Role of detergents in conformational exchange of a G protein-coupled receptor

Abstract

The G protein-coupled β(2)-adrenoreceptor (β(2)AR) signals through the heterotrimeric G proteins G(s) and G(i) and β-arrestin. As such, the energy landscape of β(2)AR-excited state conformers is expected to be complex. Upon tagging Cys-265 of β(2)AR with a trifluoromethyl probe, (19)F NMR was used to assess conformations and possible equilibria between states. Here, we report key differences in β(2)AR conformational dynamics associated with the detergents used to stabilize the receptor. In dodecyl maltoside (DDM) micelles, the spectra are well represented by a single Lorentzian line that shifts progressively downfield with activation by appropriate ligand. The results are consistent with interconversion between two or more states on a time scale faster than the greatest difference in ligand-dependent chemical shift (i.e. >100 Hz). Given that high detergent off-rates of DDM monomers may facilitate conformational exchange between functional states of β(2)AR, we utilized the recently developed maltose-neopentyl glycol (MNG-3) diacyl detergent. In MNG-3 micelles, spectra indicated at least three distinct states, the relative populations of which depended on ligand, whereas no ligand-dependent shifts were observed, consistent with the slow exchange limit. Thus, detergent has a profound effect on the equilibrium kinetics between functional states. MNG-3, which has a critical micelle concentration in the nanomolar regime, exhibits an off-rate that is 4 orders of magnitude lower than that of DDM. High detergent off-rates are more likely to facilitate conformational exchange between distinct functional states associated with the G protein-coupled receptor.

FIGURE 1.

A, overlay of x-ray crystal structures of β₂AR stabilized by either the inverse agonist carazolol (Cz; shown in green) or the stimulatory G protein Gα_s (shown in orange). B, sequence and secondary structure of β₂AR showing cysteine residues that are retained for stability and labeling (blue) and those that are removed (red) to avoid excess labeling. Solid lines indicate disulfide linkages.

FIGURE 2.

¹⁹F NMR spectra of β₂AR reconstituted in DDM micelles at 25 °C. Note that the leftmost peak represents a small molecule used as a reference, whereas the barely detectable peak to the right (indicated by a triangle) represents a residually labeled cysteine on β₂AR, the spectral area of which remains independent of ligand. The orange line designates residual error resulting from deconvoluting the spectrum. Cz, carazolol; BI, BI-167107.

FIGURE 3.

¹⁹F NMR chemical shift trends at Cys-265 as a function of activation in DDM micelles. Cz, carazolol; BI, BI-167107.

FIGURE 4.

Estimates of S and τ_e at Cys-265 as a function of activation of β₂AR (saturating concentrations of inverse agonist → apo → BI-167107 agonist → BI-167107 agonist plus Nb80). Estimates of local orientational order and dynamics were based upon measurements of ¹⁹F T₁ and T₂ at 600 MHz. Black bars represent τ_e, and white bars represent S². Cz, carazolol; BI, BI-167107.

FIGURE 5.

¹⁹F NMR spectra of β₂AR reconstituted in MNG micelles at 30 °C. Note that the peak to the right (indicated by a triangle) represents a residually labeled cysteine on β₂AR, the spectral area of which remains independent of ligand. The presence of a second partially labeled site was corroborated by subsequent ¹⁹F NMR spectra from proteinase K digests. Cz, carazolol; BI, BI-167107.

FIGURE 6.

A, isothermal titration calorimetry enthalpogram of DDM, where ΔH_demic is −2. 27 kJ/mol, and the CMC, determined from a first derivative plot of the enthalpogram, is estimated to be 0.13 mm. B, surface tension-based measurement of MNG-3 showing the CMC to be 11.3 nm.