3.3. Ligand Docking

The most stable structure of CB₁R with the whole N-terminus obtained from REST2 simulations and selected based on RMSF and energy analysis, was used for ligand docking. CBD was redocked to the allosteric site 1 [31] (positioned between the extracellular ends of TM1 and TM2, defined by residues: I105, E106, C107) after the last step of REST2 set of simulations, while Δ⁹‑THC was docked to the orthosteric binding site (defined by residues: I119, F174, L193, W356, F381, S383, M384) [34]. To perform docking of flexible ligands into protein binding sites we used Genetic Optimization for Ligand Docking (GOLD) v.5.7.3 [53]. Ligand poses were verified for stability by MD simulations. Only one stable pose was identified for CBD and it was stable for >1 μs of total MD simulation (in 5 simulation repeats). For each of the two ligand-receptor complexes (1st complex of CB₁R with CBD and THC, and 2nd complex of CB₁R with THC only) five independent all-atom and unbiased MD simulations were performed (2 × 5 × 1 μs).