Peng Li, Rasmussen Morten Ib, Chailyan Anna, Houen Gunnar, Højrup Peter
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
Department of Clinical Biochemistry, Immunology and Genetics, Statens Serum Institut, Copenhagen, Denmark.
J Proteomics. 2014 Aug 28;108:1-16. doi: 10.1016/j.jprot.2014.04.037. Epub 2014 May 2.
Protein disulfide-isomerase (PDI) is a four-domain flexible protein that catalyzes the formation of disulfide bonds in the endoplasmic reticulum. Here we have analyzed native PDI purified from human placenta by chemical cross-linking followed by mass spectrometry (CXMS). In addition to PDI the sample contained soluble calnexin and ERp72. Extensive cross-linking was observed within the PDI molecule, both intra- and inter-domain, as well as between the different components in the mixture. The high sensitivity of the analysis in the current experiments, combined with a likely promiscuous interaction pattern of the involved proteins, revealed relatively densely populated cross-link heat maps. The established X-ray structure of the monomeric PDI could be confirmed; however, the dimer as presented in the existing models does not seem to be prevalent in solution as modeling on the observed cross-links revealed new models of dimeric PDI. The observed inter-protein cross-links confirmed the existence of a peptide binding area on calnexin that binds strongly both PDI and ERp72. On the other hand, interaction sites on PDI and ERp72 could not be uniquely identified, indicating a more non-specific interaction pattern.
The present work demonstrates the use of chemical cross-linking and mass spectrometry (CXMS) for the determination of a solution structure of natural human PDI and its interaction with the chaperones ERp72 and calnexin. The data shows that the dimeric structure of PDI may be more diverse than indicated by present models. We further observe that the temperature influences the cross-linking pattern of PDI, but this does not influence the overall folding pattern of the molecule.
蛋白质二硫键异构酶（PDI）是一种具有四个结构域的柔性蛋白质，在内质网中催化二硫键的形成。在此，我们通过化学交联结合质谱分析（CXMS）对从人胎盘中纯化的天然PDI进行了分析。除了PDI，样品中还含有可溶性钙连蛋白和内质网蛋白72（ERp72）。在PDI分子内，无论是结构域内还是结构域间，以及混合物中的不同组分之间，都观察到了广泛的交联。当前实验中分析的高灵敏度，结合所涉及蛋白质可能的混杂相互作用模式，揭示了相对密集的交联热图。单体PDI已确立的X射线结构能够得到证实；然而，现有模型中呈现的二聚体在溶液中似乎并不普遍，因为基于观察到的交联进行建模揭示了二聚体PDI的新模型。观察到的蛋白质间交联证实了钙连蛋白上存在一个肽结合区域，该区域与PDI和ERp72都有强烈结合。另一方面，无法唯一确定PDI和ERp72上的相互作用位点，这表明存在一种更非特异性的相互作用模式。
本研究展示了利用化学交联和质谱分析（CXMS）来确定天然人PDI的溶液结构及其与伴侣蛋白ERp72和钙连蛋白的相互作用。数据表明，PDI的二聚体结构可能比现有模型所示的更加多样。我们还观察到温度会影响PDI的交联模式，但这并不影响分子的整体折叠模式。
