Structural plasticity and noncovalent substrate binding in the GroEL apical domain - A study using electrospray ionization mass spectrometry and fluorescence binding studies
Structural plasticity and noncovalent substrate binding in the GroEL apical domain - A study using electrospray ionization mass spectrometry and fluorescence binding studies
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DOI：
10.1074/jbc.m203398200
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发表时间：
2002-09-06
影响因子：
4.8
通讯作者：
Radford, SE
中科院分区：
文献类型：
--
作者：
Ashcroft, AE;Brinker, A;Radford, SE
报道了GroEL如何与非天然蛋白质结合的进展。GroEL顶端结构域的构象灵活性，这可以解释GroEL结合的各种底物，通过比较顶端结构域构建体的几个独立的晶体结构来说明，这些结构域构建体在螺旋H和I中显示构象可塑性。此外，ESI-MS表明，顶端结构域的构建体在中性pH值下具有共填充构象。为了评估不同的顶端结构域构象结合辅伴侣蛋白和底物的能力，研究了对应于GroES的移动的环和罗丹酸的螺旋D的模型肽。通过ESI-MS对顶端结构域-肽复合物的分析表明，只有折叠或部分折叠的顶端结构域构象形成在气相条件下存活的复合物。荧光结合研究表明，顶端结构域可以完全独立地结合两种肽。没有观察到结合的竞争，表明肽具有不同的顶端结构域结合位点。阻断GroEL中的GroES-顶端结构域结合位点使得伴侣蛋白在结合GroES和辅助变性罗丹酸的折叠中无活性，但仍然能够结合非天然蛋白质，支持GroES和底物蛋白即使在完整的GroEL十四聚体中也至少部分地具有不同的结合位点的结论。
Advances in understanding how GroEL binds to non-native proteins are reported. Conformational flexibility in the GroEL apical domain, which could account for the variety of substrates that GroEL binds, is illustrated by comparison of several independent crystallographic structures of apical domain constructs that show conformational plasticity in helices H and I. Additionally, ESI-MS indicates that apical domain constructs have co-populated conformations at neutral pH. To assess the ability of different apical domain conformers to bind cochaperone and substrate, model peptides corresponding to the mobile loop of GroES and to helix D from rhodanese were studied. Analysis of apical domain-peptide complexes by ESI-MS indicates that only the folded or partially folded apical domain conformations form complexes that survive gas phase conditions. Fluorescence binding studies show that the apical domain can fully bind both peptides independently. No competition for binding was observed, suggesting the peptides have distinct apical domain-binding sites. Blocking the GroES-apical domain-binding site in GroEL rendered the chaperonin inactive in binding GroES and in assisting the folding of denatured rhodanese, but still capable of binding non-native proteins, supporting the conclusion that GroES and substrate proteins have, at least partially, distinct binding sites even in the intact GroEL tetradecamer.