A2A Adenosine Receptor Partial Agonism Related to Structural Rearrangements in an Activation Microswitch

Abstract

In drug design, G protein-coupled receptor (GPCR) partial agonists enable one to fine-tune receptor output between basal and maximal signaling levels. Here, we add to the structural basis for rationalizing and monitoring partial agonism. NMR spectroscopy of partial agonist complexes of the A_2A adenosine receptor (A_2AAR) revealed conformations of the P-I-F activation motif that are distinctly different from full agonist complexes. At the intracellular surface, different conformations of helix VI observed for partial and full agonist complexes manifest a correlation between the efficacy-related structural rearrangement of this activation motif and intracellular signaling to partner proteins. While comparisons of A_2AAR in complexes with partial and full agonists with different methods showed close similarity of the global folds, this NMR study now reveals subtle but distinct local structural differences related to partial agonism.

Keywords: G protein-coupled receptors; NMR spectroscopy; drug efficacy; partial agonists.

Figure 1.. Global Folds of A_2AAR Complexes with a Partial Agonist, a Full Agonist and an Antagonist.

(A) 800 MHz [¹⁵N,¹H]-TROSY correlation spectrum of [u-¹⁵N, ~70% ²H]-A_2AAR in complex with the partial agonist LUF5834 (purple) superimposed with that of the complex with the full agonist NECA (black). The region framed with dotted lines corresponds to the region shown on an expanded scale in Figure 2A. On the left the chemical structures of the ligands are shown. (B) Corresponding superposition for the complexes with LUF5834 (purple) and the antagonist ZM241385 (green).

Figure 2.. Manifestation of A_2AAR Partial Agonism in the Toggle Switch Tryptophan Indole ¹⁵N─¹H NMR Signal.

(A and B) Assignment of the Trp246^6.48 indole ¹⁵N─¹H NMR signal of A_2AAR in complex with the partial agonist LUF5834 by its absence in the spectrum of A_2AAR[W246F]. (A) Superposition of the indole ¹⁵N─¹H region in contour plots of 800 MHz 2D [¹⁵N,¹H]-TROSY correlation spectra of [u-¹⁵N, ~70% ²H]-A_2AAR (blue) and [u-¹⁵N, ~70% ²H]-A_2AAR[W246F] (red). (B) 1D cross sections at the ¹⁵N chemical shift indicated by the horizontal dashed line in (A). (C) The indole ¹⁵N─¹H regions of four 800 MHz 2D [¹⁵N,¹H]-TROSY correlation spectra are superimposed, highlighting the Trp246^6.48 indole ¹⁵N─¹H NMR signal of the A_2AAR complexes with the antagonist ZM241385 (green), the partial agonist Regadenoson (black), the partial agonist LUF5834 (blue), and the full agonist NECA (red). For improved clarity, the opacity of all other signals in the displayed spectral region has been reduced.

Figure 3.. Engineered NMR Probe Trp233 at the Intracellular Surface of A_2AAR Manifests Variable Efficacies of Full and Partial Agonists.

(A) Superposition of contour plots of the Trp indole ¹⁵N─¹H region in 800 MHz 2D [¹⁵N,¹H]-TROSY correlation spectra of [u-¹⁵N, ~70% ²H]-A_2AAR[K233W] in complexes with NECA (red) and LUF5834 (blue). (B) Corresponding superposition as in (A) for the A_2AAR complexes with NECA (red) and Regadenoson (black).

Figure 4.. Location of the P–I–F Activation Microswitch in A_2AAR and Spatial Arrangement of the Toggle Switch Trp246^6.48 and Phe242^6.44 that are Compatible with Ring Current Shift Calculations Using the Atomic Coordinates of an A_2AAR Partial Agonist Complex Model.

(A) Crystal structures of A_2AAR in complexes with the full agonist NECA (purple; PDB 2YDV) and the antagonist ZM241385 (orange; PDB 3EML) are superimposed. The dotted black box shows the location of the P–I–F activation microswitch in the protein structures and the cyan-colored sphere identifies the amino acid position 233. (B) Top-down expanded view of the P–I–F activation microswitch and the nearby Trp246^6.48. The protein backbone is colored grey, and the annotated side chains for the complexes with NECA and ZM241385 are purple and orange, respectively. The protein backbone segments located above the annotated amino acids were removed for improved clarity. (C) A ring current shift-derived model of the complex with the partial agonist LUF5834 (blue) superimposed on the A_2AAR structures presented in (B). In the LUF5834 complex the Trp246^6.48 χ² angle is rotated by −20° and the F242^6.44 χ¹ angle by +60° relative to the orientations in the NECA complex (Supplemental Table 1).