Figure 1. Major structural features and internal hydration of class A GPCRs in the inactive and active state as shown by the m2r.

(A) The main structural features of class A GPCRs, as exemplified by m2r, include 7 TM helices (blue), an extracellular ligand binding site, the intracellular effector (G-protein) binding site as well as conserved and functionally important residues termed microswitches (selected ones are highlighted). The vertical axis (Z-coordinate) and all positions stated in the text use the Cα atom of D103^3.32 as a reference. (B) Conformation of inactive m2r (PDB: 3UON) during the simulations showing the presence of the hydrophobic layer separating the hydrophilic pocket and effector binding site. (C) After transition to the active state (PDB: 4MQT), and further simulation, m2r displays a continuous water channel connecting the orthosteric ligand binding site, hydrophilic pocket and effector binding site. Note that in (B) and (C) the upward conformation of the Y440^7.53 is shown in order to highlight the changes in hydration levels only; please see Fig S3 for a detailed comparison of the upward and downward tyrosine conformations. Water molecules are shown in red (surface representation); the position of the allosteric Na⁺ ion, as obtained from our initial simulations, is shown as a green sphere, and residues forming the hydrophobic layer (yellow) as well as the bound ligand (carbachol, light green) are depicted in stick representation.