Disease Area Oncology, Novartis Institutes for Biomedical Research, CH-4002 Basel, Switzerland.
Department of Medical Biochemistry and Microbiology, Uppsala University, 751 23 Uppsala, Sweden.
Biochemistry. 2020 May 19;59(19):1804-1812. doi: 10.1021/acs.biochem.0c00217. Epub 2020 Apr 30.
The Hippo pathway is an evolutionarily conserved signaling pathway that is involved in the control of organ size and development. The TEAD transcription factors are the most downstream elements of the Hippo pathway, and their transcriptional activity is regulated via the interaction with different co-regulators such as YAP. The structure of the YAP:TEAD complex shows that YAP binds to TEAD via two distinct secondary structure elements, an α-helix and an Ω-loop, and site-directed mutagenesis experiments revealed that the Ω-loop is the "hot spot" of this interaction. While much is known about how YAP and TEAD interact with each other, little is known about the mechanism leading to the formation of a complex between these two proteins. Here we combine site-directed mutagenesis with pre-steady-state kinetic measurements to show that the association between these proteins follows an apparent one-step binding mechanism. Furthermore, linear free energy relationships and a Φ analysis suggest that binding-induced folding of the YAP α-helix to TEAD occurs independently of and before formation of the Ω-loop interface. Thus, the binding-induced folding of YAP appears not to conform to the concomitant formation of tertiary structure (nucleation-condensation) usually observed for coupled binding and folding reactions. Our findings demonstrate how a mechanism reminiscent of the classical framework (diffusion-collision) mechanism of protein folding may operate in disorder-to-order transitions involving intrinsically disordered proteins.
Hippo 通路是一条进化上保守的信号通路，参与调控器官大小和发育。TEAD 转录因子是 Hippo 通路的最下游元件，其转录活性通过与不同的共调节剂（如 YAP）相互作用来调节。YAP:TEAD 复合物的结构表明，YAP 通过两个不同的二级结构元件（α-螺旋和Ω环）与 TEAD 结合，定点突变实验表明，Ω环是这种相互作用的“热点”。虽然人们已经了解了 YAP 和 TEAD 如何相互作用，但对于导致这两种蛋白质形成复合物的机制知之甚少。在这里，我们将定点突变与预稳态动力学测量相结合，表明这两种蛋白质之间的关联遵循明显的一步结合机制。此外，线性自由能关系和Φ分析表明，YAPα-螺旋与 TEAD 的结合诱导折叠独立于形成Ω环界面之前发生。因此，YAP 的结合诱导折叠似乎不符合通常观察到的偶联结合和折叠反应中观察到的三级结构（成核-凝聚）的同时形成。我们的研究结果表明，一种类似于经典构象变化（扩散-碰撞）机制的机制如何在涉及固有无序蛋白的无序到有序转变中发挥作用。
