Fig. 4.

The free energy surface for formoterol’s access and binding to the β2-adrenergic receptor from its energetically favorable location in the membrane. The two-dimensional free energy surface is characterized by the distance between the COM of the ligand and binding site residues (x-axis) and the internal angle of formoterol (y-axis). The minimum energy path, as determined by well tempered metadynamics simulations, is given in bold black line, and several representative low-energy intermediate states along the path were labeled (A–D). (A) As formoterol (licorice representation, yellow color) approaches the receptor from within the membrane, its alkoxy-aryl-alkyl tail comes into contact with TMHs 2 and 7, engaging in hydrophobic contacts with I94^2.65, L310^7.37, and W313^7.40. (B and C) Formoterol moves up over the transmembrane helices and remains in a region where it makes extensive H-bond interactions with several polar residues and bulk water molecules. Specifically, the H-bond with D192^ECL2 through its O3 appears to destabilize the salt bridge between D192^ECL2 and K305^7.32. The breakage of the salt bridge allows formoterol to slide down from this region into the binding pocket. As formoterol enters the pocket, the hydrophobic lock (between F193^ECL2 and Y308^7.35) breaks, resulting in the formation of a π-π interaction between F193’s phenyl ring and the aromatic ring at the tail end of the ligand. (D) Formoterol is seen in its final bound pose, slightly different from its X-ray structure pose (RMSD 2.1Å) bound to turkey β1-AR [PDB ID 6IBL (Lee et al., 2020)]. In its final bound pose, formoterol engages in polar H-bond and salt bridge interactions with S203^5.42, S207^5.46, D113^3.32, and N312^7.39. Formoterol (crystal pose in cyan) and the binding site residues (green) are illustrated in licorice representation. The receptor is illustrated in secondary structure representation (light golden yellow).