Activation of G proteins through nucleotide exchange initiates intracellular signaling cascades essential for life processes. Under normal conditions, nucleotide exchange is regulated by the formation of G protein–G protein–coupled receptor complexes. Single point mutations in the Gα subunit of G proteins bypass this interaction, leading to loss of function or constitutive gain of function, which is closely linked with the onset of multiple diseases. Despite the recognized significance of Gα mutations in disease pathology, structural information for most variants is lacking, potentially due to inherent protein dynamics that pose challenges for crystallography. To address this, we leveraged an integrative spectroscopic and computational approach to structurally characterize seven of the most frequently observed and clinically relevant mutations in the stimulatory Gα subunit, Gα. A previously proposed allosteric model of Gα activation linked structural changes in the nucleotide-binding pocket with functionally important changes in interactions between switch regions. We investigated this allosteric connection in Gα by integrating data from variable temperature CD spectroscopy, which measured changes in global protein structure and stability, and molecular dynamics simulations, which observed changes in interaction networks between Gα switch regions. Additionally, saturation-transfer difference NMR spectroscopy was applied to observe changes in nucleotide interactions with residues within the nucleotide binding site. These data have enabled testing of predictions regarding how mutations in Gα result in loss or gain of function and evaluation of proposed structural mechanisms. The integration of experimental and computational data allowed us to propose a more nuanced classification of mechanisms underlying Gα gain-of-function and loss-of-function mutations.

中文翻译：

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以综合生物物理方法为特征的疾病相关 GαS 变体内的相互作用网络


通过核苷酸交换激活 G 蛋白会启动生命过程所必需的细胞内信号级联。在正常情况下，核苷酸交换是通过 G 蛋白-G 蛋白偶联受体复合物的形成来调节的。 G蛋白Gα亚基的单点突变绕过了这种相互作用，导致功能丧失或组成性功能获得，这与多种疾病的发病密切相关。尽管 Gα 突变在疾病病理学中的重要性已得到公认，但大多数变异的结构信息仍然缺乏，这可能是由于固有的蛋白质动力学给晶体学带来了挑战。为了解决这个问题，我们利用综合光谱和计算方法对刺激性 Gα 亚基 Gα 中最常见和临床相关的七个突变进行结构表征。先前提出的 Gα 激活变构模型将核苷酸结合口袋的结构变化与开关区域之间相互作用的功能性重要变化联系起来。我们通过整合变温 CD 光谱（测量整体蛋白质结构和稳定性的变化）和分子动力学模拟（观察 Gα 开关区域之间相互作用网络的变化）的数据来研究 Gα 中的这种变构连接。此外，应用饱和转移差核磁共振波谱来观察核苷酸与核苷酸结合位点内残基相互作用的变化。这些数据使得能够测试有关 Gα 突变如何导致功能丧失或增强的预测，并评估所提出的结构机制。 实验和计算数据的整合使我们能够对 Gα 功能获得和功能丧失突变的机制提出更细致的分类。