Figure 2

ORG27569 and cholesterol binding mode.

(a) ORG27569 Binding mode. Key interactions included: (i) two H-bonds established by the 1H-indole-2-carboxamide group with the H^2.41 N^δ atom and the S^2.45 side chain; (ii) the 1-(4-ethylphenyl)piperidine arm accommodating in a hydrophobic region interacts with F^4.46, V^2.48, L^1.54 and L^1.58; and (iii) the 3-ethyl group plunges through a hydrophobic area establishing contacts with L^3.45 and M^4.49. (b) H^2.41 x₁ and x₂ dihedral angles distribution for the CB₁wt-ORG27569 simulation (run 1). The probability of each state was normalized. A highly represented conformer, in the CB₁wt-ORG27569 MD, for the H^2.41 residue (conformer 2), different from that observed in the CB₁wt unbound state (conformer 1, see also Supplementary Fig. S9) was noticed. Remarkably, in the CB₁wt-ORG27569 simulations, the shift from H^2.41 conformer 1 to 2 causes the H-bond loss between the H^2.41 Nε (TM2) and the R148 backbone oxygen (ICL1, Fig. 2d). (c–e) Probability distribution for the H^2.41(NHε)-R148(O), F^2.42(Cα)-T^3.46(Cα) and R150(CZ)-D^8.49(CG) distance atoms, for CB₁wt (blue lines) and the CB₁wt-ORG27569 simulations (black and red lines), respectively. The probability of each distance was normalized. (f) Probability distribution of cholesterol molecules along the x and y axes with respect to the main axis of CB₁, only cholesterol molecule into the lower leaflet of the bilayer were considered (See Appendix A in Supplementary Information for a detailed description of the calculation of the probability distribution). (g) Probability distribution of the cholesterol molecule, which binds to the CCM during the CB₁wt POPC:Chol (2:1) simulations. The probability distribution distance for the W^4.50(Cα)-CHL(C13) carbon atoms (x axis) is plotted vs. the probability distribution distance for the NH(Arg148)-O atoms (y axis). (h) Binding mode of the cholesterol molecules in P2/CCM. The cholesterol molecule interacts with L^1.54, L^1.58, H^2.41, V^2.48, L^2.52, F^4.46 and W^4.50 residues, while the R148 side chain anchors the OH apical group.