Allosteric Coupling of Drug Binding and Intracellular Signaling in the A2A Adenosine Receptor

Abstract

Signaling across cellular membranes, the 826 human G protein-coupled receptors (GPCRs) govern a wide range of vital physiological processes, making GPCRs prominent drug targets. X-ray crystallography provided GPCR molecular architectures, which also revealed the need for additional structural dynamics data to support drug development. Here, nuclear magnetic resonance (NMR) spectroscopy with the wild-type-like A_2A adenosine receptor (A_2AAR) in solution provides a comprehensive characterization of signaling-related structural dynamics. All six tryptophan indole and eight glycine backbone ¹⁵N-¹H NMR signals in A_2AAR were individually assigned. These NMR probes provided insight into the role of Asp52^2.50 as an allosteric link between the orthosteric drug binding site and the intracellular signaling surface, revealing strong interactions with the toggle switch Trp 246^6.48, and delineated the structural response to variable efficacy of bound drugs across A_2AAR. The present data support GPCR signaling based on dynamic interactions between two semi-independent subdomains connected by an allosteric switch at Asp52^2.50.

Keywords: G protein-coupled receptor; GPCR; NMR; allosteric modulation; membrane protein; nuclear magnetic resonance; signaling.

Figure 1. A_2AAR Signaling via an Allosteric Center at Asp52^2.50 Probed by an Extensive Network of Assigned NMR Signals

(A) Scheme of agonist-induced signaling in A_2AAR and A_2AAR[D52N], i.e., receptors with and without an active allosteric switch at position 52^2.50, respectively. The gray shape represents A_2AAR, the green shape the signal-inducing drug, and the upper and lower thin horizontal lines indicate the extracellular and intracellular membrane surfaces, respectively. (B) Pie chart showing the frequency of amino acid types occurring at position 52^2.50 in all class A GPCRs: Asp 84%, Glu 10%, Arg and others 6% (Munk et al., 2016). (C) Ribbon representation of the crystal structure of the antagonist ZM241385 (green) complex of A_2AAR expressed in Pichia pastoris (PDB 6AQF). The location of the fusion protein BRIL in the intracellular loop 3 (ICL3), where G218 was eliminated in the fusion protein, is indicated by a thin broken line; G218 was left intact in the A_2AAR sequence used for NMR studies (see Figure S1 for the location of G218 at the intracellular tip of trans-membrane helix VI in a structure without fusion protein). NMR-assigned tryptophan residues and their sequence positions are highlighted in blue, assigned glycine residues are orange, and the residue Asp52^2.50 is shown in red. (D) 2D [¹⁵N,¹H]-TROSY correlation spectrum of A_2AAR in complex with ZM241385. Dashed boxes highlight the Trp indole ¹⁵N–¹H and Gly backbone ¹⁵N–¹H regions, which are shown on expanded scales in (E) and (F), respectively, with sequence-specific assignments indicated next to the signals. The peaks numbered 1 to 30 were used to monitor the global folds of the A_2AAR variants used here for resonance assignments and function-related studies.

Figure 2. Sequence-specific Assignment of the Trp Indole ¹⁵N–¹H and Eight Gly Backbone ¹⁵N–¹H NMR Lines

Panels A and B document the assignments for the tryptophans, and the panels C to I those of eight glycines. Each panel displays a region of the 2D [¹⁵N,¹H]-TROSY correlation spectrum of A_2AAR in complex with ZM241385. On the left are contour plots and on the right are 1D cross sections taken at the ¹⁵N chemical shifts indicated by dashed lines in the contour plots. The spectrum of A_2AAR is shown in blue, and the spectra of the variant proteins used for the assignment of the residues indicated in the individual panels are shown in red; locations in the 3D A_2AAR structure are also indicated, where “TM” stands for “trans-membrane helix”, and “ICL” and “ECL”, respectively, for intracellular and extracellular loop. Comparison of the two spectra resulted in sequence-specific assignment of the signal identified by an asterisk, by observation of the absence in the variant protein of the signal to be assigned. In the right panel of (C), the most intense signals have been truncated.

Figure 3. NMR Assignment of the Toggle Switch Trp246^6.48 in A_2AAR and A_2AAR[D52N] and Chemical Structures of Ligands Used in this Study

(A and B) Assignment of the Trp246^6.48 indole ¹⁵N–¹H signal of A_2AAR complexes with different ligands. (A) Antagonist ligand ZM241385. (B) Agonist ligand NECA. (C and D) Corresponding assignments for Trp246^6.48 in the A_2AAR[D52N] complexes with ZM241385 (C) and NECA (D). On the left are contour plots of 2D [¹⁵N,¹H]-TROSY correlation spectra and on the right are cross sections taken at the ¹⁵N or ¹H chemical shifts indicated in the contour plots by dashed vertical or horizontal lines, respectively. The spectra of A_2AAR and A_2AAR[D52N] are shown in blue, and the spectra for the variant proteins used for the resonance assignments are shown in red. Comparison of the two spectra resulted in sequence-specific assignments for the signals identified by asterisks, by observation of the absence in the variant protein of the signal to be assigned. (E) Chemical structures of the ligands used in the current study, where the adenine moiety found in almost all A_2AAR antagonists and agonists is highlighted in green, and the ribose moiety found only in A_2AAR agonists is highlighted in orange.

Figure 4. Local Environments of Selected Trp Residues in Crystal Structures of A_2AAR and Response of Trp Indole ¹⁵N–¹H NMR Lines to Variable Efficacy of Bound Drugs

(A) Global superposition of the environment of Trp246^6.48 in the crystal structures of A_2AAR complexes with ZM241385 (red; PDB 3EML) and UK432097 (silver-blue; PDB 3QAK), and the ternary complex with NECA and a “mini-Gs” protein (grey; PDB 5G53). Trp246^6.48 and the nearby residues F242^6.44, I92^3.40 and P189^5.50 are labeled and shown in stick representation. (B and C) Environment of two Trp residues in the A_2AAR complexes with six different ligands (Figure 3E), i.e., ZM241385 (red; PDB 3EML), CGS21680 (orange; PDB 4UHR), UK432097 (silver-blue, PDB 3QAK), NECA (yellow; PDB 2YDV), caffeine (green; PDB 3PWH) and XAC (blue; PDB 3REY). Global superpositions of the crystal structures are shown, with ribbon representations of the backbone and stick representations of aromatic residues of interest. (B) Trp143. (C) Trp29^1.55. These structures were used to calculate the ring current shifts in Table S1. (D) Survey of the response of NMR probes in A_2AAR to drug efficacy. Assigned Gly and Trp residues are shown as spheres positioned in the structure of the A_2AAR-ZM241385 complex (PDB 3PWH). Red spheres indicate that a response was observed, either by different chemical shifts, variation of the signal line shapes, or both. Black spheres indicate that there was no response. The curved dashed line at the top of the receptor indicates the position of ECL2, which was not observed in the crystal structure. (E) – (G) Contour plots of the tryptophan indole ¹⁵N–¹H region of 2D [¹⁵N,¹H]-TROSY correlation spectra of [u-¹⁵N,~70% ²H]-A_2AAR bound to ligands with different efficacies, as identified in each panel. Complexes with antagonists are in the left column and those with agonists on the right. Colors of the spectra correspond to the colors of the corresponding crystal structures of A_2AAR–ligand complexes in panels (B) and (C). (H) – (K) Projections of the contents of the boxes in the corresponding contour plots along the ¹⁵N chemical shift axis onto the lower boundary of the boxes. The same colors are used as for the contour plots and peak assignments are indicated.

Figure 5. Probing Drug Efficacy-Dependent Signaling in A_2AAR by Gly Backbone ¹⁵N–¹H Resonances

Contour plots are shown of 2D [¹⁵N,¹H]-TROSY correlation spectra of [u-¹⁵N,~70% ²H]-A_2AAR complexes with ligands of different efficacies, as identified by the color code in (A) (same colors as in Figure 4B, C, E–G): (A) Gly118^4.39; (B) Gly142^4.63; (C and D) Gly158, Gly160 and Gly218; (E) G114. In (A), (B) and (E) each panel shows a superposition of the spectra of complexes with two different ligands, whereas in (C) and (D) each panel displays the spectrum of one complex.

Figure 6. NMR Response on the Extracellular and Intracellular Surfaces of A_2AAR to Perturbation of the Allosteric Center at Asp52^2.50

(A) Survey of the response of NMR Signals in A_2AAR to replacement of Asp52^2.50 in the allosteric center with Asn52^2.50. Assigned Trp and Gly residues are shown as spheres positioned in the structure of A_2AAR-ZM241385 (PDB 3PWH). Cyan coloring indicates that changes were observed either in chemical shifts or in NMR line shapes. Red coloring indicates that there was no response. The curved dashed line at the top of the receptor indicates the position of ECL2, which was not observed in the crystal structure. (B)–(E) 2D [¹⁵N,¹H]-TROSY correlation spectra documenting the results surveyed in (A). On the left and in the center are 2D contour plots, and on the right are projections along the ¹⁵N dimension onto the lower boundary of the regions indicated by the dashed boxes in the contour plots. (B) and (C) Gly218. (D) and (E) Trp29^1.55, Trp32^1.58 and Trp143. The proteins and the ligands are identified in the figure; the panels on the right show superpositions of the projections for the two proteins.

Figure 7. Visualization of Correlations Between Structural and Functional Response to Drug Efficacy and Activity of the Allosteric Center at Asp52^2.50 in A_2AAR

(A) and (B) Schematic side-views of A_2AAR and A_2AAR[D52N]. The transmembrane helices are represented by two adjoining rectangles, each indicating the extracellular and intracellular subdomains of the receptor (see text). The three helices carrying NMR reporter groups near the intracellular surface are shaded, and the residues with assigned NMR lines are indicated by yellow spheres, where framed spheres indicate that a single NMR line was observed, and unframed spheres correspond to multiple-component resonances. The helices drawn with broken lines indicate the local polymorphisms seen in the NMR spectra of the residues with unframed yellow spheres. The broken horizontal lines indicate the extracellular and intracellular membrane surfaces. The black arrow indicates the signaling pathway from the orthosteric drug binding site to the intracellular surface. In A_2AAR, signaling has been correlated in the present work with local polymorphisms at the intracellular tips of the helices I and VI. In A_2AAR[D52N], signaling to the intracellular surface is quenched (Massink et al., 2014); the broken arrow indicates that an NMR-detectable structural response to variable efficacy of the bound drug was observed. The connection between the toggle switch Trp246^6.48 and the allosteric switch at Asp52^2.50 is supported by the observation of a strong interplay between these two centers in the present work.