Plasticity of human protein disulfide isomerase: evidence for mobility around the X-linker region and its functional significance.
Plasticity of human protein disulfide isomerase: evidence for mobility around the X-linker region and its functional significance.
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DOI：
10.1074/jbc.m110.107839
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发表时间：
2010-08-27
期刊：
影响因子：
--
通讯作者：
Wang CC
中科院分区：
文献类型：
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作者：
Wang C;Chen S;Wang X;Wang L;Wallis AK;Freedman RB;Wang CC
蛋白质二硫键异构酶（PDI）是内质网中负责氧化折叠的关键酶，由abb′xa′c结构域组成。在这项工作中，我们专注于这种酶的构象可塑性。几种蛋白酶对天然人PDI（hPDI）的蛋白水解始终靶向分子C端一半（x-接头和a′结构域）中的位点，留下N端完整的大片段。对全长PDI的W111 F/W390 F突变体的荧光研究表明，其荧光由x-连接体中的Trp-347主导，该x-连接体作为内在报告分子，并表明该连接体可以在“加帽”和“去帽”构象之间移动，其中它占据或暴露hPDI的B′结构域上的主要配体结合位点。使用内源荧光、碰撞淬灭和外源探针荧光对一系列构建体和突变体进行研究（1-苯胺基-8-萘磺酸盐）的研究表明，全长hPDI中a′结构域的存在缓和了x-接头产生加帽构象的能力（与较短片段相比），但不会消除它。因此，与酵母PDI不同，全长hPDI的主要构象可塑性涉及由x-接头介导的a′结构域“臂”相对于bb′“主干”的移动性。这些构建体和突变体的伴侣和酶活性与以下解释一致：x-接头与配体结合位点的可逆相互作用介导蛋白质底物进入该位点。
Protein disulfide isomerase (PDI), which consists of multiple domains arranged as abb′xa′c, is a key enzyme responsible for oxidative folding in the endoplasmic reticulum. In this work we focus on the conformational plasticity of this enzyme. Proteolysis of native human PDI (hPDI) by several proteases consistently targets sites in the C-terminal half of the molecule (x-linker and a′ domain) leaving large fragments in which the N terminus is intact. Fluorescence studies on the W111F/W390F mutant of full-length PDI show that its fluorescence is dominated by Trp-347 in the x-linker which acts as an intrinsic reporter and indicates that this linker can move between “capped” and “uncapped” conformations in which it either occupies or exposes the major ligand binding site on the b′ domain of hPDI. Studies with a range of constructs and mutants using intrinsic fluorescence, collision quenching, and extrinsic probe fluorescence (1-anilino-8-naphthalene sulfonate) show that the presence of the a′ domain in full-length hPDI moderates the ability of the x-linker to generate the capped conformation (compared with shorter fragments) but does not abolish it. Hence, unlike yeast PDI, the major conformational plasticity of full-length hPDI concerns the mobility of the a′ domain “arm” relative to the bb′ “trunk” mediated by the x-linker. The chaperone and enzymatic activities of these constructs and mutants are consistent with the interpretation that the reversible interaction of the x-linker with the ligand binding site mediates access of protein substrates to this site.