Structural Determinants for Light-Dependent Membrane Binding of a Photoswitchable Polybasic Domain
Structural Determinants for Light-Dependent Membrane Binding of a Photoswitchable Polybasic Domain
复制标题
光可切换多碱域的光依赖性膜结合的结构决定因素。
DOI：
10.1021/acssynbio.0c00571
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发表时间：
2021-03-09
影响因子：
4.7
通讯作者：
Zhu, Lei
中科院分区：
文献类型：
--
作者：
Li, Ling;He, Lian;Zhu, Lei
OptoPB是一种将Rit1的磷脂酰肌醇(PI)结合的多碱基结构域(RIT-PB)融合到光反应光氧电压(LOV)结构域上的光遗传工具。在蓝光激发下，OptoPB选择性且可逆地与质膜结合，在黑暗中，它释放回细胞质。然而，光学调控和脂质识别的分子机制仍不清楚。在这里，使用核磁共振(核磁共振)光谱、脂质体下拉试验和表面等离子体共振(SPR)，我们发现OptoPB与含有二或三磷酸化磷脂酰肌醇的膜模拟物结合，特别是磷脂酰肌醇4，5-二磷酸(PI(4，5)P2)，这是一种主要位于真核细胞PM中的酸性磷脂在黑暗中，空间位阻阻止了这种蛋白质-膜相互作用，而470 nm蓝光照射则激活了这种相互作用。核磁共振滴定和定点突变表明，阳离子和疏水Rit-PB残基对膜相互作用都是必不可少的，表明OptoPB通过一种特殊的PI(4，5)P2依赖机制与膜结合。
OptoPB is an optogenetic tool engineered by fusion of the phosphoinositide (PI)-binding polybasic domain of Rit1 (Rit-PB) to a photoreactive light-oxygen-voltage (LOV) domain. OptoPB selectively and reversibly binds the plasma membrane (PM) under blue light excitation, and in the dark, it releases back to the cytoplasm. However, the molecular mechanism of optical regulation and lipid recognition is still unclear. Here using nuclear magnetic resonance (NMR) spectroscopy, liposome pulldown assay, and surface plasmon resonance (SPR), we find that OptoPB binds to membrane mimetics containing di- or triphosphorylated phosphatidylinositols, particularly phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), an acidic phospholipid predominantly located in the eukaryotic PM. In the dark, steric hindrance prevented this protein-membrane interaction, while 470 nm blue light illumination activated it. NMR titration and site-directed mutagenesis revealed that both cationic and hydrophobic Rit-PB residues are essential to the membrane interaction, indicating that OptoPB binds the membrane via a specific PI(4,5)P2-dependent mechanism.