1] Molecular Structure and Function Program, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada [2] Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Molecular Structure and Function Program, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada.
Nature. 2015 Mar 5;519(7541):106-9. doi: 10.1038/nature13999. Epub 2014 Dec 22.
Intrinsically disordered proteins play important roles in cell signalling, transcription, translation and cell cycle regulation. Although they lack stable tertiary structure, many intrinsically disordered proteins undergo disorder-to-order transitions upon binding to partners. Similarly, several folded proteins use regulated order-to-disorder transitions to mediate biological function. In principle, the function of intrinsically disordered proteins may be controlled by post-translational modifications that lead to structural changes such as folding, although this has not been observed. Here we show that multisite phosphorylation induces folding of the intrinsically disordered 4E-BP2, the major neural isoform of the family of three mammalian proteins that bind eIF4E and suppress cap-dependent translation initiation. In its non-phosphorylated state, 4E-BP2 interacts tightly with eIF4E using both a canonical YXXXXLΦ motif (starting at Y54) that undergoes a disorder-to-helix transition upon binding and a dynamic secondary binding site. We demonstrate that phosphorylation at T37 and T46 induces folding of residues P18-R62 of 4E-BP2 into a four-stranded β-domain that sequesters the helical YXXXXLΦ motif into a partly buried β-strand, blocking its accessibility to eIF4E. The folded state of pT37pT46 4E-BP2 is weakly stable, decreasing affinity by 100-fold and leading to an order-to-disorder transition upon binding to eIF4E, whereas fully phosphorylated 4E-BP2 is more stable, decreasing affinity by a factor of approximately 4,000. These results highlight stabilization of a phosphorylation-induced fold as the essential mechanism for phospho-regulation of the 4E-BP:eIF4E interaction and exemplify a new mode of biological regulation mediated by intrinsically disordered proteins.
无定形蛋白质在细胞信号转导、转录、翻译和细胞周期调控中发挥着重要作用。尽管它们缺乏稳定的三级结构，但许多无定形蛋白质在与伴侣结合时会发生无序到有序的转变。同样，一些折叠蛋白利用受调控的有序到无序的转变来介导生物学功能。原则上，无定形蛋白质的功能可以通过导致结构变化的翻译后修饰来控制，例如折叠，尽管尚未观察到这一点。在这里，我们表明多位点磷酸化诱导主要神经同工型 4E-BP2 的折叠，该同工型是结合 eIF4E 并抑制帽依赖性翻译起始的三种哺乳动物蛋白家族的主要神经同工型。在非磷酸化状态下，4E-BP2 使用结合时发生无序到螺旋转变的典型 YXXXXLΦ 基序（从 Y54 开始）和动态二级结合位点与 eIF4E 紧密相互作用。我们证明，T37 和 T46 的磷酸化诱导 4E-BP2 的残基 P18-R62 折叠成一个四链β-结构域，将螺旋 YXXXXLΦ 基序封闭在部分埋藏的β-链中，阻止其与 eIF4E 的可及性。pT37pT46 4E-BP2 的折叠状态是弱稳定的，与 eIF4E 结合时亲和力降低 100 倍，导致有序到无序的转变，而完全磷酸化的 4E-BP2 更稳定，亲和力降低约 4000 倍。这些结果突出了磷酸化诱导折叠的稳定性作为磷酸化调节 4E-BP:eIF4E 相互作用的关键机制，并举例说明了一种新的由无定形蛋白质介导的生物调节模式。
