ALG-2 interacts with the amino-terminal domain of annexin XI in a Ca2+-dependent manner
ALG-2 interacts with the amino-terminal domain of annexin XI in a Ca2+-dependent manner
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DOI：
10.1006/bbrc.2002.6600
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发表时间：
2002-03-15
影响因子：
3.1
通讯作者：
Maki, M
中科院分区：
文献类型：
--
作者：
Satoh, H;Shibata, H;Maki, M
凋亡相关蛋白ALG-2是一种Ca2+结合蛋白，属于penta-EF-hand蛋白家族。ALG-2与另一种五ef -hand蛋白peflin形成同型二聚体、异源二聚体，并与其相互作用的蛋白AIP1或Alix形成复合物。以人ALG-2为诱饵，通过酵母双杂交筛选，分离到一个新的与ALG-2相互作用的蛋白cDNA，该蛋白为膜联蛋白XI。缺失分析显示，ALG-2与annexin XI （AnxN）的n端结构域相互作用，该结构域的氨基酸序列与AIP1/Alix的c端区域相似。利用重组生物素标记的ALG-2与AnxN的谷胱甘肽s -转移酶（GST）融合蛋白，通过ALG-2覆盖法和表面等离子体共振（SPR）生物传感器实时相互作用分析，分析其直接相互作用。解离常数（K-d）估计约为70 nM。在疏水荧光探针2-对甲苯基萘-6-磺酸盐（TNS）存在下，与GST-AnxN混合抑制了ALG-2的Ca2+依赖性荧光变化，表明AnxN中富含Pro/Gly/Tyr/ ala的疏水区域掩盖了ALG-2的Ca2+依赖性暴露疏水表面。(C) 2002 Elsevier Science （USA）。
The apoptosis-linked protein ALG-2 is a Ca2+ binding protein that belongs to the penta-EF-hand protein family. ALG-2 forms a homodimer, a heterodimer with another penta-EF-hand protein, peflin, and a complex with its interacting protein, named AIP1 or Alix. By yeast two-hybrid screening using human ALG-2 as bait, we isolated a cDNA of a novel ALG-2-interacting protein, which turned out to be annexin XI. Deletion analysis revealed that ALG-2 interacted with the N-terminal domain of annexin XI (AnxN), which has an amino acid sequence similar to that of the C-terminal region of AIP1/Alix. Using recombinant biotin-tagged ALG-2 and the glutathione S-transferase (GST) fusion protein of AnxN, the direct interaction was analyzed by an ALG-2 overlay assay and by real-time interaction analysis with a surface plasmon resonance (SPR) biosensor. The dissociation constant (K-d) was estimated to be approximately 70 nM. The Ca2+-dependent fluorescence change of ALG-2 in the presence of the hydrophobicity fluorescent probe 2-p-toluidinylnaphthalene-6-sulfonate (TNS) was inhibited by mixing with GST-AnxN, suggesting that the Pro/Gly/Tyr/Ala-rich hydrophobic region in AnxN masked the Ca2+-dependently exposed hydrophobic surface of ALG-2. (C) 2002 Elsevier Science (USA).