Life Sciences Institute and Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Proc Natl Acad Sci U S A. 2011 Sep 20;108(38):16086-91. doi: 10.1073/pnas.1113645108. Epub 2011 Sep 13.
The active-state complex between an agonist-bound receptor and a guanine nucleotide-free G protein represents the fundamental signaling assembly for the majority of hormone and neurotransmitter signaling. We applied single-particle electron microscopy (EM) analysis to examine the architecture of agonist-occupied β(2)-adrenoceptor (β(2)AR) in complex with the heterotrimeric G protein Gs (Gαsβγ). EM 2D averages and 3D reconstructions of the detergent-solubilized complex reveal an overall architecture that is in very good agreement with the crystal structure of the active-state ternary complex. Strikingly however, the α-helical domain of Gαs appears highly flexible in the absence of nucleotide. In contrast, the presence of the pyrophosphate mimic foscarnet (phosphonoformate), and also the presence of GDP, favor the stabilization of the α-helical domain on the Ras-like domain of Gαs. Molecular modeling of the α-helical domain in the 3D EM maps suggests that in its stabilized form it assumes a conformation reminiscent to the one observed in the crystal structure of Gαs-GTPγS. These data argue that the α-helical domain undergoes a nucleotide-dependent transition from a flexible to a conformationally stabilized state.
激动剂结合的受体和无核苷酸结合的 G 蛋白之间的活性状态复合物代表了大多数激素和神经递质信号的基本信号转导组装。我们应用单颗粒电子显微镜（EM）分析来检查激动剂占据的β（2）-肾上腺素能受体（β（2）AR）与异三聚体 G 蛋白 Gs（Gαsβγ）形成的复合物的结构。去污剂溶解复合物的 EM 2D 平均值和 3D 重建揭示了与活性状态三元复合物的晶体结构非常吻合的整体结构。然而，令人惊讶的是，在没有核苷酸的情况下，Gαs 的α-螺旋结构域显得非常灵活。相比之下，焦磷酸类似物膦甲酸（磷羧基甲酸酯）的存在以及 GDP 的存在有利于 Gαs 的 Ras 样结构域上α-螺旋结构域的稳定。3D EM 图谱中α-螺旋结构域的分子建模表明，在其稳定形式下，它采用了与 Gαs-GTPγS 晶体结构中观察到的构象相似的构象。这些数据表明，α-螺旋结构域经历了从灵活到构象稳定状态的核苷酸依赖性转变。
