PDF(Pubmed)
Abstract：
Many neurotransmitter receptors activate G proteins through exchange of GDP for GTP. The intermediate nucleotide-free state has eluded characterization, due largely to its inherent instability. Here we characterize a G protein variant associated with a rare neurological disorder in humans. GαoK46E has a charge reversal that clashes with the phosphate groups of GDP and GTP. As anticipated, the purified protein binds poorly to guanine nucleotides yet retains wild-type affinity for G protein βγ subunits. In cells with physiological concentrations of nucleotide, GαoK46E forms a stable complex with receptors and Gβγ, impeding effector activation. Further, we demonstrate that the mutant can be easily purified in complex with dopamine-bound D2 receptors, and use cryo-electron microscopy to determine the structure, including both domains of Gαo, without nucleotide or stabilizing nanobodies. These findings reveal the molecular basis for the first committed step of G protein activation, establish a mechanistic basis for a neurological disorder, provide a simplified strategy to determine receptor-G protein structures, and a method to detect high affinity agonist binding in cells.
摘要：
许多神经递质受体通过将GDP交换为GTP来激活G蛋白。中间无核苷酸状态已被表征，主要是由于其固有的不稳定性。在这里，我们描述了与人类罕见神经系统疾病相关的G蛋白变体。GαoK46E具有与GDP和GTP的磷酸盐基团冲突的电荷逆转。如预期,纯化的蛋白质与鸟嘌呤核苷酸的结合能力较差，但仍保留对G蛋白βγ亚基的野生型亲和力。在具有生理核苷酸浓度的细胞中，GαoK46E与受体和Gβγ形成稳定的复合物，阻碍效应器激活。Further,我们证明了突变体可以很容易地与多巴胺结合的D2受体复合纯化，并使用低温电子显微镜来确定结构，包括Gαo的两个域，没有核苷酸或稳定的纳米抗体。这些发现揭示了G蛋白激活第一步的分子基础，为神经系统疾病建立机制基础，提供了一种简化的策略来确定受体G蛋白结构，和检测细胞中高亲和力激动剂结合的方法。
