**3. CB1 receptor crystal structures**

Two inactive state crystal structures for the hCB1 receptor have been resolved. The first structure (PDB ID: 5TGZ) was resolved at 2.8 Å; the receptor was truncated at both the N-terminus (1–98) and the C-terminus (415–472), with a flavodoxin protein fused into the IC3 loop (V306, P332), the receptor was crystalized in complex with a biaryl-pyrazole derivative (AM6358, **Figure 2**) and using thermo-stabilizing mutations (T3.46A, E5.37K, T5.47V, and R6.32E) [6]. The second structure was resolved at 2.6 Å (PDB ID: 5U09) in complex with an acyclic high affinity inverse agonist of the CB1 receptor, taranabant (**Figure 2**) [7]. In this structure, fewer amino acid residues were truncated from the N-terminus (1–76) and the C-terminus (422–472), and *P. abysii* glycogen synthase protein was fused into the IC3 loop (A301, D333) of a single point mutant (T3.46A) hCB1 receptor [7]. In both structures, resolved residues were from E100 at the N-terminus to F412 at the C-terminus of the receptor.

> Agonist bound hCB1 crystal structures (PDB IDs: 5XRA, 5XR8) were resolved at 2.80 and 2.95 Å resolution and in complex with the classical cannabinoids (AM11542, AM841) respectively (**Figure 2**). The receptor was constructed in a similar way to the AM6358-bound crystal structure. Resolved residues included D104-S414 and F102-S414 in the 2.80 and 2.95 Å resolu-

> **Figure 3.** N-terminus residues F102 and M103 (green VdW) penetrate the binding crevice in the inactive state CB1 structure (PDB ID: 5U09. This influences the positions of K3.28 (magenta tube), D2.63 and D184 (wheat tube) which form

> Inactive state CB1 structures show a transmembrane portal for antagonist entry between TMH1

CB1 receptor forms a loop that extends towards the orthosteric binding site with two amino acid residues (F102, M103) invading unpredictably the binding site in the inactive state structures and forming Van der Waal (VDW) interactions with the antagonists (**Figure 3**) [6, 7].

Active state structures show characteristic conformational changes featuring class A GPCR activation including an outward movement and a counterclockwise rotation (EC view) of the IC end of TMH6, resulting in a break in the R3.50/D6.30 inactive state "ionic lock" [19, 20]. Unlike inactive state structures, a transmembrane portal is not present in active state structures due to the packing of the EC domain of TMH1 towards TMH7. In addition, the N-terminus resides at the top of the receptor with no invasion of the orthosteric binding site. On the other hand, the active state binding site displays a profound (53%) reduction in size that is resulting from an inward kink of the EC domain of TMH2 towards the orthosteric binding site, as well

**4. Mutation and labeling studies on CB1: consistency with CB1** 

Multiple mutation studies on either mCB1 or hCB1 were aimed to study the receptor's binding site and to identify key residues for CB1 receptor activation (**Figure 4**). While different ligands where used in functional and binding affinity assessment, WIN55212, SR141716A and CP55940, were used primarily, due to the availability of tritiated versions of these compounds.

structure. However, the membrane proximal region in the

Structural Insights from Recent CB1 X-Ray Crystal Structures

http://dx.doi.org/10.5772/intechopen.80783

37

tion structures respectively [8].

an interaction with each other.

and TMH7 that is similar to the S1P1

as, rotation of TMH3 towards TMH2 [8].

**crystal structures**

**Figure 2.** Compounds discussed in this chapter.

**3. CB1 receptor crystal structures**

36 Recent Advances in Cannabinoid Research

**Figure 2.** Compounds discussed in this chapter.

at the N-terminus to F412 at the C-terminus of the receptor.

Two inactive state crystal structures for the hCB1 receptor have been resolved. The first structure (PDB ID: 5TGZ) was resolved at 2.8 Å; the receptor was truncated at both the N-terminus (1–98) and the C-terminus (415–472), with a flavodoxin protein fused into the IC3 loop (V306, P332), the receptor was crystalized in complex with a biaryl-pyrazole derivative (AM6358, **Figure 2**) and using thermo-stabilizing mutations (T3.46A, E5.37K, T5.47V, and R6.32E) [6]. The second structure was resolved at 2.6 Å (PDB ID: 5U09) in complex with an acyclic high affinity inverse agonist of the CB1 receptor, taranabant (**Figure 2**) [7]. In this structure, fewer amino acid residues were truncated from the N-terminus (1–76) and the C-terminus (422–472), and *P. abysii* glycogen synthase protein was fused into the IC3 loop (A301, D333) of a single point mutant (T3.46A) hCB1 receptor [7]. In both structures, resolved residues were from E100

**Figure 3.** N-terminus residues F102 and M103 (green VdW) penetrate the binding crevice in the inactive state CB1 structure (PDB ID: 5U09. This influences the positions of K3.28 (magenta tube), D2.63 and D184 (wheat tube) which form an interaction with each other.

Agonist bound hCB1 crystal structures (PDB IDs: 5XRA, 5XR8) were resolved at 2.80 and 2.95 Å resolution and in complex with the classical cannabinoids (AM11542, AM841) respectively (**Figure 2**). The receptor was constructed in a similar way to the AM6358-bound crystal structure. Resolved residues included D104-S414 and F102-S414 in the 2.80 and 2.95 Å resolution structures respectively [8].

Inactive state CB1 structures show a transmembrane portal for antagonist entry between TMH1 and TMH7 that is similar to the S1P1 structure. However, the membrane proximal region in the CB1 receptor forms a loop that extends towards the orthosteric binding site with two amino acid residues (F102, M103) invading unpredictably the binding site in the inactive state structures and forming Van der Waal (VDW) interactions with the antagonists (**Figure 3**) [6, 7].

Active state structures show characteristic conformational changes featuring class A GPCR activation including an outward movement and a counterclockwise rotation (EC view) of the IC end of TMH6, resulting in a break in the R3.50/D6.30 inactive state "ionic lock" [19, 20]. Unlike inactive state structures, a transmembrane portal is not present in active state structures due to the packing of the EC domain of TMH1 towards TMH7. In addition, the N-terminus resides at the top of the receptor with no invasion of the orthosteric binding site. On the other hand, the active state binding site displays a profound (53%) reduction in size that is resulting from an inward kink of the EC domain of TMH2 towards the orthosteric binding site, as well as, rotation of TMH3 towards TMH2 [8].
