Department of Chemistry, University of Florida, Gainesville, Florida, USA.
Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden.
J Biol Chem. 2024 Aug;300(8):107497. doi: 10.1016/j.jbc.2024.107497. Epub 2024 Jun 24.
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.
通过核苷酸交换激活 G 蛋白，启动对生命过程至关重要的细胞内信号级联反应。在正常情况下，核苷酸交换受 G 蛋白 -G 蛋白偶联受体复合物形成的调节。G 蛋白的 Gα亚基中的单点突变绕过这种相互作用，导致功能丧失或组成型功能获得，这与多种疾病的发生密切相关。尽管 Gα突变在疾病发病机制中具有公认的意义，但大多数变体的结构信息仍然缺乏，这可能是由于固有蛋白质动力学给晶体学带来了挑战。为了解决这个问题，我们利用综合光谱学和计算方法对刺激型 G 蛋白 Gα中最常观察到的七种且具有临床相关性的突变进行结构表征。先前提出的 Gα激活变构模型将核苷酸结合口袋中的结构变化与开关区之间功能重要变化的相互作用联系起来。我们通过整合来自变温 CD 光谱学的数据来研究 Gα中的这种变构连接，该方法测量了全局蛋白质结构和稳定性的变化，以及分子动力学模拟，该方法观察了 Gα开关区之间相互作用网络的变化。此外，饱和转移差异 NMR 光谱学被应用于观察核苷酸与核苷酸结合位点内残基相互作用的变化。这些数据使我们能够测试关于 Gα突变如何导致功能丧失或获得的预测，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提出更细致的分类机制，以解释 Gα功能获得和功能丧失突变的潜在机制。
