**6. Crystal contacts**

In the inactive and active state CB1 crystal structures, crystal packing impinges on the ligand binding site (**Figure 6**). In the first published CB1 inactive state structure [6], receptor bundles are crystallized top-to-top, forcing the N-terminus to invade the binding pocket and flattening the EC loops. In the second inactive state CB1 structure [7], adjacent bundles impinge on receptor EC loops and N-terminus around the "rim" of the receptor's EC domain (**Figure 6**). The effect on CB1 structure is similar to that discussed above for the first inactive crystal structure. Crystal packing in the active state structure [8] also causes an impact on the CB1 binding pocket. Packing causes TMH2 to hinge at G2.53/S2.54 (S2.54 has a χ1 = g−) and invade the binding pocket. Packing also impacts the N-terminus, TMH1 above N1.50, the EC top of TMH3, the EC-2 loop and the EC end of TMH4.

is positioned 2.6 Å from the secondary amine of the bound agonist. Mutation of this residue into alanine did not affect the affinity of the ligand to the receptor, suggesting that the resulting conformation of the N-terminus in MOR structure is a result of crystallization and not

Structural Insights from Recent CB1 X-Ray Crystal Structures

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

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Because X-ray crystal structures are used frequently for drug design projects, it is critical to identify any issues with these structures, such as crystal packing effects and to evaluate how consistent these structures are with the body of structural information in the literature for a given receptor, such as mutation, cross-linking and NMR studies. Results presented in this chapter show that crystal packing issues impact both of the CB1 inactive state crystal structures and the activated state CB1 crystal structures. Impacts include N-terminus insertions deep into the binding pocket seen in the CB1 inactive state structures, as well as, TMH1 and TMH2 bending into the binding pocket seen in the activated state structures. Not surprisingly, we find here that the CB1 structures have important inconsistencies with mutation data, particularly in their TMH1-2-3 regions. In addition, the CB1 crystal structures do not capture the movement of W6.48 during receptor activation, or the existence of a ligand portal in the activated state, however, X-ray structures by their very nature will not capture all transient changes. In conclusion, then, the CB1 crystal structures are an important contribution to the drug design field, but revisions are advisable before these structures are used for structure-based drug discovery.

and Patricia H. Reggio2

1 Department of Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy, Jordan

2 Department of Chemistry and Biochemistry, University of North Carolina at Greensboro,

[1] Palczewski K et al. Crystal structure of rhodopsin: A G protein-coupled receptor. Science.

\*

relevant in the real state [68].

**7. Conclusions**

**Acknowledgements**

**Author details**

Rufaida Al-Zoubi1

Greensboro, NC, USA

2000;**289**(5480):739

**References**

This work was supported by NIDA RO1 DA003934 to PHR.

, Dow P. Hurst2

\*Address all correspondence to: phreggio@uncg.edu

University of Science and Technology, Irbid, Jordan

**Figure 6.** Unit cell TM region and extracellular loop crystal contacts for hCB1 receptor crystal structures. CB1 ribbon colors: TMH1 (red), TMH2 (orange), TMH3 (yellow), TMH4 (pale green), TMH5 (green), TMH6 (cyan), TMH7 (blue), crystal mate ribbons (white). Top panel: inactive state structure (PDB ID: 5TGZ) [6]. Middle panel: inactive state structure (PDB ID: 5U09) [7]. Bottom panel: active state structure (PDB ID: 5XRA) [8]. Amino acid residues for crystal mates are colored cyan, while inactive and active state structures are shown in orange and green respectively.

Such packing issues can promote non-genuine conformations in the structure that is promoted by the crystalline low energy state. A recently published crystal structure of the μ-opioid receptor (MOR) has revealed a histidine H54 residue in the N-terminus of the receptor that is positioned 2.6 Å from the secondary amine of the bound agonist. Mutation of this residue into alanine did not affect the affinity of the ligand to the receptor, suggesting that the resulting conformation of the N-terminus in MOR structure is a result of crystallization and not relevant in the real state [68].
