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

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

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NF2WT(无模拟磷酸化突变)和突变型NF2S518D(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NF2WT和NF2S518D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NF2WTC-端蛋白质而言,NF2S518D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NF2S518D比全长NF2WT产生更强的FRET信号。结论野生型NF2WT处于"开放"状态而模拟磷酸化的突变型NF2S518D处于"闭合"状态。

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 （NF2WT） without mimic phosphorylation mutant and a phosphorylation mimic mutant protein （NF2S518D ）. 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 NF2WT and NF2S518D 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 NF2wT and NF2S518D 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 NF2s518D, compared with that within NF2wT. In FRET assay, NF2s518D generated stronger FRET signal than NF2wT at the wavelength of 525 nm. Conclusions The NF2WT protein adopted a closed form while the wild phosphorylation mimic NF2s518D mutant prefers an open form.