Figure 4:

PIP2 and intracellular calcium modulate receptor flexibility and activity. A-D. The extent of receptor conformation sampling of NECA bound A_2AR in R, R’ and R*.G states in four different environments, namely, the cell membrane(A), cell membrane with calcium ions (B) and DDM detergent (C) and pure POPC bilayer (D). The MD snapshots are projected on inter-residue distance measures. The distances are measured between the C_α-C_α atoms of the residue pairs R102^3.50-E228^6.34 on TM3 and TM6 and between R102^3.50-Y288^7.53 on TM3 and TM7. These two distances in the starting crystal structures of A_2AR in R, R’ and R*.G states are shown as red (R), yellow (R’) and green (R*.G) dots respectively. E-P. The rows in this set of figures show the representative structures in R, R’ and R*.G states. The four columns are the four environments as indicated in the heading of each column. In figures E and I, PIP2 forms bifurcated salt bridge interactions with the Lys/Arg in the intracellular region of A_2AR, bridging the TM helices, in cell membrane conditions, in the inactive R state (E) and in active intermediate R’ state (I). In figure M, PIP2 forms salt bridge interactions with basic residues both in the fully active state of A_2AR and in mini-G protein thus stapling the receptor with mini-G protein. PIP2 is shown in grey and orange sticks representation. Figures F, J and N show Calcium ions (shown in black spheres) interactions with PIP2 and yanks the PIP2 away from the Lys/Arg residues in the receptor R state (F), R’ state (J) and R*.G state (N). Calcium ions coordinate with PIP2 freeing up the TM helices thus promoting transitions between R and R’ state seen in figure B. In figures G, K and O, DDM forms weak hydrogen bonds with Arg/Lys in the intracellular region in the R state (G), R’ state (K) and R*.G state (O). This results in transitions between R and R’ states in DDM as shown in figure C. In figures H, L and P, the POPC lipid forms salt bridge interactions with the Arg/Lys in the intracellular region in the R state (H), R’ state (L) and in R*.G state (P). However, POPC does not form stapling interactions between A_2AR and mini-G protein. All the structures shown in this figure are representative structures from the most occupied conformation cluster. The conformation clustering was done using RMSD of the receptor backbone with a cutoff of 1.5Å.