Figure 4.

Free energy landscapes projected along variables of interest highlight changes in the pairwise coupling of microswitches following binding of an agonist ligand (middle column) and protonation of conserved residue Asp79^2.50 (right column). The free energy landscapes are projected along (a–c) the TM5 bulge (distance between Ser207^5.46 and Gly315^7.41, representing the ligand binding site contraction) and the distance between Leu272^6.34 and Arg131^3.50, representing the outward movement of TM6; (d–f) the TM5 bulge (distance between Ser207^5.46 and Gly315^7.41) and the difference between the RMSD of Ile121^3.40 and Phe282^6.44 heavy atoms to the active and inactive crystal structures,^5,6 representing the connector region ΔRMSD; (g–i) the connector region ΔRMSD and the number of water molecules within 0.8 nm of Asp79^2.50, representing the hydration of the Asp79^2.50 cavity; (j–l) the connector region ΔRMSD and the RMSD of the NPxxY motif relative to the inactive structure 2RH1; (m–o) the connector region ΔRMSD and the distance between the two tyrosines implicated in the Y–Y interaction (Y–Y motif), and (p–r) the Y–Y motif distance and the displacement of TM6. Active and inactive state regions are labeled for each variable pair. Low-free energy regions are colored red, and high-free energy regions light yellow. Free energies are reported in kilocalories per mole. See Table S2 for microswitch definitions. (s–v) Vignettes showing the conformation of the different microswitches in the active and inactive structures 3P0G (orange) and 2RH1 (white): (s) the TM5 bulge shifting Ser207^5.46 inward, (t) the connector region containing Ile121^3.40 and Phe282^6.44, (u) the Asp79^2.50 cavity, NPxxY motif, and the two tyrosines, Tyr219^5.58 and Tyr326^7.53, of the Y–Y motif, and (v) the outward movement of TM6 upon activation.