Heterotrimeric guanine nucleotide-binding proteins (G proteins) are composed of α, β, and γ subunits. Gα switches between guanosine diphosphate (GDP)-bound inactive and guanosine triphosphate (GTP)-bound active states, and Gβγ interacts with the GDP-bound state. The GDP-binding regions are composed of two sites: the phosphate-binding and guanine-binding regions. The turnover of GDP and GTP is induced by guanine nucleotide-exchange factors (GEFs), including G protein-coupled receptors (GPCRs), Ric8A, and GIV/Girdin. However, the key structural factors for stabilizing the GDP-bound state of G proteins and the direct structural event for GDP release remain unclear. In this study, we investigated structural factors affecting GDP release by introducing point mutations in selected, conserved residues in Gαi3. We examined the effects of these mutations on the GDP/GTP turnover rate and the overall conformation of Gαi3 as well as the binding free energy between Gαi3 and GDP. We found that dynamic changes in the phosphate-binding regions are an immediate factor for the release of GDP.

异源三聚鸟嘌呤核苷酸结合蛋白（G 蛋白）由 α、β 和 γ 亚基组成。Gα 在鸟苷二磷酸 (GDP) 结合的非活性状态和鸟苷三磷酸 (GTP) 结合的活性状态之间切换，并且 Gβγ 与 GDP 结合状态相互作用。GDP结合区由两个位点组成：磷酸盐结合区和鸟嘌呤结合区。GDP 和 GTP 的周转由鸟嘌呤核苷酸交换因子 (GEF) 诱导，包括 G 蛋白偶联受体 (GPCR)、Ric8A 和 GIV/Girdin。然而，稳定 G 蛋白 GDP 结合状态的关键结构因素和 GDP 释放的直接结构事件仍不清楚。在这项研究中，我们通过在 Gαi3 中选定的保守残基中引入点突变来研究影响 GDP 释放的结构因素。我们检查了这些突变对 GDP/GTP 周转率和 Gαi3 的整体构象以及 Gαi3 和 GDP 之间的结合自由能的影响。我们发现磷酸盐结合区域的动态变化是 GDP 释放的直接因素。