Heterotrimeric guanine nucleotide-binding proteins (G proteins) are composed of α, β, and γ subunits, and Gα has a GDP/GTP-binding pocket. When a guanine nucleotide exchange factor (GEF) interacts with Gα, GDP is released, and GTP interacts to Gα. The GTP-bound activated Gα dissociates from GEF and Gβγ, mediating the induction of various intracellular signaling pathways. Depending on the sequence similarity and cellular function, Gα subunits are subcategorized into four subfamilies: Gαi/o, Gαs, Gαq/11, and Gα12/13. Although the Gαi/o subtype family proteins, Gαi3 and GαoA, share similar sequences and functions, they differ in their GDP/GTP turnover profiles, with GαoA possessing faster rates than Gαi3. The structural factors responsible for these differences remain unknown. In this study, we employed hydrogen/deuterium exchange mass spectrometry and mutational studies to investigate the factors responsible for these functional differences. The Gα subunit consists of a Ras-like domain (RD) and an α-helical domain (AHD). The RD has GTPase activity and receptor-binding and effector-binding regions; however, the function of the AHD has not yet been extensively studied. In this study, the chimeric construct containing the RD of Gαi3 and the AHD of GαoA showed a GDP/GTP turnover profile similar to that of GαoA, suggesting that the AHD is the major regulator of the GDP/GTP turnover profile. Additionally, site-directed mutagenesis revealed the importance of the N-terminal part of αA and αA/αB loops in the AHD for the GDP/GTP exchange. These results suggest that the AHD regulates the nucleotide exchange rate within the Gα subfamily.

中文翻译：

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Gi/oα亚基α-螺旋结构域对GDP/GTP转换的影响

异三聚体鸟嘌呤核苷酸结合蛋白 (G 蛋白) 由 α、β 和 γ 亚基组成，Gα 具有 GDP/GTP 结合口袋。当鸟嘌呤核苷酸交换因子 (GEF) 与 Gα 相互作用时，GDP 被释放，GTP 与 Gα 相互作用。GTP 结合的活化 Gα 从 GEF 和 Gβγ 解离，介导各种细胞内信号通路的诱导。根据序列相似性和细胞功能，Gα 亚基分为四个亚科：Gαi/o、Gαs、Gαq/11 和 Gα12/13。尽管 Gαi/o 亚型家族蛋白 Gαi3 和 GαoA 具有相似的序列和功能，但它们的 GDP/GTP 周转谱不同，GαoA 比 Gαi3 具有更快的速率。造成这些差异的结构因素仍然未知。在这项研究中，我们采用氢/氘交换质谱和突变研究来研究导致这些功能差异的因素。Gα 亚基由一个 Ras 样结构域 (RD) 和一个 α-螺旋结构域 (AHD) 组成。RD具有GTP酶活性和受体结合区和效应结合区；然而，AHD 的功能尚未得到广泛研究。在本研究中，包含 Gαi3 的 RD 和 GαoA 的 AHD 的嵌合构建体显示出与 GαoA 相似的 GDP/GTP 周转曲线，表明 AHD 是 GDP/GTP 周转曲线的主要调节因子。此外，定点诱变揭示了 AHD 中 αA 和 αA/αB 环的 N 端部分对 GDP/GTP 交换的重要性。这些结果表明 AHD 调节 Gα 亚家族内的核苷酸交换率。