Activation of the A2A adenosine G-protein-coupled receptor by conformational selection

Abstract

Conformational selection and induced fit are two prevailing mechanisms1,2 to explain the molecular basis for ligand-based activation of receptors. G-protein-coupled receptors are the largest class of cell surface receptors and are important drug targets. A molecular understanding of their activation mechanism is critical for drug discovery and design. However, direct evidence that addresses how agonist binding leads to the formation of an active receptor state is scarce3. Here we use 19F nuclear magnetic resonance to quantify the conformational landscape occupied by the adenosine A2A receptor (A2AR), a prototypical class A G-protein-coupled receptor. We find an ensemble of four states in equilibrium: (1) two inactive states in millisecond exchange, consistent with a formed (state S1) and a broken (state S2) salt bridge (known as ‘ionic lock’) between transmembrane helices 3 and 6; and (2) two active states, S3 and S3′, as identified by binding of a G-protein-derived peptide. In contrast to a recent study of the β2-adrenergic receptor4, the present approach allowed identification of a second active state for A2AR. Addition of inverse agonist (ZM241385) increases the population of the inactive states, while full agonists (UK432097 or NECA) stabilize the active state, S3′, in a manner consistent with conformational selection. In contrast, partial agonist (LUF5834) and an allosteric modulator (HMA) exclusively increase the population of the S3 state. Thus, partial agonism is achieved here by conformational selection of a distinct active state which we predict will have compromised coupling to the G protein. Direct observation of the conformational equilibria of ligand-dependent G-protein-coupled receptor and deduction of the underlying mechanisms of receptor activation will have wide-reaching implications for our understanding of the function of G-protein-coupled receptor in health and disease.

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Figure 1: Ligand-dependent A2AR state equilibria.
Figure 2: Ligand-induced effects on conformational state lifetimes.
Figure 3: Effect of pH and Gαs-derived peptide on A2AR conformational states.
Figure 4: Model of the free energy landscape and corresponding model of A2A receptor activation.

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Acknowledgements

This work was supported by the Natural Sciences and Engineering Research Council of Canada, research discovery award grant number 261980 (to R.S.P.) and the Canada Excellence Research Chair Program (to O.P.E., who is the Anne and Max Tanenbaum Chair in Neuroscience at the University of Toronto). We thank T. Kobayashi and R. Grisshammer for providing plasmids with A2AR sequence. We thank J. Wells, S. Larda, and F. Huang from the University of Toronto, as well as S. Furness, B. K. Kobilka, and R. Sunahara for their suggestions and comments.

Author information

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Authors

Contributions

L.Y., O.P.E., and R.S.P. designed the research. L.Y. performed the molecular biology work, generated high-yield transformants, and optimized receptor expression and purification. L.Y. also performed NMR and EPR labelling, NMR experiments, and analysed spectroscopy data. N.V.E. performed and analysed data from EPR experiments. M.Z. assisted with cell culture and receptor purification. R.S.P., L.Y., and O.P.E. prepared the manuscript. O.P.E. and R.S.P. supervised the project.

Corresponding authors

Correspondence to Oliver P. Ernst or R. Scott Prosser.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Comparison of inactive and active GPCR crystal structures.

a, Inactive state A2AR (cyan, inverse agonist ZM241385 bound, PBD accession number 4EIY) and active state A2AR (brown, agonist UK432097 bound, PDB accession number 3QAK). b, Inactive state β2AR (green, inverse agonist carazolol bound, PDB accession number 2RH1) and active state β2AR (red, agonist (8-[(1R)-2-{[1,1-dimethyl-2-(2-methylphenyl)ethyl] amino}-1-hydroxyethyl]-5-hydroxy-2H-1,4-benzoxazin-3(4H)-one) bound, PDB accession number 3SN6). c, Inactive rhodopsin (purple, inverse agonist 11-cis-retinal bound, PDB accession number 1U19) and active metarhodopsin II (blue, agonist all-trans-retinal bound, PDB accession number 3PQR).

Extended Data Figure 2 Secondary structure and topology of C-terminally truncated A2AR-V229C.

Residues 2–317 of A2AR are preceded by an Ala residue resulting from TEV protease cleavage, and are succeeded by an Ala–His10 sequence. A2AR was expressed in P. pastoris (SMD1163 strain) through genomic integration of a pPIC9K vector with a leader sequence consisting of α-Factor, Flag tag (DYKDDDDK), and a TEV protease recognition domain (SNNNNNNNNNNLGENLYFQGA). During the secretion process, the signal peptide of the α-Factor gets cleaved and the domain associated with the Flag tag and TEV recognition domain is removed by TEV protease. a, The truncated wild-type receptor used in this study contains all four native disulfide bonds and six buried cysteine residues (indicated in red), none of which were perturbed by the labelling process, which was specific for the introduced (solvent-exposed) cysteine residue V229C6.31 (shown in green with yellow background; the superscript refers to the Ballesteros–Weinstein numbering37). b, A surface map suggests V229C (green, solvent exposed) should be fully labelled without perturbing the receptor. c, Structures of protein-attached labels for NMR (BTFMA; 2-bromo-N-(4-(trifluoromethyl)phenyl)acetamide) and EPR (PROXYL; 3-(2-iodoacetamido)-PROXYL) analysis. d, e, Location and topology of the labelling site associated with V229C for both the inverse agonist (inactive, grey) and agonist-bound (active, yellow) states (PDB accession numbers 4EIY and 3QAK). Two rotamers of the BTFMA label are indicated in green and purple (the phenyl moiety is shown as a sphere). Note that the size of the tags is slightly larger than that depicted in the figure. The environment around the tag is predicted to differ for inactive and active states of the receptor.

Extended Data Figure 3 Labelling efficiency of A2AR-V229C.

a, Single cysteine CW-EPR spectrum of 50 μM apo A2AR-V229C receptor, labelled with a PROXYL spin-label and reconstituted into MNG-3 detergent micelles. b, DEER measurement of 50 μM PROXYL spin-labelled apo A2AR-V229C receptor. c, 19F NMR spectra of protease-K-digested 19F-labelled A2AR-V229C, showing one dominant peak.

Extended Data Figure 4 Car–Purcell–Meiboom–Gill (CPMG) relaxation dispersion experiment to evaluate dynamics of S1–2.

a, 19F NMR CPMG relaxation series of 19F-labelled apo A2AR-V229C. Each spectrum was acquired using 10,000 scans with a constant T2-refocusing period of 3.5 ms. The spectra in the relaxation series were recorded with different refocusing frequencies (that is, different periods between the refocusing pulses as indicated above, representative of three experiments). The sample consisted of 200 μM 19F-labelled apo A2AR-V229C in 50 mM HEPES buffer (pH 7.4) and 100 mM NaCl. b, CPMG curve for the S1–2 peak (red diamonds) and reference peak (black triangles). S1–2 undergoes millisecond timescale exchange while the reference peak exhibits no dispersion. c, Cartoon illustrating S1 and S2 exchange in addition to the activation intermediates.

Extended Data Figure 5 Comparison of two- and three-state models of 19F-labelled A2AR-V229C.

a, 19F NMR T2 relaxation series of the 19F-labelled apo A2AR-V229C receptor. b, Exponential fit to T2 for the downfield and upfield resonances, A and B in a. c, Deconvolution of the 19F NMR spectrum for 19F-labelled apo A2AR-V229C receptor assuming a two-state model. The fitted line width of the upfield resonance is roughly twice that estimated from the T2 measurement, suggesting the upfield resonance may be better represented as a superposition of two Lorentzian lines, associated with S3 and S3′, as discussed in the Supplementary Information. d, Spectral deconvolution of the 19F NMR spectrum of the 19F-labelled apo A2AR-V229C receptor assuming three states. Note that the most downfield peak is ascribed to S1–2, which results from the rapid flickering of the ionic lock from ‘on’ (S1) to ‘off’ (S2), as evidenced by the CPMG measurements in Extended Data Fig. 4. Thus, we propose a total of four states, three of which may be spectroscopically resolved. The resonance frequencies chosen in the fit for S3 and S3′ were based on the observed peaks seen in the presence of agonists and those identified at pH 6, where S3 and S3′ are better resolved. The fitted line widths are also comparable to the homogeneous line widths, estimated from the above T2 experiment. Note that the difference spectrum (that is, the experimental spectrum minus spectral deconvolution) associated with the fit is shown in blue in c and d.

Extended Data Figure 6 19F NMR spectra of 19F-labelled A2AR-V229C in the presence of 50- or 100-fold excess of different ligands.

Representative (N = 3) 19F NMR spectra as a function of ligands (inverse agonist (ZM241385), partial agonist (LUF5834), and full agonists UK432097, as shown in Fig. 1a. The downfield peak represents a reference peak resulting from the addition of 10 μM bendroflumethazide. Note that a difference spectrum (shown in dark blue), corresponding to the difference between the sum of the three deconvolved resonances and the observed spectrum, is shown in each case. Note that the chemical shifts in the deconvolutions were referenced to the standard (−59.050 ppm) and estimated to be −61.08 ppm (S1–2 (red)), −61.60 ppm (S3 (green)), and −61.85 ppm (S3′ (blue)), respectively. Corresponding line widths were estimated to be 220 Hz, 230 Hz, and 260 Hz, respectively.

Extended Data Figure 7 The role of HMA in the receptor activation process.

a, 19F NMR spectra of 19F-labelled apo A2AR-V229C and 19F-labelled A2AR-V229C in the presence of saturating amounts of the amiloride ligand 5-(N,N-hexamethylene) amiloride (HMA). Addition of 50-fold excess of HMA results in an increase in the S3 fraction and an apparent exchange broadening and slight coalescence of S1–2 and S3, which are represented by the deconvolutions in lavender and green, respectively. After accounting for the exchange process between S1–2 and S3 by assuming kex = 600 Hz, the simulated spectrum (shown in red) compares favourably with the observed spectrum. If we assume that exchange between S1–2 is slow, we then obtain the ‘rigid’ lattice spectrum, shown in black. bd, 19F NMR spectra of 19F-labelled A2AR-V229C showing the effect of the addition of 50-fold excess of HMA to saturating amounts of inverse agonist (100 × ZM241385) and agonist (50 × UK432097 or 100 × NECA). In all cases, addition of HMA competes with the bound ligand and establishes a greater fraction of the S3 state. The three deconvolved resonances are shown in red, green, and blue.

Extended Data Figure 8 Saturation transfer experiments of 19F-labelled A2AR-V229C.

a, 19F NMR spectra of 19F-labelled apo A2AR-V229C with corresponding decay curves associated with continuous wave saturation of either the active state ensemble, S3 + S3′, or the inactive state ensemble, S1–2, are provided in the left and right columns, respectively. To account for off-resonant saturation effects, a control experiment was performed at a frequency, νc, such that the peak of interest was equidistant to the saturation frequency, νS, and the control frequency, νc. The response of the peak of interest (that is, S1–2 and S3 + S3′ in the left and right panels, respectively) to saturation at the control frequency, νc, is represented by black squares. Similarly, the response of the peak of interest to saturation at vS is shown in violet while the effective responses, accounting for off-resonant saturation, are shown in red (S1–2) and green (S3 + S3′). On the basis of the effective decay profiles, and using a two-site exchange model, the lifetime of the inactive state ensemble and active states is estimated to be 1.6 s and 9 s. Spectral deconvolutions allow us to estimate the populations, p(S1–2) and p(S3 + S3′), to be 0.28 and 0.72, respectively. Using the fitted forward rate constant, kAB = 0.62 s−1, the reverse rate constant is estimated to be kBA = 0.24 s−1, assuming kAB × p(S1–2) = kBA × p(S3 + S3′). In contrast, the response to the saturation of S1–2 provided an estimate of kBA = 0.11 ± 0.03 s−1. b, Saturation transfer experiments of full agonist UK432097-bound 19F-labelled A2AR-V229C. The effective decay curve (blue dashed line), associated with saturation of S1–2 is consistent with a process where S3′ magnetization is exchanged with S1–2 via S3, as suggested by the figure in c. c, Model for presumed exchange pathway between S1–2, S3, and S3′.

Extended Data Table 1 Primers/gene fragments used to construct plasmids for this study

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Ye, L., Van Eps, N., Zimmer, M. et al. Activation of the A2A adenosine G-protein-coupled receptor by conformational selection. Nature 533, 265–268 (2016). https://doi.org/10.1038/nature17668

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