Identification of two distinct inactive conformations of the β2-adrenergic receptor reconciles structural and biochemical observations
Identification of two distinct inactive conformations of the β2-adrenergic receptor reconciles structural and biochemical observations
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DOI：
10.1073/pnas.0811065106
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发表时间：
2009-03-24
影响因子：
11.1
通讯作者：
Shaw, David E.
中科院分区：
文献类型：
--
作者：
Dror, Ron O.;Arlow, Daniel H.;Shaw, David E.
充分理解信号蛋白如G蛋白偶联受体（GPCR）的机制将需要表征其构象状态和连接这些状态的途径。最近，β（2）-和β（1）-肾上腺素能受体在名义上无活性状态下的晶体结构构成了朝着这一目标的重大进展，但也提出了新的问题。虽然早期的生物化学观察表明，这些受体在螺旋3和6之间具有一组接触，称为离子锁，被认为是形成受体激活的分子开关，但晶体结构缺乏这些接触。出乎意料地破坏的离子锁引起了关于非活性状态的真实构象以及离子锁在受体活化和信号传导中的作用的问题。为了解决这些问题，我们对多种野生型和突变型的β 2肾上腺素能受体（β 2 AR）进行了微秒时间尺度的分子动力学模拟。在野生型模拟中，离子锁可重复地形成，将螺旋3和6的细胞内末端结合在一起，以采用类似于在非活性视紫红质中发现的构象。我们的研究结果表明，无论是否存在共结晶配体，无活性的β（2）AR都存在于形成锁和打破锁的构象之间的平衡中。这些发现，沿着在我们的模拟过程中在β 2 AR环中形成了几个二级结构元件，可以提供β肾上腺素能受体非活性状态的更全面的图片，使晶体结构与生化研究相一致。
Fully understanding the mechanisms of signaling proteins such as G protein-coupled receptors (GPCRs) will require the characterization of their conformational states and the pathways connecting those states. The recent crystal structures of the beta(2)- and beta(1)-adrenergic receptors in a nominally inactive state constituted a major advance toward this goal, but also raised new questions. Although earlier biochemical observations had suggested that these receptors possessed a set of contacts between helices 3 and 6, known as the ionic lock, which was believed to form a molecular switch for receptor activation, the crystal structures lacked these contacts. The unexpectedly broken ionic lock has raised questions about the true conformation(s) of the inactive state and the role of the ionic lock in receptor activation and signaling. To address these questions, we performed microsecond-timescale molecular dynamics simulations of the beta(2)-adrenergic receptor (beta(2)AR) in multiple wild-type and mutant forms. In wild-type simulations, the ionic lock formed reproducibly, bringing the intracellular ends of helices 3 and 6 together to adopt a conformation similar to that found in inactive rhodopsin. Our results suggest that inactive beta(2)AR exists in equilibrium between conformations with the lock formed and the lock broken, whether or not the cocrystallized ligand is present. These findings, along with the formation of several secondary structural elements in the beta(2)AR loops during our simulations, may provide a more comprehensive picture of the inactive state of the beta-adrenergic receptors, reconciling the crystal structures with biochemical studies.