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.2019 May 24;364(6442):775-778.
doi: 10.1126/science.aau5595. Epub 2019 May 9.

Molecular basis for high-affinity agonist binding in GPCRs

Affiliations

Molecular basis for high-affinity agonist binding in GPCRs

Tony Warne et al. Science..

Abstract

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.

Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

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Conflict of interest statement

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

Figures

Fig. 1
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-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
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.
Fig. 3
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.
Fig. 4
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 radioligand3H-DHA on to β1AR (blue circles), β1AR(F325Y) (red triangles) and β1AR(F325A) (orange circles). The inset shows the affinities of3H-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-Gs, 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.
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