Ligand-dependent activation and deactivation of the human adenosine A(2A) receptor

Abstract

G-protein-coupled receptors (GPCRs) are membrane proteins with critical functions in cellular signal transduction, representing a primary class of drug targets. Acting by direct binding, many drugs modulate GPCR activity and influence the signaling pathways associated with numerous diseases. However, complete details of ligand-dependent GPCR activation/deactivation are difficult to obtain from experiments. Therefore, it remains unclear how ligands modulate a GPCR's activity. To elucidate the ligand-dependent activation/deactivation mechanism of the human adenosine A2A receptor (AA2AR), a member of the class A GPCRs, we performed large-scale unbiased molecular dynamics and metadynamics simulations of the receptor embedded in a membrane. At the atomic level, we have observed distinct structural states that resemble the active and inactive states. In particular, we noted key structural elements changing in a highly concerted fashion during the conformational transitions, including six conformational states of a tryptophan (Trp246(6.48)). Our findings agree with a previously proposed view that, during activation, this tryptophan residue undergoes a rotameric transition that may be coupled to a series of coherent conformational changes, resulting in the opening of the G-protein binding site. Further, metadynamics simulations provide quantitative evidence for this mechanism, suggesting how ligand binding shifts the equilibrium between the active and inactive states. Our analysis also proposes that a few specific residues are associated with agonism/antagonism, affinity, and selectivity, and suggests that the ligand-binding pocket can be thought of as having three distinct regions, providing dynamic features for structure-based design. Additional simulations with AA2AR bound to a novel ligand are consistent with our proposed mechanism. Generally, our study provides insights into the ligand-dependent AA2AR activation/deactivation in addition to what has been found in crystal structures. These results should aid in the discovery of more effective and selective GPCR ligands.

Fig. 1. C_α RMSDs of the 7 transmembrane helices to the active and inactive crystal structures (PDBIDs: 3QAK and 3REY)

For helix 6, as a guide to the eye the three distinct regions are highlighted by red (agonist-bound), black (apo), and blue (inverse agonist-bound) ellipses. We determined the positions and shapes of the ellipses by maximizing the inclusion of points from the target state while minimizing the inclusion of points from the other states. In addition to the distinct states in TM6, RMSDs of helix 7 also display two distinct states corresponding to agonist-bound/apo and the inverse agonist-bound complexes.

Fig. 2. Volume of the G protein-binding site in AA2AR simulations

(A) Graph of the average volume (bars, left scale) of the G protein-binding site and the fraction of conformations with volume greater than 800 ų (plot, right scale), calculated from the longest trajectory of each system. (B) A cartoon to illustrate our definition of the G protein-binding site. An example of the superimposed open and occluded conformations (red and cyan) is shown. These two conformations were snapshots from the longest simulations of R*-ADN and R-XAC taken at 350 ns. The yellow dots indicate the space needed to accommodate the terminal helix of G_αs.

Fig. 3

Comparison of the key structural elements in the active (red) and inactive (cyan) conformations from simulations of R*-ADN and R-XAC, respectively. (A) Superposition of two snapshots at 350 ns. (B) Ligand-AA2AR interactions. The ligand is shown using a space-filling representation. (C) Trp246^6.48 and key residues involved in the packing between TM3 and TM6. (D) The salt bridge network.

Fig. 4

Trp246^6.48 χ1 and χ2 distributions and their representative conformations. The regions enclosed by the contour lines have a normalized probability higher than 0.005. The shortest heavy atom distance between the Trp246^6.48 side chain and the TM3 backbone is labeled for the states T1 and T2. Table S2 contains the details of each simulation regarding the fraction of time spent in each rotameric state. The original scatter plot is shown as Fig. S5. The color scheme is consistent with Fig. 2.

Fig. 5

Metadynamics results of R*-ADN, apo-R, R-XAC simulations. (A1–3) The free energy surface as a function of Trp246^6.48 dihedral angles χ1 and χ2. (B1–3) The minimum free-energy path between states X and A.

Fig. 6

Metadynamics results of R*-ADN, R*-NEC, R*-UKA simulations. (A1–3) The free energy surface as a function of Trp246^6.48 dihedral angles χ1 and χ2. (B1–3) The minimum free-energy path between states X and A.

Fig. 7

Cartoon of the ligand-binding pocket projected into two dimensions. The chemical structures of the endogenous agonist adenosine (red) and the selective inverse agonist ZM-241385 (green) are superimposed by overlapping the adenine ring and the triazolotriazine ring.

Fig. 8

Snapshots of AA2AR-LUF contacts when LUF approaches Trp246^6.48. (A) is from 74ns into the first simulation while (B) if from 150ns into the second simulation. Brown dash lines represent hydrogen bonds, while the blue ones represent the LUF-Trp246^6.48 closest contacts within 4.4 Å.