Figure 8.

Role of water in EMPA (8) OX₁/OX₂ selectivity. (a) EMPA ligand in OX₂ mimicking OX₁ A127^3.33T mutant with a WaterFLAP-computed network (small spheres, color-coded by energy) and X-ray crystallographic waters (large green spheres). The interstitial water hydrogen bonding to the two pyridine nitrogens and Q126^3.32 can be clearly seen. (b) Suvorexant (2) and lemborexant (4) ligand poses from OX₁ crystal structures overlaid with an EMPA water network in OX₂. The carbon atoms of the ligands are colored cyan (EMPA), green (suvorexant), and purple (lemborexant). GRID maps are contoured (transparent solid) and colored in the following manner: C1 is the probe (lipophilic) in yellow at −2.8 kcal/mol, and the CH₃ methyl group probe is in gray at 1 kcal/mol, which defines the pocket surface in terms of how close a ligand carbon atom can reside. WaterFLAP water networks calculated on the pseudo-apo structure (shown as large spheres) have been color-coded in red if predicted to have a free energy (ΔG) >3.5 kcal/mol, in yellow if ΔG is between 2.0 and 3.5 kcal/mol, in gray if ΔG is between −1.0 and 2.0 kcal/mol, and in blue if ΔG < −1.0 kcal/mol. All WaterFLAP free energy estimations are relative to bulk solvent. (c, d) Comparison of the binding site surfaces of the OX₂ mimicking the OX₁ A127^3.33T mutant structure (solid) and back mutated T127^3.33A/wild-type (WT) OX₁ (dark gray mesh) indicates that in wild-type OX₁, an energetically unhappy water molecule will be trapped by the OX₂-selective EMPA antagonist. WaterMap water network calculations of the complex with OX₁ (A127^3.33) with a very unhappy (high relative energy to bulk solvent, 4 kcal/mol) water trapped in the larger OX₁ binding site, shown as a large red sphere. The water stabilized by the two pyridines is also shown as a large blue sphere (stabilized, 2 kcal/mol); in the pseudo-apo structure, this water is calculated by WaterFLAP to be unstable relative to bulk water (small yellow sphere in panel (b)).