β₂-adrenergic receptor activation by agonists studied with ¹⁹F NMR spectroscopy.

G-protein-coupled receptors (GPCRs) recognize a wide array of orthosteric ligands in their binding site on the periplasmic cell membrane surface, initiating signal transmission through the cellular membrane to cytoplasmic partner proteins. Crystal structures of several human GPCRs in complexes with antagonists and agonists provide insights into activation-related structural rearrangements,[1] and fluorescence spectroscopy experiments indicated activation-related conformational changes in detergent-solubilized receptors.[2] 19F-NMR spectroscopy and site-specific mutagenesis, as applied previously with mammalian rhodopsin,[3] more recently revealed an equilibrium between an activated state A and an inactive state I in the β2-adrenergic receptor (β2AR).[4] This communication now presents thermodynamic and kinetic data for this conformational equilibrium in β2AR. In our earlier experiments,[4] the β2AR complexes were reconstituted in mixed micelles of n-dodecyl-β-D-maltoside (DDM) and cholesteryl hemisuccinate (CHS), with DDM:CHS = 5:1, and 19F-labels were introduced by conjugation of 2,2,2-trifluoroethanethiol (TET) with cysteines near the cytoplasmic ends of the helices VI (Cys265) and VII (Cys327), and at the C-terminus (Cys341). Ligand binding assays showed that the labeled proteins retained the biological activity.[4] Sequence-specific 19F-NMR assignments were based on comparison of β2AR variants with single-residue TET-labeling, and the signal I was assigned from its high intensity in the apo-form of β2AR and its complexes with inverse agonists.[4] Observation of the TET labels in β2AR-complexes with different pharmacological ligands then enabled to distinguish between the activation of two different signaling pathways.[4] A first extension of the previous work was to analyze the temperature dependence of the 1D 19F-NMR spectra of β2AR (TETC265, C327S, C341A) and β2AR (C265A, TETC327, C341A) in terms of the thermodynamic parameters that characterize the conformational equilibrium between the states A and I. This analysis was focused on complexes with agonists, i.e., norepinephrine and formoterol, since for the complexes with antagonists or inverse agonists the amplitude of the signal A is near the noise level and its volume cannot reliably be quantified. Based on the observation that the temperature dependence over the range 280 K to 310 K of the NMR spectra recorded with the agonist complexes was reversible, the relative populations of the conformations represented by the signals A and I, pA and pI, were determined by fits to a double-Lorentzian function (Figure 1), yielding an apparent equilibrium constant, K = pI/ pA. ln K was found to depend linearly on the inverse of the temperature, T (Figure 2), which is in agreement with the van’t Hoff relationship between K, the molar enthalpy difference, ΔH0, and the molar entropy difference, ΔS0.[5] lnK=−(ΔH0−TS0)/RT, (1) where R is the gas constant. ΔH0 values near 40 kJ/mol were obtained for both labeling sites at Cys265 and Cys327, and for both agonists used (Table 1), suggesting that the structural differences between the states A and I observed in the 19F-NMR spectra (Figure 1) represent more extensive conformational rearrangements than, for example, reorientation of a single amino acid side chain. For the different systems in Table 1 the Gibbs free energy, ΔG0 = −ln(K)RT, is between 0 and 3 kJ/mol, which shows that the entropy and enthalpy terms (Table 1) nearly cancel each other. This observation is in line with the widely observed entropy–enthalpy compensation in biological systems.[6] Figure 1 1D 19F-NMR spectra at 280 K, 298 K and 310 K of the complexes with the partial agonist norepinephrine (upper row) and the full agonist formoterol (lower row) of β2AR(TETC265, C327S, C341A) and β2AR(C265A, TETC327, C341A) in mixed micelles of ... Figure 2 van’t Hoff plots for the interconversion between the active state (A) and the inactive state (I) of β2AR(TETC265, C327S, C341A) and β2AR(C265A, TETC327, C341A) in the complexes with norepinephrine and formoterol. The apparent equilibrium constants, ... Table 1 Molar enthalpy differences, ΔH0, and molar entropy differences, ΔS0, for the interconversion between the activated and inactive states of the formoterol and norepinephrine complexes of β2AR (TETC265, C327S, C341A) and β ... To investigate the exchange rates between the states A and I within the framework of a 2-state model, where k1 and k−1 are the forward and reverse rate constants, A⇄k−1k1I, (2) we used 2D 19F–19F exchange spectroscopy (EXSY)[7] and 1D 19F saturation transfer NMR experiments.[8] The overall exchange rate constant, kex, is given by kex=k1+k−1=k1/pI=k−1/pA, (3) where pA and pI are the relative populations of the states A and I. The observation of two distinct signals A and I in the 19F-NMR spectra of β2AR (Figure 1) showed that the conformational exchange is slow on the 19F-NMR chemical shift time scale, so that kex satisfies the inequality, kex≪Δω2pApI, (4) where Δω is the chemical shift between I and A in rad/sec.[9] For TET-labeled Cys265 and Cys327 the Δω values are 2×103 rad/sec and 4×103 rad/sec, respectively, and pA = 1−pI is between 0.2 and 0.9.[4] An upper limit of kex ≤ 103 s−1 was thus previously established, and additional support for this limit was obtained from experiments with paramagnetic shift reagents.[4] Here, 2D [19F,19F]-EXSY experiments with a TET β2AR–isoproterenol complex were performed with mixing times of 300 and 600 ms. For kex values of 10 s−1 or larger, 2D [19F,19F]-cross-peaks between the signals A and I are predicted to be of similar size as the diagonal peaks in these experiments (Figure 3c). The absence of [19F,19F]-cross-peaks (Figure 3) then enabled us to establish a new upper limit of kex < 10 s−1 at 280 K. 19F-NMR saturation-transfer experiments with apo- β2AR(C265A, TETC327, C341A) at 280 K further indicated that the exchange rate is significantly slower than 10 s−1. Considering the spectral overlap of the two signals (Figure 4), we applied selective off-resonance continuous wave (cw) pre-irradiation in these experiments (Figure 4), and analyzed the resulting intensity variations of the signals A and I with model simulations based on the Bloch equations for two-site exchange (Equations (5) to (10) in the Appendix).[10] The longitudinal and transverse spin relaxation times, T1 and T2, used in these model computations were determined with an inversion–recovery experiment (see the Experimental Section), and from the line shapes of the signals in the 1D 19F-NMR spectrum (top trace in Figure 4), respectively. Comparison of the experimental data with the simulations (Figure 5) showed that the observed decay of the signal I was due to direct saturation by the off-resonance irradiation, and that there was no measureable contribution due to coherence transfer from signal A to signal I by conformational exchange (Figure 5). Figure 3 2D [19F,19F]-EXSY experiments with the isoproterenol complex of wild type β2AR (TETC265, TETC327, TETC341) in mixed micelles of DDM and CHS 5:1. (a) Contour plot. At the top the chemical shifts of five previously assigned peaks[4] are indicated, ... Figure 4 1D 19F-NMR saturation transfer experiments with apo-β2AR(C265A, TETC327, C341A) in mixed micelles of DDM and CHS 5:1 used to measure the exchange rate between activated state (A) and inactive state (I) of β2AR. Top trace: 1D 19F-NMR spectrum, ... Figure 5 Model simulations of the attenuation of the intensity of signal I in the NMR spectra of Figure 4 by off-resonance continuous wave (cw) pre-irradiation at the chemical shifts i to v. The relative peak volumes of the signal I in 1D 19F-NMR saturation transfer experiments, ... In conclusion, this paper used TET 19F-NMR probes attached to three cysteine residues near the cytoplasmic surface to determine thermodynamic and kinetic parameters for the equilibrium between an active state, A, and an inactive state, I, of β2AR, which both represent an ensemble of rapidly interconverting conformers. Slow exchange, on the TET 19F-NMR chemical shift timescale, between the states I and A enabled a quantitative characterization of this rate process. Large values for ΔH0 (Table 1) and an exchange rate slower than 10 S−1 (Figures 3–5) indicate that the interconversion entails major structural rearrangements, which likely involve polypeptide backbone segments.[11] Furthermore, the near-identical values of ΔH0 for different ligands bound to the receptor (Table 1) indicate that the equilibrium between the two states is an intrinsic property of the receptor, so that binding of different orthosteric ligands, allosteric effectors, and possibly of cytoplasmic partner proteins would result in shifts of this pre-existing equilibrium. Comparison with recent related studies of β2AR in DDM micelles shows that TET-labeling provides different information from NMR experiments using either a different 19F-label, 3-bromo-1,1,1-trifluoroacetone (BTFA), on Cys 265[12] or 13C-labeled Met 82,[13] which both provided evidence for two or multiple states of β2AR in fast exchange on the respective chemical shift time scales. Results obtained by combining BTFA-labeling of Cys 265 with the use of the detergent maltose-neopentyl-glycol (MNG-3) were interpreted in terms of slow exchange between at least three states of β2AR.[12] Different experimental approaches thus appear to provide complementary information on the β2AR system, and one can look forward to continued studies of the dynamics of GPCRs with a variety of different reporter groups, including investigations of possible modulation of the protein conformational equilibria by allosteric effectors.