Center for Biomolecular Structure and Dynamics, The University of Montana, Missoula, Montana, United States of America.
PLoS One. 2011;6(8):e23197. doi: 10.1371/journal.pone.0023197. Epub 2011 Aug 11.
Heterotrimeric G protein α subunits are activated upon exchange of GDP for GTP at the nucleotide binding site of Gα, catalyzed by guanine nucleotide exchange factors (GEFs). In addition to transmembrane G protein-coupled receptors (GPCRs), which act on G protein heterotrimers, members of the family cytosolic proteins typified by mammalian Ric-8A are GEFs for Gi/q/12/13-class Gα subunits. Ric-8A binds to Gα•GDP, resulting in the release of GDP. The Ric-8A complex with nucleotide-free Gαi1 is stable, but dissociates upon binding of GTP to Gαi1. To gain insight into the mechanism of Ric-8A-catalyzed GDP release from Gαi1, experiments were conducted to characterize the physical state of nucleotide-free Gαi1 (hereafter referred to as Gαi1[ ]) in solution, both as a monomeric species, and in the complex with Ric-8A. We found that Ric-8A-bound, nucleotide-free Gαi1 is more accessible to trypsinolysis than Gαi1•GDP, but less so than Gαi1[ ] alone. The TROSY-HSQC spectrum of [(15)N]Gαi1[ ] bound to Ric-8A shows considerable loss of peak intensity relative to that of [(15)N]Gαi1•GDP. Hydrogen-deuterium exchange in Gαi1[ ] bound to Ric-8A is 1.5-fold more extensive than in Gαi1•GDP. Differential scanning calorimetry shows that both Ric-8A and Gαi1•GDP undergo cooperative, irreversible unfolding transitions at 47° and 52°, respectively, while nucleotide-free Gαi1 shows a broad, weak transition near 35°. The unfolding transition for Ric-8A:Gαi1[ ] is complex, with a broad transition that peaks at 50°, suggesting that both Ric-8A and Gαi1[ ] are stabilized within the complex, relative to their respective free states. The C-terminus of Gαi1 is shown to be a critical binding element for Ric-8A, as is also the case for GPCRs, suggesting that the two types of GEF might promote nucleotide exchange by similar mechanisms, by acting as chaperones for the unstable and dynamic nucleotide-free state of Gα.
三聚体 G 蛋白 α 亚基在 Gα 上的核苷酸结合位点处通过鸟嘌呤核苷酸交换因子（GEFs）将 GDP 交换为 GTP 而被激活。除了作用于 G 蛋白异三聚体的跨膜 G 蛋白偶联受体（GPCRs）之外，以哺乳动物 Ric-8A 为代表的细胞质蛋白家族成员也是 Gi/q/12/13 类 Gα 亚基的 GEF。Ric-8A 与 GDP 结合的 Gα•GDP 结合，导致 GDP 释放。Ric-8A 与无核苷酸的 Gαi1 复合物是稳定的，但在 GTP 与 Gαi1 结合时会解离。为了深入了解 Ric-8A 催化的 GDP 从 Gαi1 释放的机制，进行了实验以表征溶液中无核苷酸的 Gαi1（以下简称 Gαi1[ ]）的物理状态，无论是作为单体物种，还是与 Ric-8A 的复合物。我们发现，与 Ric-8A 结合的无核苷酸 Gαi1 比 Gαi1•GDP 更容易被胰蛋白酶水解，但比单独的 Gαi1[ ]更难。与 Ric-8A 结合的 [(15)N]Gαi1[ ]的 TROSY-HSQC 谱与 [(15)N]Gαi1•GDP 的谱相比，峰强度明显降低。与 Ric-8A 结合的 Gαi1[ ]中的氢氘交换比 Gαi1•GDP 扩展了 1.5 倍。差示扫描量热法显示，Ric-8A 和 Gαi1•GDP 分别在 47°和 52°处均经历协同、不可逆的展开转变，而无核苷酸的 Gαi1 则在 35°附近显示出宽而弱的转变。Ric-8A:Gαi1[ ]的展开转变很复杂，具有 50°的宽峰，表明 Ric-8A 和 Gαi1[ ]在复合物中相对于其各自的游离状态都得到了稳定。Gαi1 的 C 末端被证明是 Ric-8A 的关键结合元件，与 GPCR 也是如此，这表明这两种类型的 GEF 可能通过类似的机制促进核苷酸交换，充当 Gα 不稳定和动态的无核苷酸状态的伴侣。
