A RasGTP-Induced Conformational Change in C-RAF Is Essential for Accurate Molecular Recognition
A RasGTP-Induced Conformational Change in C-RAF Is Essential for Accurate Molecular Recognition
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DOI：
10.1016/j.bpj.2009.05.048
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发表时间：
2009-09-02
影响因子：
3.4
通讯作者：
Sako, Yasushi
中科院分区：
文献类型：
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作者：
Hibino, Kayo;Shibata, Tatsuo;Sako, Yasushi
Ras-RAF信号的失调与许多类型的人类癌症相关。然而，Ras和RAF的相互分子识别的动力学和动力学特征仍然未知。在这里，我们开发了一种技术，用于成像单对荧光共振能量转移活细胞中，并将此技术耦合到单分子动力学分析，以研究C-RAF（RAF的一种亚型）分子如何区分活性形式的Ras（RasGTP）从非活性形式（RasGDP）。C-RAF的功能片段含有Ras结合结构域没有检测到开关的Ras活性在活细胞中的C-RAF一样有效。单分子分析表明，RasGDP与闭合构象的C-RAF缔合，而C-RAF与RasGTP缔合立即触发开放RAF构象，这诱导C-RAF与RasGTP之间的有效相互作用。从封闭的C-RAF到开放形式的自发构象变化很少发生在静止细胞中。C-RAF的构象变化对Ras-RAF的分子识别非常重要，缺乏构象变化的C-RAF突变体不能区分RasGDP和RasGTP。操纵效应分子的构象是RasGTP的一个新发现的功能。
The dysregulation of Ras-RAF signaling is associated with many types of human cancer. However, the kinetic and dynamic features of the mutual molecular recognition of Ras and RAF remain unknown. Here, we developed a technique for imaging single-pair fluorescence resonance energy transfer in living cells, and coupled this technique to single-molecule kinetic analysis to investigate how C-RAF (a subtype of RAF) molecules distinguish the active form of Ras (RasGTP) from the inactive form (RasGDP). Functional fragments of C-RAF containing the Ras-binding domains did not detect the switch in Ras activity in living cells as efficiently as did C-RAF. Single-molecule analysis showed that RasGDP associates with closed-conformation C-RAF, whereas the association of C-RAF with RasGTP immediately triggers the open RAF conformation, which induces an effective interaction between C-RAF and RasGTP. Spontaneous conformational changes from closed C-RAF to the open form rarely occur in quiescent cells. The conformational change in C-RAF is so important to Ras-RAF molecular recognition that C-RAF mutants lacking the conformational change cannot distinguish between RasGDP and RasGTP. The manipulation of the conformation of an effector molecule is a newly identified function of RasGTP.