Fig. 3. Molecular dynamics simulations of functionally selective KOR agonists.

a Chemical structure of G protein biased agonist nalfurafine, balanced agonist U50,488, and arrestin-biased agonist WMS-X600. U50,488 was used as a reference agonist that has a bias factor = 1; G protein bias factor of nalfurafine (95% confidence interval) = 6 (4.5–8.2); arrestin2 bias factor of WMS-X600 (95% confidence interval) = 10 (6.5–15.4). Calculation of bias factor was described in the “Methods”. b Functional characterization of KOR agonists in G protein-mediated cAMP inhibition and arrestin-mediated recruitment. Data are expressed as the mean ± SEM of three independent experiments (n = 3 experiments each done in duplicate). c Overlay of nalfurafine, U50,488, and WMS-X600 in the binding pocket of KOR. d Differences in Q115^2.60 rotamer orientations favored by different ligands. In the left panel, Q115^2.60 points towards TM3, keeping the pocket close to its starting configuration (nalfurafine). In the right panel, the rotation of Q115^2.60 out of the pocket depresses Y66^1.39, allowing Y320^7.43 to move forward and the top of TM7 to rotate counter-clockwise (WMS-X600). The Q115^2.60 rotamer is quantified as the dihedral angle formed by the C, Ca, Cg, and Cd atoms of Q115^2.60. e Differences in TM5 and TM6 conformation favored by different ligands. The distribution of K227^5.39 amine nitrogen to E297^6.58 carboxylate oxygen distances shows that the two residues generally do form a salt bridge when WMS-X600 or U50,488 is bound but do not when nalfurafine is bound. Values in each plot were summarized in Supplemental Table 6.