Mapping the oligomeric interface of diacylglycerol kinase by engineered thiol cross-linking: Homologous sites in the transmembrane domain
Mapping the oligomeric interface of diacylglycerol kinase by engineered thiol cross-linking: Homologous sites in the transmembrane domain
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DOI：
10.1021/bi991781n
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发表时间：
2000-04-11
期刊：
影响因子：
2.9
通讯作者：
Sanders, CR
中科院分区：
文献类型：
--
作者：
Nagy, JK;Lau, FW;Sanders, CR
这项工作代表了基于巯基的交联研究的第一阶段，以映射的同源三聚体膜蛋白二酰甘油激酶（DAGK）的低聚界面。共53个单半胱氨酸突变体跨越DAGK的三个跨膜段和第一部分的胞质结构域进行纯化，并在混合胶束中进行催化氧化。四头异种（A52 C、I53 C、A74 C和I75 C）进行三聚体内二硫键形成。为了确定同源位点在DAGK的基态构象中是否是近端的，或者二硫键是否是由于使正常远端位点短暂接近的运动而形成的，另外的交联实验在三种不同流动性的不同环境中进行[混合胶束、1-棕榈酰基-2-油酰基-sn-甘油基-3-磷酸胆碱（POPC）囊泡和大肠杆菌膜]。交联实验包括在三种不同的催化条件下形成二硫键[Cu（II）-菲咯啉氧化，I-2氧化。和基于硫代硝基苯甲酸酯的硫醇交换]和与一组双官能硫醇反应性化学交联剂的反应，所述双官能硫醇反应性化学交联剂呈现两种不同的反应性化学和几种间隔基长度。基于这些研究，残基53和75被判断为在DAGK同源三聚体内稳定接近，而位置52似乎更远，并且仅由于蛋白质运动而形成二硫键。位置74的结果是不明确的，在脂质囊泡和混合胶束DAGK似乎执行的议案，不存在于天然膜，流动性也较高的DAGK在混合胶束比POPC囊泡。
This work represents the first stage of thiol-based cross-linking studies to map the oligomeric interface of the homotrimeric membrane protein diacylglycerol kinase (DAGK). A total of 53 single-cysteine mutants spanning DAGK's three transmembrane segments and the first part of a cytoplasmic domain were purified and subjected to catalytic oxidation in mixed micelles. Four mutants (A52C, I53C, A74C, and I75C) were observed to undergo intratrimer disulfide bond formation between homologous sites on adjacent subunits, To establish whether the homologous sites are proximal in the ground-state conformation of DAGK or whether the disulfide bonds formed as a result of motions that brought normally distal sites into transient proximity, additional cross-linking experiments were carried out in three different milieus of varying fluidity [mixed micelles, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles, and Escherichia coli membranes]. Cross-linking experiments included disulfide bond formation under three different catalytic conditions [Cu(II)-phenanthroline oxidation, I-2 oxidation. and thionitrobenzoate-based thiol exchange] and reactions with a set of bifunctional thiol-reactive chemical cross-linkers presenting two different reactive chemistries and several spacer lengths. On the basis of these studies, residues 53 and 75 are judged to be in stable proximity within the DAGK homotrimer, while position 52 appears to be more distal and forms disulfide bonds only as a result of protein motions. Results for position 74 were ambiguous, In lipid vesicles and mixed micelles DAGK appears to execute motions that are not present in native membranes, with mobility also being higher for DAGK in mixed micelles than in POPC vesicles.