光凝素(Pdc),一个高度保守的磷蛋白参与调节视网膜光转导级联,转录控制,和血压的调节,以依赖磷酸化的方式控制,包括与14-3-3蛋白的结合。然而,这种调节的分子机制在很大程度上是未知的。这里,使用小角度X射线散射研究了Pdc的溶液结构及其与14-3-3蛋白的相互作用,时间分辨荧光光谱,和氢-氘交换耦合到质谱。14-3-3蛋白二聚体使用其中央通道内外的表面与Pdc相互作用。Pdc的N端结构域,磷酸化位点和14-3-3结合基序都位于其中,是一种内在无序的蛋白质,在与14-3-3结合后,在几个区域降低其灵活性,而不会发生剧烈的无序到有序的转变。我们的数据还表明Pdc的C末端结构域通过参与Gtβγ结合的区域与14-3-3二聚体的外表面相互作用。总之,我们显示14-3-3蛋白与磷酸化Pdc的N端和C端Gtβγ结合界面相互作用并在空间上闭塞,从而为Pdc功能的14-3-3依赖性抑制提供了机械解释。
Phosducin (Pdc), a highly conserved phosphoprotein involved in the regulation of retinal phototransduction cascade, transcriptional control, and modulation of blood pressure, is controlled in a phosphorylation-dependent manner, including the binding to the 14-3-3 protein. However, the molecular mechanism of this regulation is largely unknown. Here, the solution structure of Pdc and its interaction with the 14-3-3 protein were investigated using small angle x-ray scattering, time-resolved fluorescence spectroscopy, and hydrogen-deuterium exchange coupled to mass spectrometry. The 14-3-3 protein dimer interacts with Pdc using surfaces both inside and outside its central channel. The N-terminal domain of Pdc, where both phosphorylation sites and the 14-3-3-binding motifs are located, is an intrinsically disordered protein that reduces its flexibility in several regions without undergoing dramatic disorder-to-order transition upon binding to 14-3-3. Our data also indicate that the C-terminal domain of Pdc interacts with the outside surface of the 14-3-3 dimer through the region involved in Gtβγ binding. In conclusion, we show that the 14-3-3 protein interacts with and sterically occludes both the N- and C-terminal Gtβγ binding interfaces of phosphorylated Pdc, thus providing a mechanistic explanation for the 14-3-3-dependent inhibition of Pdc function.