Molecular basis for dramatic changes in cannabinoid CB1 G protein-coupled receptor activation upon single and double point mutations.
Molecular basis for dramatic changes in cannabinoid CB1 G protein-coupled receptor activation upon single and double point mutations.
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单点和双点突变后大麻素 CB1 G 蛋白偶联受体激活发生巨大变化的分子基础。
DOI：
10.1002/pro.2192
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Goddard3rd,WilliamA
中科院分区：
文献类型：
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作者：
Scott,CaitlinE;Abrol,Ravinder;Ahn,KwangH;Kendall,DebraA;Goddard3rd,WilliamA
G蛋白偶联受体(GPCRs)是细胞间信号转导最重要的蛋白质之一，其激活机制的研究颇受关注。最近，对于大麻素CB1GPCR，研究表明，单个突变T210A可以使CB1完全失活，而T210I使其本质上具有结构性活性。为了了解这种活性对突变的戏剧性依赖，我们使用基于第一性原理的方法预测了野生型(WT)和突变体(T210A和T210I)的低能七螺旋构象的集合。我们发现跨膜(TM)螺旋包装明显依赖于这些突变，导致T210A在细胞质表面发生TM3+TM6和TM2+TM6的盐桥耦合，这解释了该突变体的不活性。相比之下，T210I在受体上没有这样的偶联，这解释了激活这个突变体是多么容易。WT只有TM3+TM6偶联，已知在GPCR激活时会被破坏。为了验证这一关于活性的假设，我们预测了两个突变体，它们将不活跃的突变体转换为正常的活性，然后通过实验证实了这一点。这种CB1激活机制或与之类似的机制有望在其他成分活跃的GPCR中发挥作用。
There is considerable interest in determining the activation mechanism of G protein‐coupled receptors (GPCRs), one of the most important types of proteins for intercellular signaling. Recently, it was demonstrated for the cannabinoid CB1 GPCR, that a single mutation T210A could make CB1 completely inactive whereas T210I makes it essentially constitutively active. To obtain an understanding of this dramatic dependence of activity on mutation, we used first‐principles‐based methods to predict the ensemble of low‐energy seven‐helix conformations for the wild‐type (WT) and mutants (T210A and T210I). We find that the transmembrane (TM) helix packings depend markedly on these mutations, leading for T210A to both TM3+TM6 and TM2+TM6 salt‐bridge couplings in the cytoplasmic face that explains the inactivity of this mutant. In contrast T210I has no such couplings across the receptor explaining the ease in activating this mutant. WT has just the TM3+TM6 coupling, known to be broken upon GPCR activation. To test this hypothesis on activity, we predicted double mutants that would convert the inactive mutant to normal activity and then confirmed this experimentally. This CB1 activation mechanism, or one similar to it, is expected to play a role in other constitutively active GPCRs as well.