Molecular basis for high affinity agonist binding in GPCRs

Abstract

G protein-coupled receptors (GPCRs) in the G protein-coupled active state have higher affinity for agonists compared to 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 24-42% 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.

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 sub-states can also exist between these two extremes (3–7). 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 bound to different ligands, such as for the β₂-adrenoceptor (β₂AR) (8–11). Here we present structures of β₁AR in the active state and compare them to 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 Å - 3.2 Å (Table S1) were determined of β₁AR bound to either Nb80 or Nb6B9, and the overall structures were virtually identical (Fig. 1; 0.2-0.3 Å RMSD for Cα atoms). Nb80 and Nb6B9 are nanobodies originally developed to stabilize the active state of β₂AR (8, 10) and bind to β₁AR due to the high sequence conservation of the receptors. Structures were determined bound to a full agonist (isoprenaline), partial agonists (salbutamol, 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-G_s, whereas cyanopindolol bound with similar high affinity in both the presence and absence of mini-G_s (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-G_s, H, for the ligands co-crystallised with the receptor. Data are in Tables S2 and S3, and Fig. S6. Results are the mean of 2-4 experiments performed in duplicate with error bars representing the SEM. (C) Structures of the ligands co-crystallised in the β₁AR complexes. (D) Disposition of the ligands after superposition of the receptors, using the same colour coding as in (B). See Fig. S9 for ligand densities.

Fig. 2. Conformational changes in isoprenaline-bound β₁AR.

(A), Superposition of isoprenaline-bound β₁AR in the inactive state (grey, PDB ID 2Y03) with isoprenaline-bound β₁AR in the active state (rainbow colouration). Arrows (magenta) indicate the transitions from the inactive to active state. Alignment was performed based on the isoprenaline molecules using PyMol (magenta, isoprenaline bound to active state β₁AR). (B) View of the orthosteric binding site from the extracellular surface with atoms shown as space filling models: isoprenaline (magenta, carbon atoms); 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 to the high-affinity active state (H, green bars). (D) Number of atomic contacts (Database S1) between the respective ligands and β₁AR in the low-affinity inactive state (L, pink bars) compared to 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 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, 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 due primarily to the inward movement of the extracellular ends of H6 and H7, an inward movement and an increase in the H5 bulge at Ser215^5.46 and the reorientation of residues Phe201^ECL2 and Phe325^7.35. The magnitude of these changes was greatest for the full agonist isoprenaline and smallest for the weak partial agonist cyanopindolol. The pincer-like movement of Phe201^ECL2 and Phe325^7.35 towards 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 Phe201^ECL2 and 2.5 Å for Phe325^7.35 (measured at the CZ atom of the side chain). The movement of Phe201^ECL2 appeared to correlate with the structure of the ligand bound, because in all cases it formed van der Waals contacts with the ligand. In contrast, Phe325^7.35 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 to the ligands are indicated and coloured according to where they are in β₁AR (blue, H2; red, H3; orange, ECL2; grey, 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 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 significant rotamer change between the inactive and active states. For the details of additional contacts made by each side chain, see 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 0.1-0.3 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 to which regions of the receptor changed most significantly (Fig. 3, Fig. S2). Side chains were up to 1.2 Å closer to the ligand in the active state compared to the inactive state. In a number of instances, changes resulted in the strengthening of hydrogen bonds. For example, Asn310^6.55 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 Ser215^5.46 was 0.8 Å shorter for both ligands allowing hydrogen bond formation; the hydrogen bond to Ser211^5.42 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 significant shortening of the hydrogen bond between Ser211^5.42 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 amongst 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, 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). It was particularly notable that 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 significant factor in increasing agonist affinity upon G protein coupling (14). In particular, Tyr308^7.35 was within van der Waals distance of Phe193^ECL2 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 to the inactive state (14). The β₂AR residues Phe193^ECL2/Tyr308^7.35 are equivalent to Phe201^ECL2/Phe325^7.35 in β₁AR, which are not in van der Waals contact (Fig. 4). Thus the mutation F325Y^7.35 in β₁AR was predicted to occlude the entrance to the orthosteric binding pocket and decrease the rate of ligand association, and conversely, F325A^7.35 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. Comparisons of affinities (Fig. 4) for the inactive states of β₁AR and β₁AR(F325Y) showed a large decrease in affinities for norepinephrine (14.8-fold), epinephrine (11.2-fold) and isoprenaline (7.6-fold), which implied that the greater agonist affinity shift observed in β₁AR(F325Y) compared to β₁AR was due to destabilisation of the inactive state and not stabilisation 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 colouration) and β₂AR (grey, 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). The inset shows the affinities of ³H-DHA (same colour code). (C, D) Affinities of β₁AR, β₂AR and their respective mutants in the low affinity state, L, and high affinity state coupled to mini-G_s, H. All data are in Tables S2 and S3 and representative graphs of affinity shifts are in Figure S6. Results are the mean of 2-6 experiments performed in duplicate with error bars representing the SEM.

The destabilising 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; see Methods for the rationale of the four mutations used: Y174W^ECL2, H296N^6.58, K305D^7.32 and Y308F^7.35). 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 behaviour 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 due to 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 (15, 16), these considerations are likely to apply to other GPCRs that bind diffusible ligands.

One Sentence Summary.

High affinity agonist binding to G protein-coupled GPCRs results from an increase in the number and strength of protein-ligand interactions.