Molecular basis for high-affinity agonist binding in GPCRs

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Science  24 May 2019:
Vol. 364, Issue 6442, pp. 775-778
DOI: 10.1126/science.aau5595

A GPCR seen in the active state

G protein–coupled receptors (GPCRs) are exceptionally good targets for drug development. Warne et al. describe four crystal structures of complexes of a GPCR—the β1-adrenergic receptor—in its active state. They used nanobodies (recombinant variable domains of heavy-chain antibodies) and engineered G protein to stabilize the β1-adrenergic receptor bound to a full agonist, two partial agonists, and a weak partial agonist. Comparison of these structures to the inactive state elucidates how agonist binding is altered in the active conformation.

Science, this issue p. 775


G protein–coupled receptors (GPCRs) in the G protein–coupled active state have higher affinity for agonists as compared with when they are in the inactive state, but the molecular basis for this is unclear. We have determined four active-state structures of the β1-adrenoceptor (β1AR) bound to conformation-specific nanobodies in the presence of agonists of varying efficacy. Comparison with inactive-state structures of β1AR bound to the identical ligands showed a 24 to 42% reduction in the volume of the orthosteric binding site. Potential hydrogen bonds were also shorter, and there was up to a 30% increase in the number of atomic contacts between the receptor and ligand. This explains the increase in agonist affinity of GPCRs in the active state for a wide range of structurally distinct agonists.

G protein–coupled receptors (GPCRs) exist in an ensemble of conformations that can be selectively stabilized by the binding of a ligand and through interactions with signaling molecules such as G proteins (1, 2). Pharmacology has characterized at least two distinct states of GPCRs, an active state with high affinity for agonists when coupled to G proteins and an inactive state with low affinity for agonists in the absence of G proteins (1), although a plethora of substates can also exist between these two extremes (37). The reason why the active state has a high affinity for agonists is unclear because receptor structures in the inactive and active states have been determined with different ligands bound, such as for the β2-adrenoceptor (β2AR) (811). Here, we present structures of β1AR in the active state and compare them with inactive-state structures (12) bound to the identical ligand to define the structural differences in the orthosteric binding site.

Four crystal structures with overall resolutions between 3.0 and 3.2 Å (table S1) were determined of β1AR bound to either Nb80 or Nb6B9, and the overall structures were virtually identical [0.2 to 0.3 Å root mean square deviation (RMSD) for Cα atoms] (Fig. 1). Nb80 and Nb6B9 are nanobodies originally developed to stabilize the active state of β2AR (8, 10) and bind to β1AR because of the high sequence conservation of the receptors. Structures were determined bound to a full agonist (isoprenaline), partial agonists (salbutamol and dobutamine), and a weak partial agonist (cyanopindolol). Isoprenaline, salbutamol, and dobutamine showed an increase in affinity when β1AR was coupled to the engineered G protein mini-Gs, whereas cyanopindolol bound with similar high affinity in both the presence and absence of mini-Gs (Fig. 1). The overall structure of the β1AR-nanobody complexes bound to either agonist or partial agonists is virtually identical to that of the agonist-bound Nb6B9-β2AR complex (0.5 Å RMSD of 1601 atoms), and the overall conformational changes are virtually identical. These changes result in the partial occlusion of the orthosteric binding pocket (Fig. 2), which is consistent with observations on nanobody-bound β2AR (8, 10).

Fig. 1 Structure of the active state of agonist-bound β1AR-nanobody complex.

(A) Superposition of four structures of β1AR-nanobody complexes bound to ligands shown in (C). (B) Affinities of β1AR in the low-affinity state (L) and high-affinity state coupled to mini-Gs (H) for the ligands co-crystallized with the receptor. Data are in tables S2 and S3 and fig. S6. Results are the mean of two to four experiments performed in duplicate, with error bars representing the SEM. (C) Structures of the ligands cocrystallized in the β1AR complexes. (D) Disposition of the ligands after superposition of the receptors, using the same color coding as in (B). Ligand densities are provided in fig. S9.

Fig. 2 Conformational changes in isoprenaline-bound β1AR.

(A) Superposition of isoprenaline-bound β1AR in the inactive state (gray, PDB ID 2Y03) with isoprenaline-bound β1AR in the active state (rainbow coloration). Arrows (magenta) indicate the transitions from the inactive to active state. Alignment was performed based on the isoprenaline molecules by using PyMol (magenta, isoprenaline bound to active state β1AR). (Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. In the mutants, other amino acids were substituted at certain locations; for example, F325Y indicates that phenylalanine at position 325 was replaced by tyrosine.) (B) View of the orthosteric binding site from the extracellular surface, with atoms shown as space filling models. For isoprenaline, magenta, carbon atoms. For β1AR, H1, dark blue; H2, light blue; H5, yellow; ECL2, green; ECL3 and parts of H6 and H7, red. (C) Volumes of the orthosteric binding site in the low-affinity inactive state (L, pink bars) compared with the high-affinity active state (H, green bars). (D) Number of atomic contacts (data file S1) between the respective ligands and β1AR in the low-affinity inactive state (L, pink bars) compared with the high-affinity active state (H, green bars). The dark shades represent the number of polar interactions. Ligand abbreviations are shown in Fig. 1.

Detailed comparisons were made between the inactive-state structures of β1AR with the respective active-state structures bound to the same ligand (Figs. 2 and 3). In all cases, there was a decrease in the volume (13) of the orthosteric binding site that varied depending on the ligand (Fig. 2 and fig. S1). The largest decrease was observed for the full agonist isoprenaline (42%), and the smallest decrease was observed for the weak partial agonist cyanopindolol (24%). The decrease in the volume of the orthosteric binding site when isoprenaline was bound was primarily due to the inward movement of the extracellular ends of H6 and H7, an inward movement and an increase in the H5 bulge at Ser2155.46, and the reorientation of residues Phe201ECL2 and Phe3257.35 [superscripts refer to the Ballesteros-Weinstein numbers (14)]. The magnitude of these changes was greatest for the full agonist isoprenaline and smallest for the weak partial agonist cyanopindolol. The pincerlike movement of Phe201ECL2 and Phe3257.35 toward the ligand has the largest effect on reducing the volume of the orthosteric binding pocket, with the maximal shift observed in the isoprenaline structure of 3.1 Å for Phe201ECL2 and 2.5 Å for Phe3257.35 (measured at the CZ atom of the side chain). The movement of Phe201ECL2 appeared to correlate with the structure of the ligand bound because in all cases it formed van der Waals contacts with the ligand. By contrast, Phe3257.35 was not within van der Waals contact with any of the four ligands and moved as a consequence of the inward tilt of H7.

Fig. 3 Changes in β1AR-ligand contact distances.

The maximal changes in contact distances between ligands and atoms in β1AR from the inactive to active states are depicted. Amino acid side chains making contact with the ligands are indicated and colored according to where they are in β1AR (blue, H2; red, H3; orange, ECL2; gray, H5; green, H6; purple, H7), with the diameter of the circle representing the magnitude of the distance change (shown as numbers below the amino acid residue). Numbers next to the lines indicate the change in length of polar contacts (blue dashed lines) and hydrogen bonds (red dashed lines; determined by using HBPLUS). Negative numbers imply a decrease in distance between the ligand and receptor in the transition from the inactive state to the active state. An asterisk indicates a rotamer change between the inactive and active states. The details of additional contacts made by each side chain are provided in fig. S2.

The reduction in the volume of the orthosteric binding pocket correlated with an overall reduction in the average distance between atoms in the ligand and receptor by 0.1 to 0.3 Å. Amino acid residues in H3, H5, H6, H7, and ECL2 (and H2 for dobutamine) were all involved in contributing to ligand-receptor contacts, but there was no clear pattern as to which regions of the receptor changed most substantially (Fig. 3 and fig. S2). Side chains were up to 1.2 Å closer to the ligand in the active state compared with the inactive state. In a number of instances, changes resulted in the strengthening of hydrogen bonds. For example, Asn3106.55 was predicted to make a weak hydrogen bond to the para-hydroxyl group of isoprenaline (3.5 Å between donor and acceptor) in the inactive state, which changed to 2.8 Å in the active state. In the active-state structures containing dobutamine and salbutamol, the distance to Ser2155.46 was 0.8 Å shorter for both ligands, allowing hydrogen bond formation; the hydrogen bond to Ser2115.42 also shortened by 0.7 Å to the para hydroxyl in salbutamol but remained unchanged to the meta hydroxyl in dobutamine. Most of the observed differences are due to the contraction of the binding pocket, whereas the substantial shortening of the hydrogen bond between Ser2115.42 and salbutamol is due to a rotamer change. Although all the ligand binding pockets contracted upon receptor activation, the changes in ligand-receptor contacts were not conserved, despite the similarity in chemotypes among the four ligands studied. There was a weak correlation between the decrease in volume of the orthosteric binding site and ligand efficacy (fig. S3), particularly if the most similar chemtoypic ligands were compared (cyanopindolol, salbutamol, and isoprenaline). However, there was no correlation between efficacy and the magnitude of ligand affinity increase on receptor activation or the increase in the number of ligand-receptor atomic contacts (fig. S3). Cyanopindolol bound to β1AR with similar affinity in both the presence and absence of a coupled G protein (Fig. 1) despite the contraction of the binding pocket and increase in receptor ligand contacts upon activation. This may be a consequence of constraints on the possible conformation change imposed by the rigidity of cyanopindol that prevents the full contraction of the ligand binding pocket by preventing the movement of H7 and the bulge in H5 observed in the other structures (fig. S1).

The role of the partial occlusion of the orthosteric binding site upon activation of β1AR was tested by mutagenesis inspired from the active state structure of β2AR (8, 10). In β2AR, it was proposed that the occlusion of the binding site was a major factor in increasing agonist affinity upon G protein coupling (15). In particular, Tyr3087.35 was within van der Waals distance of Phe193ECL2 on the opposite side of the entrance to the orthosteric binding pocket and had a major effect on decreasing the rates of association and dissociation of ligands in the active state compared with the inactive state (15). The β2AR residues Phe193ECL2/Tyr3087.35 are equivalent to Phe201ECL2/Phe3257.35 in β1AR, which are not in van der Waals contact (Fig. 4). Thus, the mutation F325Y7.35 in β1AR was predicted to occlude the entrance to the orthosteric binding pocket and decrease the rate of ligand association, and conversely, F325A7.35 was predicted to make the entrance wider and increase the rate of ligand association. When the initial rate of 3H-dihydroalprenolol (3H-DHA) association was measured (Fig. 4), β1AR(F325A) had the same rate as β1AR, but β1AR(F325Y) had a considerably slower rate of association. However, the affinities (Fig. 4) of epinephrine and isoprenaline for the high-affinity state of β1AR and β1AR(F325Y) were identical, and there was only a minor difference with norepinephrine. The inactive state of β1AR and the inactive state of β1AR(F325Y) were compared for their affinity to agonists (Fig. 4), and it was found that β1AR had higher affinity than β1AR(F325Y) for norepinephrine (8.1 nM versus 120 nM), epinephrine (56 nM versus 630 nM), and isoprenaline (6.9 nM versus 52 nM). This implied that the greater agonist affinity shift observed in β1AR(F325Y) compared with β1AR was due to destabilization of the inactive state and not stabilization of the active state. This suggested that partial occlusion of the ligand binding pocket in β1AR(F325Y) during formation of the active state played little role in the increase of agonist affinity on G protein coupling.

Fig. 4 Comparisons between β1AR and β2AR.

(A) Alignment of the active-state structures of β1AR (rainbow coloration) and β2AR (gray, PDB ID 4LDO). Ligands are shown as sticks. Isoprenaline, yellow; adrenaline, grey. (B) Rate of association of the radioligand 3H-DHA on to β1AR (blue circles), β1AR(F325Y) (red triangles), and β1AR(F325A) (orange circles). (Inset) The affinities of 3H-DHA (same color code). (C and D) Affinities of β1AR, β2AR, and their respective mutants in the low-affinity state (L) and high-affinity state coupled to mini-Gs (H). All data are in tables S2 and S3, and representative graphs of affinity shifts are in fig. S6. Results are the mean of two to six experiments performed in duplicate, with error bars representing the SEM.

The destabilizing effect of the F325Y mutation in β1AR on the agonist-bound inactive state suggested that converting the extracellular surface of β2AR to make it similar to β1AR would increase the affinity of the inactive state and leave the affinity of the G protein–coupled activated state approximately unchanged. The β2AR mutant constructed, β2AR(β1LBP), did indeed show these characteristics (fig S4; the rationale of the four mutations used—Y174WECL2, H296N6.58, K305D7.32, and Y308F7.35—is provided in the supplementary materials, materials and methods). In addition, the accessibility of the β1AR orthosteric binding pocket in the G protein–coupled state to 125I-cyanopindolol was greater than that observed for β2AR (fig. S5). The four mutations in β2AR(β1LBP) converted the behavior of β2AR to that of β1AR. Adding the converse residues from β2AR into β1AR, to make the mutant β1AR(β2LBP), converted the accessibility of the orthosteric binding site in β1AR to that of β2AR (fig. S5).

The increase in affinity of agonist binding to the active state of β1AR arises from the increase in the number and/or strength of ligand-receptor contacts (a thermodynamic effect). This is consequently associated with a decrease in the rate of ligand dissociation. Where a native ligand is a large peptide that interacts with extracellular domains, this thermodynamic effect may be the major contributor to the decrease in the rate of ligand dissociation. However, where the ligand is small, such as adrenaline or acetylcholine, the rate of ligand dissociation may also be decreased because of a purely steric blockage of the entrance to the ligand binding pocket (a kinetic effect) (14). Given the high conservation of both the structure and function of GPCRs (16, 17), these considerations are likely to apply to other GPCRs that bind diffusible ligands.

Supplementary Materials

Materials and Methods

Figs. S1 to S9

Tables S1 to S4

References (1729)

Data Files S1 and S2

References and Notes

Acknowledgments: We thank the beamline staff at the European Synchrotron Radiation Facility (beamlines ID23-2, ID30-A3, ID29, ID30B, and MASSIF-1) and at Diamond Light Source (beamline I24). Funding: This work was supported by core funding from the Medical Research Council (MRC U105197215 and U105184325) and a grant from the European Research Council (EMPSI 339995). Author contributions: T.W. performed receptor and nanobody expression, purification, crystallization, cryo-cooling of the crystals, data collection, data processing, and structure refinement. T.W. also performed the pharmacological analyses. P.C.E. purified mini-Gs, and A.S.D. assisted with structure solution. A.G.W.L. was involved in data processing and structure solution, refinement, and analysis. Manuscript preparation was performed by T.W., A.G.W.L., and C.G.T. The overall project management was by C.G.T. Competing interests: C.G.T. is a shareholder, consultant, and member of the Scientific Advisory Board of Heptares Therapeutics, who also partly funded this work. Data and materials availability: The coordinates and structure factors for all the structures determined have been deposited at the Protein Data Bank (PDB) with the following accession codes (ligand cocrystallized in parentheses): 6H7J (isoprenaline), 6H7L (dobutamine), 6H7M (salbutamol), and 6H7O (cyanopindolol).
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