Casajuana-Martin, NilNavarro Brugal, GemmaGonzález Ureña, A.Llinàs Del Torrent Masachs, ClàudiaGómez-Autet, MarcQuintana García, AleixFranco Fernández, RafaelPardo, Leonardo2023-01-102023-11-282022-11-281549-9596https://hdl.handle.net/2445/191975Molecular dynamic (MD) simulations have become a common tool to study the pathway of ligand entry to the orthosteric binding site of G protein-coupled receptors. Here, we have combined MD simulations and site-directed mutagenesis to study the binding process of the potent JWH-133 agonist to the cannabinoid CB2 receptor (CB2R). In CB2R, the N-terminus and extracellular loop 2 fold over the ligand binding pocket, blocking access to the binding cavity from the extracellular environment. We, thus, hypothesized that the binding pathway is a multistage process consisting of the hydrophobic ligand diffusing in the lipid bilayer to contact a lipid-facing vestibule, from which the ligand enters an allosteric site inside the transmembrane bundle through a tunnel formed between TMs 1 and 7 and finally moving from the allosteric to the orthosteric binding cavity. This pathway was experimentally validated by the Ala2827.36Phe mutation that blocks the entrance of the ligand, as JWH-133 was not able to decrease the forskolin-induced cAMP levels in cells expressing the mutant receptor. This proposed ligand entry pathway defines transient binding sites that are potential cavities for the design of synthetic modulators.9 p.application/pdfeng(c) American Chemical Society , 2022http://creativecommons.org/licenses/by/3.0/es/Dinàmica molecularBicapes lipídiquesMutació (Biologia)Molecular dynamicsLipid bilayersMutation (Biology)info:eu-repo/semantics/article7276892023-01-10info:eu-repo/semantics/openAccess