NF2蛋白518位丝氨酸磷酸化状态对其分子内相互作用机制的结构生物学及生物化学研究

 目的  揭示NF2 (neurofibromatosis type 2)蛋白518位丝氨酸的磷酸化如何对其结构与功能进行调节。  方法  解析野生型NF2^WT(无模拟磷酸化突变)和突变型NF2^S518D(模拟518位丝氨酸被磷酸化的突变体)蛋白的晶体结构,进一步利用体外Pull-down实验及荧光共振能量转移(fluorescence resonance energy transfer,FRET)实验检测了NF2的分子内相互作用。  结果  由于NF2的C-端在结晶过程中降解,未能观察到NF2^WT和NF2^S518D两种蛋白质构象的差异,结构分析发现NF2的N-端FERM(Four-point one,Ezrin,Radixin,Moesin)结构域与已有结构报道的FERM结构域具有相似的空间构象。体外Pull-down实验证明生化实验结果提示,相对NF2^WTC-端蛋白质而言,NF2^S518D的C-端蛋白质与NF2的N-端蛋白质结合更强。FRET实验在波长525 nm处,检测到全长NF2^S518D比全长NF2^WT产生更强的FRET信号。  结论  野生型NF2^WT处于“开放”状态而模拟磷酸化的突变型NF2^S518D处于“闭合”状态。

Structural and biochemical insights into the mechanism for intramolecular interaction in NF2 regulated by phosphorylation of serine 518

Objective  To reveal the structural and biochemical mechanism for intramolecular interaction in neurofibromatosis type 2 (NF2) regulated by phosphorylation of serine 518.  Methods  We resolved the crystal structures of wild-type NF2 (NF2^WT) without mimic phosphorylation mutant and a phosphorylation mimic mutant protein (NF2^S518D). Further, in vitro Pull-down assay and fluorescence resonance energy transfer (FRET) assay were executed to detect the intramolecular interaction of NF2.  Results  No obvious conformational difference was observed between the structures of NF2^WT and NF2^S518D because of the unexpected C-terminal degradation of NF2 during crystallization. Structural analysis indicated that the FERM (Four-point one,Ezrin,Radixin,Moesin) domains of NF2^WT and NF2^S518D adopted similar conformation to that of the reported FERM domains. In vitro Pull-down assay showed stronger intramolecular interaction between the N-terminus and C-terminus of NF2^S518D, compared with that within NF2WT. In FRET assay, NF2^S518D generated stronger FRET signal than NF2^WT  at the wavelength of 525 nm.  Conclusions  The NF2^WT protein adopted a closed form while the wild phosphorylation mimic NF2^S518D mutant prefers an open form.