Agonist alkyl tail interaction with cannabinoid CB1 receptor V6.43/I6.46 groove induces a helix 6 active conformation

Our modeling studies have suggested that β branched amino acids Val, lle, or Thr located (i, i + 3) or (i, i + 4) apart on an alpha helix can form a groove into which a ligand alkyl chain can fit. Experimental support forthis idea comes from the crystal structure of adipocyte lipid binding protein complexed with stearic acid (Xu et al. J Biol Chem 1993, 268, 7874). We hypothesized that the transmembrane helix 6 (TMH 6) βXXβ motif of the CB1/CB2 receptors (V6.43/I6.46), which immediately precedes the conserved TMH 6 CWXP motif, serves as an interaction site for the alkyl tail of cannabinoid (CB) ligands and that interaction with this motif may trigger receptor activation. Conformational memories (CM) calculations on TMH 6 of CB1 and CB2 were used to explore the conformation of TMH 6 in unbound and complexed states. The conserved Pro 6.50 generated a kink in the α-helical structure that behaved as a flexible hinge. In the context of a three-dimensional model of the CB1 receptor, a helix from the more kinked family of CB1 TMH 6 conformers calculated by CM brought the intracellular portion of TMH 6 in proximity to TMH 3, analogous to the inactive state TMH 3/6 conformation seen in the X-ray crystal structure of rhodopsin (Palczewski et al. Science 2000, 289, 739). A CM study of CB1 TMH 6 in which a pentane molecule (as a model system for the CB ligand side chain) interacts with the V6.43/I6.46 groove was also conducted. The results of this calculation showed that alkyl chain interaction with the V6.43/I6.46 groove directly modulates the overall conformation of TMH 6, biasing the population of TMH 6 conformers toward the family of less kinked CB1 TMH 6 conformations calculated by CM. In the context of the CB1 bundle, this conformational change would cause the intracellular end of TMH 6 to move away from TMH 3. Such a movement is consistent with recent experimental results for agonist induced conformational changes at the intracellular side of TMH 6 in the β 2 -adrenergic receptor (Jensen et al. J Biol Chem 2001, 276, 9279). In contrast to results for CB1, CB2 TMH 6 showed a smaller range of kink angles possible for unbound TMH 6, with no significant shift in the populations of TMH 6 when the V6.43/I6.46 groove was occupied by pentane. We hypothesized that the profound flexibility differences between wild-type (WT) CB1 vs. WT CB2 TMH 6 revealed by CM calculations may be due to the size of residue 6.49 which immediately precedes P6.50 of the CWXP motif (G6.49 in WT CB1 and F6.49 in WT CB2). To test this hypothesis, using CM, we compared the flexibilities of WT CB1 and CB2 TMH 6 with those of the switch mutants, CB1 G6.49F and CB2 F6.49G. Consistent with results reported above, the degree of kinking (average of 100 conformers) was distinctly different between CB1 (40.9°; std. dev. ′16.9°) and CB2 (24.6°; std. dev. ′ 4.3°), with CB1 TMH 6 exhibiting a noticeably wider range of kink angles than CB2. These flexibilities were essentially switched in the mutants [CB1 G6.49F mutant (25.3°; std. dev. ′ 5.7°) and a CB2 F6.49G mutant (44.3°; std. dev. ′ 21.4°]. Taken together, these results suggest that TMH 6 in CB1, but not in CB2, is sensitive to conformational modulation by an alkyl chain bound in the V6.43/I6.46 groove. Furthermore, results suggest that the small size of residue 6.49 in CB1 facilitates the P6.50 flexible hinge motion of CB1 TMH 6.