Binding of the PX domain of p47phox to phosphatidylinositol 3,4-bisphosphate and phosphatidic acid is masked by an intramolecular interaction
Binding of the PX domain of p47phox to phosphatidylinositol 3,4-bisphosphate and phosphatidic acid is masked by an intramolecular interaction
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DOI：
10.1093/emboj/cdf519
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发表时间：
2002-10-01
期刊：
影响因子：
11.4
通讯作者：
Williams, RL
中科院分区：
文献类型：
--
作者：
Karathanassis, D;Stahelin, RV;Williams, RL
P47(Phox)是吞噬细胞NADPH氧化酶激活所需的关键胞浆亚基。P47(Phox)Px结构域的X射线结构显示，在膜结合表面有两个不同的碱性口袋，每个口袋都被硫酸盐占据。这两个口袋有不同的特异性：一个优先结合磷脂酰肌醇3，4-二磷酸[PtdIns(3，4)P-2]，类似于p40(Phox)的磷脂酰肌醇3-磷酸(PtdIns3P)结合口袋，而另一个结合阴离子磷脂，如磷脂酸(PtdOH)或磷脂酰丝氨酸。PtdOH对这个第二位点的偏好可能与先前观察到的PtdOH激活NADPH氧化酶有关。同时占据两个磷脂结合口袋，从根本上增加了膜的亲和力。值得注意的是，全长P47(Phox)的测量表明，Px结构域与C-末端SH3结构域(C-SH3)的分子内结合掩盖了膜的相互作用。C-SH3(W263R)上的定点突变或P47(Phox)的模拟磷酸化形式[Ser(303,304,328,359,370)Glu]会导致从封闭构象到开放构象的转变，这种构象与膜结合的亲和力比分离的Px结构域更大。
p47(phox) is a key cytosolic subunit required for activation of phagocyte NADPH oxidase. The X-ray structure of the p47(phox) PX domain revealed two distinct basic pockets on the membrane-binding surface, each occupied by a sulfate. These two pockets have different specificities: one preferentially binds phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P-2] and is analogous to the phophatidylinositol 3-phosphate (PtdIns3P)-binding pocket of p40(phox), while the other binds anionic phospholipids such as phosphatidic acid (PtdOH) or phosphatidylserine. The preference of this second site for PtdOH may be related to previously observed activation of NADPH oxidase by PtdOH Simultaneous occupancy of the two phospholipid-binding pockets radically increases membrane affinity. Strikingly, measurements for full-length p47(phox) show that membrane interaction by the PX domain is masked by an intramolecular association with the C-terminal SH3 domain (C-SH3). Either a site-specific mutation in C-SH3 (W263R) or a mimic of the phosphorylated form of p47(phox) [Ser(303, 304, 328, 359, 370)Glu] cause a transition from a closed to an open conformation that binds membranes with a greater affinity than the isolated PX domain.