Two Closed Conformations of CRAF Require the 14-3-3 Binding Motifs and Cysteine-Rich Domain to be Intact in Live Cells
Two Closed Conformations of CRAF Require the 14-3-3 Binding Motifs and Cysteine-Rich Domain to be Intact in Live Cells
复制标题
CRAF 的两个闭合构象需要 14-3-3 结合基序和富含半胱氨酸的结构域在活细胞中保持完整
DOI：
10.1016/j.jmb.2023.167989
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发表时间：
2023
影响因子：
5.6
通讯作者：
Sako Yasushi
中科院分区：
文献类型：
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作者：
Okamoto Kenji;Sako Yasushi
蛋白快速加速纤维肉瘤（RAF）是细胞信号转导系统中膜蛋白RAS下游的一种激酶。在RAF的结构中，N端和c端结构域由一个柔性连接体连接。RAF的开/闭动力学和二聚化被认为调节其活性，尽管这些构象的细节尚不清楚，特别是在活细胞中。在这项工作中，我们使用交替激光激发来测量活HeLa细胞的胞质CRAF，并获得了结构态的单分子Förster共振能量转移（smFRET）分布。我们比较了野生型(WT)-CRAF在表皮生长因子（EGF）刺激前后的结果，包括14-3-3结合位点和富含半胱氨酸结构域的突变，以及n端截断。采用三种beta分布进行全局拟合，分析了全长craft的smFRET分布。我们的研究结果表明，14-3-3二聚体结合到单个CRAF分子的两个位点上，诱导形成自抑制封闭构象。有两种封闭构象，大多数wt - craft采用。这两种构象对EGF刺激表现出不同的反应性。
The protein rapidly accelerated fibrosarcoma (RAF) is a kinase downstream of the membrane protein RAS in the cellular signal transduction system. In the structure of RAF, the N- and C-terminus domains are connected with a flexible linker. The open/close dynamics and dimerization of RAF are thought to regulate its activity, although the details of these conformations are unknown, especially in live cells. In this work, we used alternating laser excitation to measure cytosolic CRAF in live HeLa cells and obtained single-molecule Förster resonance energy transfer (smFRET) distributions of the structural states. We compared the results for wild-type (WT)-CRAF before and after epidermal growth factor (EGF) stimulation, with mutations of the 14-3-3 binding sites and cysteine-rich domain, and an N-terminus truncation. The smFRET distributions of full-length CRAFs were analyzed by global fitting with three beta distributions. Our results suggested that a 14-3-3 dimer bound to two sites on a single CRAF molecule and induced the formation of the autoinhibitory closed conformation. There were two closed conformations, which the majority of WT-CRAF adopted. These two conformations showed different responsiveness to EGF stimulation.