A Single Point Mutation Blocks the Entrance of Ligands to the Cannabinoid CB₂ Receptor via the Lipid Bilayer

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 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.

The Leu/Val6.51 Side Chain of Cannabinoid Receptors Regulates the Binding Mode of the Alkyl Chain of Δ9-Tetrahydrocannabinol

“…Among the ∼350 GPCRs for nonsensory functions, ∼35 are activated by hormone-like signaling molecules derived from lipid species with long hydrophobic chains. , Some of these receptors possess distinctive structural signatures relative to other class A GPCRs such as the N-terminus and ECL-2 folding over the binding site, , which causes the entry of the ligand to the orthosteric site through a tunnel formed between TMs 1 and 7; , or lacking the highly conserved Pro 5.50 , part of the PIF motif that transmits the signal from the orthosteric ligand binding site to the G protein binding site. , In PIF-containing GPCRs, the interaction of agonists with TM 5 triggers an inward movement of TM 5 at P 5.50 , a rotation of TM 3 at I 3.40 , and an outward movement of TM 6 at F 6.44 . , In GPCRs lacking P 5.50 , agonists can alter the rotamer of the amino acid at position 3.36 to trigger the rotation of TM 3 at I 3.40 and outward movement of TM 6 at F 6.44 . For instance, in the active crystal structure of S1P 3 bound to the endogenous agonist sphingosine-1-phosphate (S1P), the long hydrophobic side chain of d18:1 S1P binds in an extended conformation (I-shape) between TMs 4 and 5 (Figure a).…”

“…Among the ∼350 GPCRs for nonsensory functions, ∼35 are activated by hormone-like signaling molecules derived from lipid species with long hydrophobic chains. 6 , 49 Some of these receptors possess distinctive structural signatures relative to other class A GPCRs such as the N-terminus and ECL-2 folding over the binding site, 50 , 51 which causes the entry of the ligand to the orthosteric site through a tunnel formed between TMs 1 and 7; 52 , 53 or lacking the highly conserved Pro 5.50 , part of the PIF motif that transmits the signal from the orthosteric ligand binding site to the G protein binding site. 54 , 55 In PIF-containing GPCRs, the interaction of agonists with TM 5 triggers an inward movement of TM 5 at P 5.50 , a rotation of TM 3 at I 3.40 , and an outward movement of TM 6 at F 6.44 .…”

Novel Cannabinoid Receptor 2 (CB2) Low Lipophilicity Agonists Produce Distinct cAMP and Arrestin Signalling Kinetics without Bias

“…This could feasibly manifest as lipophilic ligands producing more prolonged responses in comparison with polar ligands that will tend to diffuse away from the plasma membrane. It is well established that lipophilic CB2 ligands enter the binding site via a membrane-embedded channel/vestibule, and that low affinity interactions along this channel are involved in facilitating orthosteric binding [ 30 , 31 , 32 ]. It would be interesting to investigate further whether relatively polar ligands, such as those studied here, can enter CB2 via this membrane-embedded channel or must enter directly from the extracellular milieu.…”

“…Therefore, if an allosteric mechanism is contributing to the kinetic signalling profiles observed, the ligands might be acting “dualsterically” with an allosteric site in close spatial proximity to the orthosteric site, have transient allosteric interactions, or be acting via dual binding at the orthosteric site and a distant allosteric site on the same or dimerised receptor protomer [ 34 , 35 , 36 , 37 ]. Various allosteric sites in CB2 have been predicted, including at least one near the orthosteric site [ 31 , 38 , 39 , 40 , 41 ]. Interestingly, all the test compounds we studied had a considerably higher signalling potency:affinity ratio than CP55,940, which could also indicate involvement of an allosteric mechanism in the transduction of signalling for the test compounds [ 37 ].…”

Homodimerization of CB2cannabinoid receptor triggered by a bivalent ligand enhances cellular signaling

“…This is the case of cannabinoid receptors, type 1 (CB1R) and type 2 (CB2R), that respond to endocannabinoids with long hydrophobic tails such as anandamide and 2-arachidonoyl glycerol, as well as phytocannabinoids like Δ⁹tetrahydrocannabinol [37,38]. The pathway of ligand entry to CB2R, determined by MD simulations, defines two transient binding sites [39]: a membrane-facing pocket between TMs 1 and 7 [40] and a bundle-facing allosteric pocket located near the orthosteric site [41].…”