A2A adenosine receptor functional states characterized by 19F-NMR

Abstract

The human proteome contains 826 G protein-coupled receptors (GPCR), which control a wide array of key physiological functions, making them important drug targets. GPCR functions are based on allosteric coupling from the extracellular orthosteric drug binding site across the cell membrane to intracellular binding sites for partners such as G proteins and arrestins. This signaling process is related to dynamic equilibria in conformational ensembles that can be observed by NMR in solution. A previous high-resolution NMR study of the A_2A adenosine receptor (A_2AAR) resulted in a qualitative characterization of a network of such local polymorphisms. Here, we used ¹⁹F-NMR experiments with probes at the A_2AAR intracellular surface, which provides the high sensitivity needed for a refined description of different receptor activation states by ensembles of simultaneously populated conformers and the rates of exchange among them. We observed two agonist-stabilized substates that are not measurably populated in apo-A_2AAR and one inactive substate that is not seen in complexes with agonists, suggesting that A_2AAR activation includes both induced fit and conformational selection mechanisms. Comparison of A_2AAR and a constitutively active mutant established relations between the ¹⁹F-NMR spectra and signaling activity, which enabled direct assessment of the difference in basal activity between the native protein and its variant.

Keywords: GPCR; NMR spectroscopy; adenosine receptor; dynamics; signaling.

Fig. 1.

Location of ¹⁹F-NMR probes in the crystal structure of A_2AAR and NMR response to variable drug efficacy. (A) Side view of a superposition of the antagonist complex A_2AAR–ZM241385 (brown, PDB-ID: 3ELM) with the agonist complex A_2AAR–UK432097 (cyan, PDB-ID: 3QAK); the extracellular membrane surface is at the top. The three sequence positions selected for the ¹⁹F labels are highlighted by spheres and identified. (B) Same as A, view onto the intracellular surface; Roman numerals indicate the TM numbering. (C) The 1D ¹⁹F-NMR spectra of A_2AAR[A289C^TET] complexes with ligands of varying efficacy, as indicated in the Center. On the Right, the NMR spectra shown on the Left are interpreted by Lorentzian deconvolutions with the minimal number of components that provided a good fit, i.e., P₁ to P₄. The chemical shift positions of P₁ to P₄ are indicated by colored broken vertical lines. (D) The 1D ¹⁹F-NMR observation of ligand exchange in A_2AAR[A289C^TET]. The agonist NECA was added at saturating concentration to the apo-A_2AAR and then displaced by the more strongly binding antagonist ZM241385.

Fig. 2.

Conformational exchange in the A_2AAR complex with the antagonist ZM241385 by ¹⁹F-NMR saturation transfer. (A, Left) The 1D ¹⁹F-NMR spectrum. The Lorentzian deconvolution introduced in Fig. 1 is indicated. A red arrow indicates the carrier position for the preirradiation, and a black arrow indicates the position for the reference measurement. The observed peak is indicated by “detection.” (A, Right) Plots of the normalized intensity of the observed peak, P₃, versus the saturation pulse length after irradiation on P₁ (cyan) and at the control (black). (B) Same as A, with inverted direction. (C) Survey of the conformational exchange rates in the A_2AAR[A289C^TET]–ZM241385 complex.

Fig. 3.

Conformational exchange in the A_2AAR complex with the full agonist NECA by ¹⁹F-NMR saturation transfer. (A–D) Four individual measurements linking P₁, P₂, and P₄, same presentation as in Fig. 2 A and B. (E) Survey of the conformational exchange rates in the A_2AAR[A289C^TET]–NECA complex. Please note in C and D that the saturation and control saturation are not symmetrical to the detection position, and this arrangement was chosen to prevent falsification of the data by direct irradiation of the tail of the observed signal.

Fig. 4.

Conformational exchange in the A_2AAR complex with the full agonist NECA observed by 2D exchange spectroscopy. A contour plot is shown of a 2D [¹⁹F, ¹⁹F]-EXSY spectrum collected at 280 K with a mixing time of 100 ms. The diagonal peak positions of P₁, P₂, and P₄ are labeled, and a dashed box indicates crosspeaks observed between P₁ and P₂.

Fig. 5.

Dynamic processes in the ¹⁹F-labeled constitutively active mutant A_2AAR[S91A, A289C^TET] observed by 1D ¹⁹F-NMR. (A) Same ligand exchange experiment as in Fig. 1D. (B) Lorentzian deconvolutions of the spectra shown in A.

Fig. 6.

Biological response versus ligand concentration as manifested in the ¹⁹F NMR spectra. A plot is shown of the biological activity (i.e., G protein signaling) versus ligand concentration for A_2AAR and A_2AAR[S91A], as labeled on the right of the individual sigmoidal response curves. Relative biological activity was determined by observation of the intensity of the peak P₄ in ¹⁹F-NMR spectra of A_2AAR[A289C^TET] and A_2AAR[S91A, A289C^TET]. The dashed lines represent the basal signaling level of the two proteins.