A Single Point Mutation Blocks the Entrance of Ligands to the Cannabinoid CB2Receptor via the Lipid Bilayer

Nil Casajuana-Martin, Gemma Navarro, Angel Gonzalez, Claudia Llinas Del Torrent, Marc Gómez-Autet, Aleix Quintana García, Rafael Franco, Leonardo Pardo*

*Corresponding author for this work

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Molecular 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 CB2receptor (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.

Original languageEnglish
Article number22
Pages (from-to)5771-5779
Number of pages9
JournalJournal of Chemical Information and Modeling
Volume62
Issue number22
DOIs
Publication statusPublished - 27 Oct 2022

Keywords

  • Binding Sites
  • Cannabinoids
  • Ligands
  • Lipid Bilayers/chemistry
  • Point Mutation
  • Protein Binding
  • Receptor, Cannabinoid, CB1/metabolism
  • Receptor, Cannabinoid, CB2/genetics
  • Receptors, Cannabinoid/metabolism

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