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

(−)-Δ9-trans-tetrahydrocannabinol (THC), which is the principal psychoactive constituent of Cannabis, mediates its action by binding to two members of the G-protein-coupled receptor (GPCR) family: the cannabinoid CB1 (CB1R) and CB2 (CB2R) receptors. Molecular dynamics simulations showed that the pentyl chain of THC could adopts an I-shape conformation, filling an intracellular cavity between Phe3.36 and Trp6.48 for initial agonist-induced receptor activation, in CB1R but not in CB2R. This cavity opens to the five-carbon chain of THC by the conformational change of the γ-branched, flexible, Leu6.51 side chain of CB1R, which is not feasible by the β-branched, mode rigid, Val6.51 side chain of CB2R. In agreement with our computational results, THC could not decrease the forskolin-induced cAMP levels in cells expressing mutant CB1RL6.51V receptor but could activate the mutant CB2RV6.51L receptor as efficiently as wild-type CB1R. Additionally, JWH-133, a full CB2R agonist, contains a branched dimethyl moiety in the ligand chain that bridges Phe3.36 and Val6.51 for receptor activation. In this case, the substitution of Val6.51 to Leu in CB2R makes JWH-133 unable to activate CB2RV6.51L. In conclusion, our combined computational and experimental results have shown that the amino acid at position 6.51 is a key additional player in the initial mechanism of activation of GPCRs that recognize signaling molecules derived from lipid species.


INTRODUCTION
Cannabinoids are naturally occurring compounds found in the Cannabis sativa plant (more commonly known as marijuana).There are over 180 cannabinoids out of the 1600 chemical compounds that have been isolated from Cannabis, with a characteristic oxygen containing C 21 aromatic hydrocarbons. 1hese exogenous cannabinoids can be further classified into 11 subclasses: cannabichromene (CBC), cannabidiol (CBD), cannabielsoin (CBE), cannabigerol (CBG), cannabicyclol (CBL), cannabinol (CBN), cannabinodiol (CBND), cannabitriol (CBT), (−)-Δ 8 -trans-tetrahydrocannabinol (Δ 8 -THC), (−)-Δ 9 -trans-tetrahydrocannabinol (Δ 9 -THC), and miscellaneous-type cannabinoids. 2The Δ 9 -THC subclass contains 25 compounds with common structural features such as a dibenzopyran ring and a hydrophobic alkyl chain.This class includes the most abundant phytocannabinoids: (−)-Δ 9 -transtetrahydrocannabinol (THC), which is the principal psychoactive constituent of Cannabis, and (−)-Δ 9 -trans-tetrahydrocannabivarin (THCV), which is homologous to THC but has a 3-carbon (propyl chain) instead of a 5-carbon (pentyl chain) in the alkyl chain (Figure 1).THCV lacks the psychoactive effects of THC and upregulates energy metabolism, converting it a clinically useful remedy for weight loss, obesity management, and type 2 diabetic patients. 3,4In addition, THCV can produce beneficial antipsychotic effects. 5Endogenous cannabinoids are N-arachidonylethanolamide (anandamide) and 2-arachidonoylglycerol (2-AG) that possess long hydrophobic moieties. 6he effects of cannabinoids are primarily mediated through two members of the G-protein-coupled receptor (GPCR) family, 7 the cannabinoid CB 1 (CB 1 R) and CB 2 (CB 2 R) receptors.CB 1 R is one of the most abundant GPCRs in the central nervous system, whereas CB 2 R is mainly expressed in the immune system. 8However, other molecular targets for certain cannabinoids, aside from CB 1 R and CB 2 R, have also been identified. 9Some authors propose that THCV is a CB 1 R and CB 2 R antagonist, 10 whereas others suggest that THC and THCV are partial agonists at both receptors. 11We have recently shown that THC acts as a partial agonist in CB 1 R and as an antagonist in CB 2 R, whereas THCV acts as an antagonist on both receptors. 12ere, we have used the recently released structures of CB 1 R 13−15 and CB 2 R 16−18 in their inactive and active, G ibound, conformations to delineate the individual signaling contributions of THC and THCV to modulate both receptors.−22 However, these amino acids are conserved in CB 1 R and CB 2 R (44% sequence identity between receptors), thus they cannot explain the different pharmacological profile of THC and THCV.In this manuscript, a combination of molecular dynamics (MD) simulations and site-directed mutagenesis have permitted to propose residue at position 6.51, which is Leu at CB 1 R and Val at CB 2 R, as an additional player capable to selectively recognize the alkyl chain of these ligands, further supporting the yin-yang functional relationship already described for CB 1 and CB 2 receptors. 16This knowledge could be of great use to facilitate the future design of selective drugs in the endocannabinoid system.

Initial CB 1 R and CB 2 R Models.
The CB 1 R-AM841-G i (PDB id 6KPG) and CB 2 R-AM12033-G i (6KPF) cryo-EM structures 18 were used in docking studies and MD simulations.Missing residues 55−180 of α i in the CB 1 R-AM841-G i structure were built from the structure of G i (6CRK); 23 and missing residues 55−181 and 233−239 of α i in the CB 2 R-AM12033-G i structure were built from the CB 2 R-WIN55,212−2-G i structure (6PT0), 17 using AutoModel class 24 of MODELER v10.1. 25Protonation states were assigned with the PDB 2PQR tool 26 using PROPKA to predict the pK a values of ionizable groups in the proteins at pH 6.5. 27Disulfide bonds between cysteines were built using the tleap module of Ambertools19.Internal water molecules were added to CB 1 R and CB 2 R using HomolWat. 28THC and THCV were docked into the orthosteric binding cavity of CB 1 R and CB 2 R and JWH-133 into CB 2 R by using AM841 in 6KPG and AM12033 in 6KPF structures as a reference.Thus, the alkyl chains of THC, THCV, and JWH-133 were initially modeled in the L-shape conformation above Trp279 5.43 and Trp194 5.43 of CB 1 R and CB 2 R, respectively, as observed in the cryo-EM structures.These systems were oriented by the Orientations of Proteins in Membranes (OPM) database, 29 and embedded in a lipid bilayer box, constructed using PACKMOL-memgen, 30 containing 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC), cholesterol (CHL) (10:1 POPC:CHL ratio), water molecules (TIP3P), and monatomic Na + and Cl − ions (0.15 M).The resulting systems comprise between 225 and 250k atoms in a box of ∼120 Å × 120 Å × 140 Å (see the Supporting Information in the Zenodo repository for detailed values).
2.2.Molecular Dynamics Simulations.MD simulations of these models were performed with GROMACS2018.5. 31he amber14sb-ildn force field was used for the protein, solvent, and ions, 32 a GROMACS adaptation of lipid14 for lipids, 33 and the general Amber force field (GAFF2) with HF/ 6-31G*-derived RESP atomic charges for THC, THCV, and JWH-133. 34Molecular systems were subjected to 5000 steps of energy minimization, using the steepest descent algorithm, PME electrostatics, with the Verlet cutoff scheme.This was followed by a 25 ns equilibration protocol consisting of six steps, in which positional restraints are progressively removed, from all heavy atoms to only helix Cα carbons being restricted, meanwhile gradually reducing the applied forces, from 1000 kJ mol −1 nm −2 to 0 kJ mol −1 nm −2 .After equilibration, three replicas of a 1 μs unrestrained MD trajectory were generated at a constant temperature of 300 K using separate v-rescale thermostats for the receptor, ligand, lipids, and solvent molecules.Initial velocities were randomly generated for each replica from a Maxwell distribution, using different random seeds.A time step of 2.0 fs was used for the integration of equations of motions using the leapfrog algorithm.Bonds involving hydrogen atoms were kept frozen by using the LINCS algorithm.Lennard-Jones interactions were computed using a cutoff of 1.1 nm under the Verlet cutoff scheme for neighbor searching, and the electrostatic interactions were treated using PME with the same real-space cutoff under periodic boundary conditions.Center of mass motion was removed from all systems.The Berendsen pressure control algorithm was chosen for equilibration and Parrinello− Rahman for production MDs.For complete details, see the Supporting Information in the Zenodo repository.

MD Analysis and Data
Visualization.The analysis of the trajectories was performed using MDAnalysis; 35 visualization and image rendering were performed with PyMOL 36 and VMD, 37 and graphical representations were obtained with the Seaborn Package. 38.4.CB 1 R and CB 2 R Mutants.Mutations were produced using the QuikChange Site-Directed Mutagenesis Kit.The cDNA for hCB 1 R and hCB 2 R, cloned into pcDNA3.1,was amplified using sense and antisense primers harboring the triplets for the desired point mutation (Pfu turbo polymerase was used).The nonmutated DNA template was digested for 1 h with DpnI.PCR products were used to transform XL1-blue supercompetent cells.Finally, positive colonies were tested by sequencing to select those expressing the correct DNA sequence.
2.5.cAMP Determination Assays.Determination of cAMP levels in HEK-293T cells transiently expressing CB 1 R or CB 2 R (1 μg of cDNA) or the mutant version of the receptor was performed by using the Lance-Ultra cAMP kit (PerkinElmer).Two hours before initiating the experiment, the medium was substituted by a serum-free medium.Then, transfected cells were dispensed in white 384-well microplates at a density of 4000 cells per well and incubated for 15 min at room temperature with compounds, followed by 15 min incubation with forskolin, and 1 h more with homogeneous time-resolved fluorescence (HTRF) assay reagents.Fluorescence at 665 nm was analyzed on a PHERAstar Flagship Journal of Chemical Information and Modeling microplate reader equipped with an HTRF optical module (BMG Labtech).Data analysis was made based on the fluorescence ratio emitted by the labeled cAMP probe (665 nm) over the light emitted by the europium cryptate-labeled anti-cAMP antibody (620 nm).A standard curve was used to calculate cAMP concentration.Forskolin-stimulated cAMP levels were normalized to 100%.

THC Adopts Two Distinct Binding Modes in CB 1 R
But Not in CB 2 R. To understand the different molecular signatures of THC and THCV, at CB 1 R and CB 2 R, we first performed three replicate runs of unbiased 1 μs MD simulations of these compounds bound to the CB 1 R-G i and CB 2 R-G i complexes (see the Methods section).We have used G i -bound active states, instead of inactive structures, despite its higher computational cost, because agonists alone are not capable to stabilize the fully active conformation in the absence of the G protein, as shown by NMR experiments. 40Similarly, MD simulations of agonist bound to the inactive state of the receptor are not capable of reaching active-like conformations in the absence of the G protein.Moreover, MD simulations of active, G protein-bound, conformations have permitted to identify additional cavities to accommodate hydrophobic chains of ligands in sphingosine-1-phosphate 41 and muscarinic 42 receptors, which were not identified in similar simulations of inactive structures.
THC and THCV were docked into these structures with the hydrophobic alkyl tail in the L-shape conformation, as observed in the cryo-EM structures of structurally similar ligands (see the Methods section).Root-mean-square deviations (rmsd) of the ligand heavy atoms show that THC visited during the MD simulations two different poses in the binding pocket of CB 1 R but not in CB 2 R (Figure 2c).In CB 1 R, THC adopts the initial L-shape conformation, in which the hydrophobic alkyl tail occupies a cavity between TMs 3 and 5 (Figure 2a), and an I-shape conformation, in which the alkyl tail occupies an intracellular cavity between TMs 3 and 6 (Figure 2b).The structure−function of the alkyl chain of THC has been reviewed, 43 and this dual orientation is consistent with previous studies by others. 15,44The different conforma- tion of the pentyl chain of THC is achieved by a change of a single dihedral angle in the chain, from − anticlinal (dihedral 1−2−3−4 around −90°) in the L-shape to + anticlinal (around 90°) in the I-shape (Figure 2g).Heatmaps showing when THC adopts the − anticlinal (dihedral <0°) or + anticlinal (>0°) conformation correlates with large (I-shape) and small (L-shape) rmsd values, respectively (Figures 2c and 2e).Notably, the pentyl chain of THC in CB 2 R rarely adopts the + anticlinal conformation (Figure 2e), thus no large rmsd values could be observed from the initial L-shape conformation during the MD simulations (Figure 2c).The shorter 3-carbon propyl chain of THCV has fewer steric constraints and can visit the + anticlinal conformation in both CB 1 R and CB 2 R simulations (Figures 2f−h).
It is not clear why the five-carbon pentyl chain of THC can also adopt the I-shape conformation, in which the alkyl tail occupies an intracellular cavity between TMs 3 and 6, in CB 1 R but not in CB 2 R.This intracellular cavity is delineated by the amino acid at position 6.51 (Figure 3a), which is Leu at CB 1 R and Val at CB 2 R (Figure 3b).The probability to undergo a side chain conformational change in Val is smaller than in Leu, 45 due to the β-branched side chain that is shorter than the γ-branched side chain of Leu.Val is generally found with the γcarbons flanking the small Hα in the trans (t, χ 1 = 180°) rotamer conformation, whereas Leu can adopt the more stable trans (t, χ 1 = 180°) and less stable gauche+ (g+, χ 1 = −60°) rotamer conformations, as observed in the dynameomics rotamer library. 46Figure 3c shows the histogram distributions of the χ 1 dihedral angle of Leu 6.51 along the MD simulations of CB 1 R.These panels illustrate that the three-carbon propyl chain of THCV maintains Leu 6.51 in the more stable t conformation during the simulation time, whereas the fivecarbon pentyl chain of THC in the I-shape conformation triggers or stabilizes the g+ conformation of Leu 6.51 in CB 1 R, opening the access to the intracellular cavity between TMs 3 and 6 (Figure 3d).In contrast, the conformation of the bulky, β-branched, and more rigid side chain of Val 6.51 in CB 2 R cannot be modified by the pentyl chain of THC (not shown), closing the access to the intracellular cavity.
3.2.The CB 1 R L6.51V and CB 2 R V6.51L Mutations Reverse the Pharmacology of THC.To experimentally validate the proposed different conformations of THC in CB 1 R and CB 2 R, we mutated Leu 6.51 to Val in CB 1 R (CB 1 R L6.51V ) and Val 6.51 to Leu in CB 2 R (CB 2 R V6.51L ), and we measured cAMP production in HEK-293T cells (Figure 3e).The nonselective CP-55940 agonist (100 nM) decreased, as expected for a G icoupled receptor, cAMP formation induced by forskolin (500 nM), in a statistically significant manner, in wild-type CB 1 R  6.51 along the MD simulations of CB 1 R bound to THC or THCV.Leu 6.51 adopted the t conformation with THCV and visited both the t and g+ conformations with THC.Representative structures of these conformations are also shown on the top panels.(d) Molecular representation of the different conformations of the five-carbon pentyl chain of THC (L-and I-shape) and Leu 6.51 in CB 1 R (t and g+).The transition of the alkyl chain of THC from the L-shape to the I-shape conformation (black arrow), to fill the intracellular cavity between TMs 3 and 6 delineated by Phe 3.36 , Trp 6.48 , and Leu 6.51 , requires the conformational transition of Leu 6.51 from t to g+ (black arrow).THC in the I-shape conformation and Leu 6.51 are shown in VdW spheres to visualize the narrow size of the intracellular cavity.Val 6.51 of CB 2 R (in orange) is superimposed to perceive that the β-branched character of the side chain blocks the access of the ligand chain to the intracellular cavity.(e) CAMP levels determined in HEK-293T cells transfected with CB 1 R, CB 2 R, CB 1 R L6.51V , or CB 2 R V6.51L .Cells were pretreated with vehicle (basal) or with THC (10 μM) or CP-55940 (100 nM) upon exposure to forskolin (Fk, 500 nM).Values are means ± SD (n = 4 with sixtiplicates in all experiments) of the percentage of forskolin-induced cAMP formation.These values were analyzed statistically with one-way ANOVA, followed by Bonferroni's multiple comparison test (*: p < 0.05 compared with Fk). and CB 2 R and mutant CB 1 R L6.51V and CB 2 R V6.51L .In contrast, THC (10 μM) can significantly decrease forskolin-induced cAMP accumulation in CB 1 R but not in CB 2 R. We used high concentrations of THC to evaluate the greatest attainable response (ceiling effect).These results suggest that, in cAMP measurements, THC acts as a weak partial agonist only in CB 1 R. Remarkably, the pharmacological profile of changes in the mutant receptors.THC can significantly decrease forskolin-induced cAMP accumulation in CB 2 R V6.51L but not in CB 1 R L6.51V .Moreover, cAMP accumulation induced by THC is not statistically different between CB 1 R and CB 2 R V6.51L .These experimental results, together with computational simulations, suggest that the residue at position 6.51, which is Leu at CB 1 R and Val at CB 2 R, is an additional element in the mechanism of receptor activation (see the Discussion section).

JWH-133
Activates CB 2 R via the Substituted Methyl Groups.JWH-133 is a potent CB 2 R agonist, with little affinity for CB 1 R. 47 The structure of JWH-133 is like THC and THCV with a 4-carbon butyl chain, instead of the 3carbon propyl chain of THCV or the 5-carbon pentyl chain of THC (Figure 1).A significant difference between JWH-133 and either THC or THCV is the methyl substitutions on the chain (Figure 1).Branching close to the aromatic ring might restrict the dimethylbutyl chain conformation of JWH-133.Thus, it seems reasonable to study the molecular properties of JWH-133, as a full agonist, in complex with CB 2 R-G i to challenge our proposed molecular models of THC and THCV (see section 3.1).Consequently, we performed simulations similar to those with THC and THCV to evaluate the binding mode of JWH-133 in CB 2 R (see the Methods section).The alkyl chain of JWH-133 always adopts the L-shape conformation in the + anticlinal conformation, filling the cavity between TMs 3 and 5 (Figure 4a).The dimethyl moiety of the dimethylbutyl chain mediates hydrophobic interactions with Phe 3.36 and Val 6.51 , during the simulation time (Figure 4c).To experimentally validate the key role of Val 6.51 in JWH-133induced CB 2 R activation, we measured the level of production of cAMP in CB 1 R L6.51V and CB 2 R V6.51L mutant receptors expressed in HEK-293T cells (Figure 4b).JWH-133 (100 nM) was unable to decrease forskolin-induced cAMP formation in CB 1 R but was statistically significantly lower in CB 2 R, as expected for a CB 2 R selective agonist.Substitution of Leu 6.51 with Val in CB 1 R does not facilitate activation of CB 1 R L6.51V by JWH-133.However, substitution of the single Val 6.51 amino acid with Leu in CB 2 R makes JWH-133 unable to activate CB 2 R V6.51L .This points to both the bulky, β-branched, and rigid Val 6.51 in CB 2 R and the bulky, branched dimethyl group of the dimethylbutyl chain of JWH-133 as key elements for CB 2 R activation (see the Discussion section).It was recently shown that the CB 2 R V6.51L mutation also impeded HU308 and CP-55940, both containing the branched dimethyl group in the alkyl chain, to activate the G protein at CB 2 R. 48

DISCUSSION AND CONCLUSIONS
Among the ∼350 GPCRs for nonsensory functions, ∼35 are activated by hormone-like signaling molecules derived from lipid species with long hydrophobic chains. 6,49Some 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. 54,55In PIFcontaining 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 . 56,57n GPCRs lacking P 5.50 , agonists can alter the rotamer of the amino acid at position 3.36 20 to trigger the rotation of TM 3 at I 3.40 and outward movement of TM 6 at F 6.44 . 41For instance, in the active crystal structure of S1P 3 bound to the endogenous agonist sphingosine-1-phosphate (S1P), 58 the long hydrophobic side chain of d18:1 S1P binds in an extended conformation (I-shape) between TMs 4 and 5 (Figure 5a).S1P triggers conformational changes of Leu122 3.36 from t to g+, among others (see quartet core in 58 ), to accommodate the alkyl chain.Similar results were observed in S1P bound to S1P 1 59 and S1P 2 60 (Figure 5a).In the cryo-EM structure of active LPA 1 bound to the lysophosphatidic acid (LPA), 61 the alkyl chain of LPA cannot extend to the cleft between TMs 4 and 5 as S1P, due to the presence of the bulky Trp210 5.43 in LPA 1 (S1P 1−3 possess the less bulky Cys206 5.43 , Val194 5.43 , or Cys200 5.43 ), blocking the access.LPA adopts an U-shaped conformation bending backward and triggering the g+ conformation of Leu132 3.36 (Figure 5b).The long acyl chain of the ONO-0740556 agonist, a more rigid and potent LPA analog, binds LPA 1 in a different bent conformation than LPA 62 (Figure 5c).In this case, the aromatic ring of ONO-0740556 triggers the g+ conformation of Leu132 3.36 .The lack of side chain in Gly274 6.51 (LPA 1−3 possess Gly at this key position) permits LPA 1 to have a small pocket in front of Leu132 3.36 encaging the terminus of LPA or the phenyl ring of ONO-0740556. 61,62B 1 R and CB 2 R possess Trp279 5.43 and Trp194 5.43 , respectively, thus blocking the TMs 4 and 5 cleft created in S1P binding to S1P 1−3 ; and contain Phe200 3.36 and Phe117 3.36 , respectively, as conformational toggle or trigger switch involved in the initial agonist-induced receptor activation.Notably, the indazole ring of MDMB-Fubinaca triggers the active g+ conformation of Phe200 3.36 in CB 1 R by an aromatic− aromatic interaction (Figure 5c), 15 and the aromatic core of WIN 55,212−2 also forms aromatic−aromatic interactions with Phe117 3.36 in g+ of CB 2 R (Figure 5c).17 In other known structures of active CB 1 R and CB 2 R bound to agonists, the bulky and branched dimethyl groups of the alkyl chain of AM841 and AM12033 bridge Phe200 3.36 in g+ and Leu359 6.51 of CB 1 R and Phe117 3.36 in g+ and Val261 6.51 of CB 2 R (Figure 5d), respectively. 18The conformational change in Phe 3.36 , from pointing toward TM 6 in t to pointing toward TM 7 in g+, permits Trp 6.48 to move toward TM 5 for receptor activation.
These data indicate that the hydrophobic alkyl chain of the signaling molecule is key in the process of ligand-induced receptor activation.Thus, in this paper, we have studied the conformation of the alkyl chain of THC, THCV, and JWH-133 bound to CB 1 R and CB 2 R and calculated their distances to Phe 3.36 along the MD simulations.Among them, the distances between the terminal methyl group of the alkyl chain of THC and the centroid of the aromatic ring of Phe 3.36 and either the δor γcarbon of Leu/Val 6.51 are important to highlight (see Figures 4c and 4d).They fluctuated from >5 Å to <5 Å in CB 1 R and are always >5 Å in CB 2 R, indicating a dual orientation of the alkyl chain in CB 1 R, which either occupies a cavity above Trp 5.43 (between TMs 3 and 5) in an L-shape conformation, or an intracellular cavity between Phe 3.36 and Trp 6.48 (TMs 3 and 6) in an I-shape conformation (Figure 3d).The main achievement of this work is the discovery that THC in CB 1 R, but not in CB 2 R, can adopt this I-shape conformation.The intracellular cavity between Phe 3.36 and Trp 6.48 is also delineated by the amino acid at position 6.51, which is the γ-branched, flexible Leu side chain in CB 1 R and the β-branched, model rigid Val side chain in CB 2 R (Figure 3b).We have shown that the five-carbon pentyl chain of THC can trigger the conformational change of Leu 6.51 from t, blocking the access of the chain to the intracellular cavity, to g +, opening the access (Figures 3c and 3d).This opening of the chain access to the intracellular cavity is not feasible with the rigid Val 6.51 side chain of CB 2 R. The binding mode of THC in the I-shape conformation positions the alkyl chain between Phe 3.36 in the active g+ conformation and Trp 6.48 that is involved in the initial mechanism of agonist-induced receptor activation.Thus, these computational results are compatible with our experiments, showing that THC acts as a partial agonist in CB 1 R and as an antagonist in CB 2 R (Figure 3e). 12In agreement with our computational results, THC could not activate the mutant CB 1 R L6.51V receptor and activated the mutant CB 2 R V6.51L receptor as efficiently as wild type CB 1 R (Figure 3e).We have recently shown that the alkyl chain of cannabidiol, in the allosteric binding mode, also expands toward the intracellular cavity modulating the conformation of Phe 3.36 . 63he predicted binding mode of the dimethylbutyl chain conformation of the full agonist JWH-133 at CB 2 R is always in the L-shape conformation, filling the cavity between TMs 3 and 5 (Figure 4a).However, the branched dimethyl moiety of the ligand chain mediates hydrophobic interactions with Phe 3.36 in the active g+ conformation and Val 6.51 (Figure 4c).Notably, substitution of Val 6.51 for Leu in CB 2 R makes JWH-133 unable to activate CB 2 R V6.51L (Figure 4b).This supports the concept that the branched dimethylbutyl chain conformation of JWH-133 needs a foothold on the rigid Val 6.51 to move Phe 3.36 to the active g+ conformation for receptor activation.
In conclusion, our findings have shown that, in cannabinoid receptors and probably other receptors that recognize signaling molecules derived from lipid species with long hydrophobic chains, the amino acid at position 6.51 defines the size and shape of the cavity near Phe 3.36 and Trp 6.48 and is a key additional player in the mechanism of activation of this type of GPCRs.

Data Availability Statement
Input coordinates (.gro), topology files (.top), ligand parameters (.itp), input files, and representative structures collected during three replicas of MD simulations (in a PyMol session) of THC and THCV bound to CB 1 R and CB 2 R and JWH-133 bound to CB 2 R (the color code of the structures is as given in Figures 2−4) are available at https://zenodo.org/record/8114762.PACKMOL-Memgen, distributed with Am-berTools, is free of charge; the Seaborn Package, MDAnalysis and GROMACS are open source; VMD is available to noncommercial users under a distribution-specific license; and PyMOL is commercial software with paid license.

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Corresponding Author

Figure 2 .
Figure 2. Conformational analysis of the alkyl chain of THC and THCV bound to CB 1 R and CB 2 R. Views parallel and perpendicular to the membrane plane of THC bound to CB 1 R in (a) an L-shape and (b) an I-shape conformation pointing toward cavities between either TMs 3 and 5 or TMs 3 and 6, respectively.Red and blue rectangles correspond to the structure in red and blue circles in panels (c) and (e).Rmsd values of ligand heavy atoms (c, d), heatmaps (e, f) showing the conformation of the alkyl chain (dihedral angle 1−2−3−4) in the − anticlinal (angle around −90°< 0°) or the + anticlinal (around 90°> 0°), and histogram distribution of the dihedral angle 1−2−3−4 (g, h) during MD simulations of THC bound to CB 1 R (green lines or panels) or CB 2 R (brown) and THCV bound to CB 1 R (purple) or CB 2 R (blue).Three replicas of 1 μs of each complex were run.

Figure 3 .
Figure 3. Conformational analysis of the Leu/Val 6.51 side chains of CB 1 R and CB 2 R. (a) Evolution of the terminal methyl group (color spheres) of the alkyl chain of THC or THCV during MD simulations of THC bound to CB 1 R (green) or CB 2 R (brown) and THCV bound to CB 1 R (purple) or CB 2 R (blue).The black arrow represents the conformational change of the side chain of Leu 6.51 from t to g+ that is triggered by the I-shaped conformation of THC in CB 1 R. (b) Sequence comparison of TM 6 between CB 1 R and CB 2 R and the position of these amino acids in CB 1 R (THC in the I-shaped conformation is shown as a reference).(c) Histogram distributions of the χ 1 dihedral angle of Leu6.51  along the MD simulations of CB 1 R bound to THC or THCV.Leu 6.51 adopted the t conformation with THCV and visited both the t and g+ conformations with THC.Representative structures of these conformations are also shown on the top panels.(d) Molecular representation of the different conformations of the five-carbon pentyl chain of THC (L-and I-shape) and Leu 6.51 in CB 1 R (t and g+).The transition of the alkyl chain of THC from the L-shape to the I-shape conformation (black arrow), to fill the intracellular cavity between TMs 3 and 6 delineated by Phe 3.36 , Trp6.48  , and Leu 6.51 , requires the conformational transition of Leu 6.51 from t to g+ (black arrow).THC in the I-shape conformation and Leu6.51  are shown in VdW spheres to visualize the narrow size of the intracellular cavity.Val 6.51 of CB 2 R (in orange) is superimposed to perceive that the β-branched character of the side chain blocks the access of the ligand chain to the intracellular cavity.(e) CAMP levels determined in HEK-293T cells transfected with CB 1 R, CB 2 R, CB 1 R L6.51V , or CB 2 R V6.51L .Cells were pretreated with vehicle (basal) or with THC (10 μM) or CP-55940 (100 nM) upon exposure to forskolin (Fk, 500 nM).Values are means ± SD (n = 4 with sixtiplicates in all experiments) of the percentage of forskolin-induced cAMP formation.These values were analyzed statistically with one-way ANOVA, followed by Bonferroni's multiple comparison test (*: p < 0.05 compared with Fk).

Figure 4 .
Figure 4. (a) The binding of JWH-133 to CB 2 R. Leu 6.51 of CB 1 R (in yellow) is superimposed to perceive the longer side chain of Leu in t compared to Val.(b) cAMP levels determined in HEK-293T cells transfected with CB 1 R, CB 2 R, CB 1 R L6.51V , or CB 2 R V6.51L .Cells were pretreated with vehicle (basal) or with JWH-133 (100 nM) upon exposure to forskolin (Fk, 500 nM).Values are means ± SD (n = 4 with sixtiplicates in all experiments) of the percentage of forskolin-induced cAMP formation.These values were analyzed statistically with two-way ANOVA, followed by Bonferroni's multiple comparison test ((***) p < 0.001 compared with Fk, (###) p < 0.001).(c) Distances between the terminal methyl group of the alkyl chain of THC and the dimethyl moiety of the dimethylbutyl chain of JWH-133 and the centroid of the aromatic ring of Phe 3.36 and either the δor γcarbon of Leu/Val 6.51 of CB 1 R or CB 2 R (matching color arrows in panel (d) obtained during three replicas of MD simulations.(d) Comparison of the proposed binding modes of JWH-133 and THC in CB 2 R and THC in CB 1 R.