Singh R, Hurst D P, Barnett-Norris J, Lynch D L, Reggio P H, Guarnieri F
Department of Chemistry and Biochemistry, Kennesaw State University, Kennesaw, Georgia, USA.
J Pept Res. 2002 Dec;60(6):357-70. doi: 10.1034/j.1399-3011.2002.21065.x.
The cannabinoid CB1 receptor, a member of the Rhodopsin (Rho) family of G protein coupled receptors (GPCRs), exhibits high levels of constitutive activity. In contrast, Rho exhibits an exquisite lack of constitutive activity. In Rho, W6.48(265) on transmembrane helix 6 (TMH6) is flanked by aromatic residues at positions i-4 (F6.44) and i + 3 (Y6.51), while in CB1 the residues i-4 and i + 3 to W6.48 are leucines (L6.44 and L6.51). Based upon spectroscopic evidence, W6.48 has been proposed to undergo a rotamer switch (chi1 g+ -->trans) upon activation of Rho. In the work reported here, the biased Monte Carlo method, Conformational Memories (CM) was used to test the hypothesis that the high constitutive activity exhibited by CB1 may be due, in part, to the lack of aromatic residues i-4 and i + 3 from W6.48. In this work, the W6.48 rotamer shift (chi1 g+ -->trans) was used as the criterion for activation. Conformational Memories (CM) calculations on WT CB1 TMH6 and L6.44F and L6.51Y mutant TMH6s revealed that an aromatic residue at 6.44 tends to disfavor the W6.48 chi1 g+ -->trans transition and an aromatic residue at 6.51 would require a concomitant movement of the Y6.51 chi1 from trans-->g+ when the W6.48 chi1 undergoes a g+ -->trans shift. In contrast, CM calculations on WT CB1 TMH6 revealed that the presence of leucines at 6.44 and 6.51 provide W6.48 with greater conformational mobility, with a W6.48 transchi1 preferred. Conformational Memories calculations also revealed that the W6.48 chi1 g+ -->trans transition in WT CB1 TMH6 is correlated with the degree of kinking in TMH6. The average proline kink angles for TMH6 were higher for helices with a W6.48 g+ chi1 than for those with a W6.48 transchi1. These results are consistent with experimental evidence that TMH6 straightens during activation. Transmembrane helix (TMH) bundle models of the inactive (R) and active (R*) states of CB1 were then probed for interactions that may constrain W6.48 in the inactive state of CB1. These studies revealed that F3.36 (transchi1) helps to constrain W6.48 in a g+ chi1 in the inactive (R) state of CB1. In the R* state, these studies suggest that F3.36 must assume a g+ chi1 in order to allow W6.48 to shift to a transchi1. These results suggest that the W6.48/F3.36 interaction may act as the 'toggle switch' for CB1 activation, with W6.48 chi1 g+/F3.36 chi1 trans representing the inactive (R) and W6.48 chi1 trans/F3.36 chi1 g+ representing the active (R*) state of CB1.
大麻素CB1受体是视紫红质（Rho）家族G蛋白偶联受体（GPCRs）的成员之一，具有高水平的组成性活性。相比之下，Rho几乎没有组成性活性。在Rho中，跨膜螺旋6（TMH6）上的W6.48（265）两侧分别是i-4位（F6.44）和i + 3位（Y6.51）的芳香族残基，而在CB1中，W6.48的i-4和i + 3位残基是亮氨酸（L6.44和L6.51）。基于光谱学证据，有人提出在Rho激活时W6.48会发生旋转异构体转换（chi1 g+ -->反式）。在本文报道的研究中，采用了偏向蒙特卡罗方法“构象记忆”（CM）来检验这一假设，即CB1表现出的高组成性活性可能部分归因于W6.48缺乏i-4和i + 3位的芳香族残基。在这项研究中，将W6.48旋转异构体转换（chi1 g+ -->反式）用作激活的标准。对野生型CB1 TMH6以及L6.44F和L6.51Y突变体TMH6进行的构象记忆（CM）计算表明，6.44位的芳香族残基往往不利于W6.48的chi1 g+ -->反式转变，而当W6.48的chi1发生g+ -->反式转变时，6.51位的芳香族残基会要求Y6.51的chi1从反式伴随移动到g+。相比之下，对野生型CB1 TMH6的CM计算表明，6.44和6.51位亮氨酸的存在使W6.48具有更大的构象灵活性，且更倾向于W性6.48反式chi1。构象记忆计算还表明，野生型CB1 TMH6中W6.48的chi1 g+ -->反式转变与TMH6的弯曲程度相关。W6.48 g+ chi1的螺旋的TMH6平均脯氨酸弯曲角度高于W6.48反式chi1的螺旋。这些结果与TMH6在激活过程中伸直的实验证据一致。然后探究了CB1非活性（R）和活性（R*）状态的跨膜螺旋（TMH）束模型中可能在CB1非活性状态下限制W6.48的相互作用。这些研究表明，F3.36（反式chi1）有助于在CB1的非活性（R）状态下将W6.48限制在g+ chi1状态。在R状态下，这些研究表明F3.36必须处于g+ chi1状态，以便W6.48转变为反式chi1。这些结果表明，W6.48/F3.36相互作用可能充当CB1激活的“切换开关”，W6.48 chi1 g+/F3.36 chi1反式代表CB1的非活性（R）状态，而W6.48 chi1反式/F3.36 chi1 g+代表CB1的活性（R）状态。
