Electrostatic Interaction between Oxysterol-binding Protein and VAMP-associated Protein A Revealed by NMR and Mutagenesis Studies
Electrostatic Interaction between Oxysterol-binding Protein and VAMP-associated Protein A Revealed by NMR and Mutagenesis Studies
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DOI：
10.1074/jbc.m109.082602
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发表时间：
2010-04-23
影响因子：
4.8
通讯作者：
Kojima, Chojiro
中科院分区：
文献类型：
--
作者：
Furuita, Kyoko;Jee, JunGoo;Kojima, Chojiro
氧甾醇结合蛋白 (OSBP) 是胆固醇和氧甾醇的胞质受体，通过与完整内质网蛋白 VAMP 相关蛋白-A (VAP-A) 的细胞质主要精子蛋白 (MSP) 结构域结合而被募集到内质网，这是刺激鞘磷脂合成所必需的过程 由25-羟基胆固醇。为了描述VAP-A和OSBP之间的相互作用机制，我们通过NMR确定了VAP-A MSP结构域（VAP-A(MSP)）和包含VAP-A结合基序FFAT（OSBPF）的OSBP片段之间的复杂结构。该解决方案结构解释了 FFAT 基序中六个保守残基中的五个是稳定复合物形成所必需的，而五个中的三个，包括三个关键的分子间静电相互作用，之前没有得到解释。通过将NMR弛豫和滴定、等温滴定量热法以及诱变实验与结构信息相结合，我们进一步阐明了FFAT基序的详细作用以及VAP-A(MSP)、OSBPF和复合物的潜在运动。我们的结果表明，OSBPF 在游离状态下是无序的，VAP-A(MSP) 和 OSBPF 通过中间体形成最终的复合物，其中通过酸性残基（包括 FFAT 基序之前的酸性补丁）的静电相互作用可能发挥集体作用。此外，我们报告导致家族运动神经元疾病的突变降低了 MSP 结构域的稳定性。
Oxysterol-binding protein (OSBP), a cytosolic receptor of cholesterol and oxysterols, is recruited to the endoplasmic reticulum by binding to the cytoplasmic major sperm protein (MSP) domain of integral endoplasmic reticulum protein VAMP-associated protein-A (VAP-A), a process essential for the stimulation of sphingomyelin synthesis by 25-hydroxycholesterol. To delineate the interaction mechanism between VAP-A and OSBP, we determined the complex structure between the VAP-A MSP domain (VAP-A(MSP)) and the OSBP fragment containing a VAP-A binding motif FFAT (OSBPF) by NMR. This solution structure explained that five of six conserved residues in the FFAT motif are required for the stable complex formation, and three of five, including three critical intermolecular electrostatic interactions, were not explained before. By combining NMR relaxation and titration, isothermal titration calorimetry, and mutagenesis experiments with structural information, we further elucidated the detailed roles of the FFAT motif and underlying motions of VAP-A(MSP), OSBPF, and the complex. Our results show that OSBPF is disordered in the free state, and VAP-A(MSP) and OSBPF form a final complex by means of intermediates, where electrostatic interactions through acidic residues, including an acid patch preceding the FFAT motif, probably play a collective role. Additionally, we report that the mutation that causes the familial motor neuron disease decreases the stability of the MSP domain.