The structural basis for agonist and partial agonist action on a β(1)-adrenergic receptor.

β-adrenergic receptors (βARs) are G-protein-coupled receptors (GPCRs) that activate intracellular G proteins upon binding catecholamine agonist ligands such as adrenaline and noradrenaline. Synthetic ligands have been developed that either activate or inhibit βARs for the treatment of asthma, hypertension or cardiac dysfunction. These ligands are classified as either full agonists, partial agonists or antagonists, depending on whether the cellular response is similar to that of the native ligand, reduced or inhibited, respectively. However, the structural basis for these different ligand efficacies is unknown. Here we present four crystal structures of the thermostabilized turkey (Meleagris gallopavo) β(1)-adrenergic receptor (β(1)AR-m23) bound to the full agonists carmoterol and isoprenaline and the partial agonists salbutamol and dobutamine. In each case, agonist binding induces a 1 Å contraction of the catecholamine-binding pocket relative to the antagonist bound receptor. Full agonists can form hydrogen bonds with two conserved serine residues in transmembrane helix 5 (Ser(5.42) and Ser(5.46)), but partial agonists only interact with Ser(5.42) (superscripts refer to Ballesteros-Weinstein numbering). The structures provide an understanding of the pharmacological differences between different ligand classes, illuminating how GPCRs function and providing a solid foundation for the structure-based design of novel ligands with predictable efficacies.

β-Adrenergic receptors (βARs) are G protein-coupled receptors (GPCRs) that activate intracellular G proteins upon binding catecholamine agonist ligands such as adrenaline and noradrenaline1,2. Synthetic ligands have been developed that either activate or inhibit βARs for the treatment of asthma, hypertension or cardiac dysfunction. These ligands are classified as either full agonists, partial agonists or antagonists, depending on whether the cellular response is similar to that of the native ligand, reduced or inhibited, respectively. However, the structural basis for these different ligand efficacies is unknown. Here we present four crystal structures of the thermostabilised turkey (Meleagris gallopavo) β1-adrenergic receptor (β1AR-m23) bound to the full agonists carmoterol and isoprenaline and the partial agonists salbutamol and dobutamine. In each case, agonist binding induces a 1 Å contraction of the catecholamine binding pocket relative to the antagonist bound receptor. Full agonists can form hydrogen bonds with two conserved serine residues in transmembrane helix 5 (Ser5.42 and Ser5.46), but partial agonists only interact with Ser5.42 (superscripts refer to Ballesteros-Weinstein numbering3). The structures provide an understanding of the pharmacological differences between different ligand classes, illuminating how GPCRs function and providing a solid foundation for the structure-based design of novel ligands with predictable efficacies.

Determining how agonists and antagonists bind to the β receptors has been the goal of research for more than 20 years4-11. Although the structures of the homologous β1 and β2 receptors12-15 show how some antagonists bind to receptors in the inactive state16, structures with agonists bound are required to understand subsequent structural transitions involved in activation. GPCRs exist in an equilibrium between an inactive state (R) and an activated state (R*) that can couple and activate G proteins17. The binding of a full agonist, such as adrenaline or noradrenaline, is thought to increase the probability of the receptor converting to R*, with a conformation similar to that of opsin18,19. In the absence of any ligand, the βARs exhibit a low level of constitutive activity, indicating that there is always a small proportion of the receptor in the activated state, with the β2AR showing a 5-fold higher level of basal activity than the β1AR20. Basal activity of β2AR is important physiologically, as shown by the T164I4.56 human polymorphism that reduces the basal activity of β2AR to levels similar to β1AR21 and whose expression has been associated with heart disease22.

As a first step towards understanding how agonists activate receptors, we have determined the structures of β1AR bound to 4 different agonists. Native turkey β1AR is unstable in detergent23, so crystallization and structure determination relied on using a thermostabilised construct (β1AR-m23) that contained six point mutations, which dramatically improved its thermostability24. In addition, the thermostabilising mutations altered the equilibrium between R and R*, so that the receptor was preferentially in the R state24. However, it could still couple to G proteins after activation by agonists13 (Supplementary Fig. 1, Supplementary Tables 1-3), although the activation energy barrier is predicted to be considerably higher than for the wild-type receptor25. Here we report structures of β1AR-m23 (see Methods) bound to r-isoprenaline (2.85 Å resolution), r,r-carmoterol (2.6 Å resolution), r-salbutamol (3.05 Å resolution) and r-dobutamine (two independent structures at 2.6 Å and 2.5 Å resolution) (Supplementary Table 5). The overall structures of β1AR-m23 bound to the agonists are very similar to the structure with the bound antagonist cyanopindolol13, as expected for a receptor mutant stabilised preferentially in the R state. None of the structures show the outward movement of the cytoplasmic end of transmembrane helix H6 by 5-6 Å that is observed during light activation of rhodopsin18,19,26. This suggests that the structures represent an inactive, non-signaling state of the receptor formed on initial agonist binding.

All four agonists bind in the catecholamine pocket in a virtually identical fashion (Fig. 1). The secondary amine and β-hydroxyl groups shared by all the agonists (except for dobutamine, which lacks the β-hydroxyl; see Supplementary Figure 4) form potential hydrogen bonds with Asp1213.32 and Asn3297.39, while the hydrogen bond donor/acceptor group equivalent to the catecholamine meta-hydroxyl (m-OH) generally forms a hydrogen bond with Asn3106.55. In addition, all the agonists can form a hydrogen bond with Ser2115.42, as seen for cyanopindolol13, and they also induce the rotamer conformation change of Ser2125.43 so that it makes a hydrogen bond with Asn3106.55. The major difference between the binding of full agonists compared to the partial agonists is that only full agonists make a hydrogen bond to the side chain of Ser2155.46 as a result of a change in side chain rotamer. All of these amino acid residues involved in the binding of the catecholamine headgroups to β1AR are fully conserved in both β1 and β2 receptors (Fig. 2). Furthermore, the role of many of these amino acid residues in ligand binding is supported by extensive mutagenesis studies on β2AR that were performed before the first β2AR structure was determined27. Thus it was predicted that Asp1133.32, Ser2035.42, Ser2075.46, Asn2936.55 and Asn3127.39 in β2AR were all involved in agonist binding4,5,7-9 (Fig. 3). Inspection of the region outside the catecholamine binding pocket in the structures with bound dobutamine and carmoterol allows the identification of non-conserved residues that interact with these ligands (Fig. 2 and Supplementary Figure 7), which may contribute to the subtype specificity of these ligands10,28.

Figure 1

Structure of the β1-adrenergic receptor bound to agonists. (a) Structure of β1AR shown in cartoon representation with the intracellular side at the bottom of the figure. The ligand carmoterol is shown as a space filling model (C, yellow; O, red; N, blue). The N-terminus (N), C-terminus (C), extracellular loop 2 (EL2), and transmembrane helices 1-4 (H1-4) are labeled. The same orientation of receptor is shown in panels (b-f); (b) the antagonist cyanopindolol; (c-d) the partial agonists dobutamine and salbutamol; (e-f) the full agonists isoprenaline and carmoterol. The colour scheme of the ligand and labeling of the receptor is identical in all panes, with amino acid sidechains that make hydrogen bonds to the ligands depicted (C, green; O, red; N, blue). For clarity, residues 171-196 and 94-119 have been removed in B-F, which correspond to the C-terminal region of H4 and EL2, and EL1 with the C-terminal region of H2 and N-terminal region of H3, respectively. All structures shown are of monomer B (Supplementary Figure 2) and were generated using Pymol (DeLano Scientific Ltd). For a comparison of the positions of the ligands when bound to the receptor, see Supplementary Figure 5.

Figure 2

Polar and non-polar interactions involved in agonist binding to β1-adrenergic receptor. Amino acid residues within 3.9 Å of the ligands are depicted, with residues highlighted in blue making van der Waals contacts (blue rays) and residues highlighted in red making potential hydrogen bonds with favourable geometry (red dashed lines) or hydrogen bonds with unfavourable geometry (blue dashed lines). Amino acid residues labeled with an asterisk make the indicated contact either in monomer A (A*) or in monomer B (B*) only; for dobutamine, some contacts, labelled , are found only in monomer B of dob92, whereas another contact, labeled [B*], is found only in monomer B of dob102 (Supplementary Figure 6 and also see Supplementary Table 6 for further details and for the Ballesteros-Weinstein numbering). If specific van der Waals interactions or polar interactions are found only in monomer A or B, then the interaction is labeled a* or b*, respectively. Where the amino acid residue differs between the turkey β1AR and the human β1AR, β2AR and β3AR, the equivalent residue is shown highlighted in orange, purple or green, respectively (see also Supplementary Table 7).

Figure 3

Comparison of the ligand binding pockets of the β1 and β2 adrenergic receptors. The ligand binding pockets are shown as viewed from the extracellular surface with EL2 removed for clarity (same colour scheme as in Fig. 1). (a) β2AR with the antagonist carazolol bound (PDB code 2RH1); (b) β1AR with the antagonist cyanopindolol bound (PDB code 2VT4); (c) β1AR with the agonist isoprenaline bound.

There are three significant differences in the β1AR catecholamine binding pocket when full agonists are bound compared to when an antagonist is bound, namely the rotamer conformation changes of side chains Ser2125.43 and Ser2155.46 (Fig.3) and the contraction of the catecholamine binding pocket by ~1 Å, as measured between the Cα atoms of Asn3297.39 and Ser2115.42 (Fig. 4). So why should these small changes increase the probability of R* formation? Agonist binding has not changed the conformation of transmembrane helix H5 below Ser2155.46, although significant changes in this region are predicted once the receptor has reached the fully activated state18,19. The only effect that the agonist-induced rotamer conformation change of Ser2155.46 appears to have is to break the van der Waals interaction between Val1724.56 and Ser2155.46, thus reducing the number of interactions between H4 and H5. As there is only a minimal interface between transmembrane helices H4 and H5 in this region (Supplementary Table 8 and Supplementary Fig. 8), this loss of interaction may be significant in the activation process. In this regard, it is noteworthy that the naturally occurring polymorphism in β2AR at the H4-H5 interface, T164I4.56, converts a polar residue to a hydrophobic residue as seen in β1AR (Val1724.56), which results in both reduced basal activity and reduced agonist stimulation21. This supports the hypothesis that the extent of interaction between H4 and H5 could affect the probability of a receptor transition into the activated state.

Figure 4

Differences in the ligand binding pocket between antagonist- and agonist-bound β1-adrenergic receptor. An alignment was performed (see Online Methods) between the structures of β1AR-m23 bound to either cyanopindolol (grey) or isoprenaline (orange) and the relative positions of the ligands and the transmembrane helices H5 and H7 are depicted. The 1 Å contraction of the ligand binding pocket between H5 and H7 is clear.

In contrast to the apparent weakening of helix-helix interactions by the agonist-induced rotamer conformation change of Ser2155.46, the agonist-induced rotamer conformation change of Ser2125.43 probably results in the strengthening of interactions between H5 and H6. Upon agonist binding, Ser2125.43 forms a hydrogen bond with Asn3106.55 (Fig. 3) and, in addition, hydrogen bond interactions to Ser2115.43 and Asn3106.55mediated by the ligand serve to bridge H5 and H6. The combined effects of strengthening the H5-H6 interface and weakening the H4-H5 interface could facilitate the subsequent movements of H5 and H6, as observed in the activation of rhodopsin.

Stabilisation of the contracted catecholamine binding pocket is probably the most important role of bound agonists in the activation process (Fig. 4). This probably requires strong hydrogen bonding interactions between the catechol (or equivalent) moiety and both H5 and H6, and strong interactions between the secondary amine and β-hydroxyl groups in the agonist and the amino acid side chains in helices H3 and H7. Reduction in the strength of these interactions is likely to reduce the efficacy of a ligand29. Both salbutamol and dobutamine are partial agonists of β1AR-m23 (Supplementary Table 3) and human β1AR. In the case of salbutamol, there are only two predicted hydrogen bonds between the headgroup and H5/H6, compared to 3-4 potential hydrogen bonds for isoprenaline and carmoterol. Dobutamine lacks the β-hydroxyl group, which similarly reduces the number of potential hydrogen bonds to H3/H7 from 3-4 seen in the other agonists to only 2. We propose that this weakening of agonist interactions with H5/H6 for salbutamol and H3/H7 for dobutamine is a major contributing factor in making these ligands partial agonists rather than full agonists.

The agonist-bound structures of β1AR suggest there are three major determinants that dictate the efficacy of any ligand; ligand-induced rotamer conformational changes of (i) Ser2125.43 and (ii) Ser2155.46 and (iii) stabilization of the contracted ligand binding pocket. The full agonists studied here achieve all three. The partial agonists studied here do not alter the conformation of Ser2155.46 and may be less successful than isoprenaline or carmoterol at stabilizing the contracted catecholamine binding pocket due to reduced numbers of hydrogen bonds between the ligand and the receptor. The antagonist cyanopindolol acts as a very weak partial agonist and none of the three agonist-induced changes are observed. In contrast to partial agonists, neutral antagonists or very weak partial agonists such as cyanopindolol may also have a reduced ability to contract the binding pocket due to the greater distance between the secondary amine and the catechol moiety (or equivalent). For example, the number of atoms in the linker between the secondary amine and the headgroup of cyanopindolol is 4 whereas the agonists in this study only contain 2 (Fig. 1 and Supplementary Fig. 4). A ligand with a sufficiently bulky headgroup that binds with high-affinity and which actively prevents any spontaneous contraction of the binding pocket and/or Ser5.46 rotamer change, would be predicted to act as a full inverse agonist. This is indeed what is observed in the recently determined structure15 of β2AR bound to the inverse agonist ICI 118,551.

The significant structural similarities amongst GPCRs suggests that similar agonist-induced conformational changes to those we have observed here may also be applicable to many other members of the GPCR superfamily, though undoubtedly there will be many subtle variations on this theme.

METHODS SUMMARY

Expression, purification and crystallization

The β44-m23 construct was expressed in insect cells, purified in the detergent Hega-10 and crystallized in the presence of cholesterol hemisuccinate (CHS), following previously established protocols30. Crystals were grown by vapour diffusion, with the conditions shown in Supplementary Table 4.

Data collection, structure solution and refinement

Diffraction data were collected from a single cryo-cooled crystal (100 K) of each complex in multiple wedges at beamline ID23-2 at ESRF, Grenoble. The structures were solved by molecular replacement using the β1AR structure13 (PDB code 2VT4) as a model (see Online Methods). Data collection and refinement statistics are presented in Supplementary Table 5.