factors might be partially attributed by the unmodeled discontinuous density maps in the gaps between protein molecule layers, which could not be fitted into any ordered lipid molecules or solvents due to the resolution limit of 3.5 Å. The interfaces of two β₁AR dimers were analyzed using the EBI PDBe PISA web server⁶⁶. Global alignment of various structural models of β₁-ARs was performed using PyMOL (super_align) (DeLano Scientific LLC) and all structural model figures were created with PyMOL as well.

Cysteine crosslinking

Disulfide trapping experiments were performed as described ^30,41.