Molecular characterization of the principal substrate binding site of the ubiquitous folding catalyst protein disulfide isomerase
Molecular characterization of the principal substrate binding site of the ubiquitous folding catalyst protein disulfide isomerase
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DOI：
10.1074/jbc.m312193200
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发表时间：
2004-03-12
影响因子：
4.8
通讯作者：
Ruddock, LW
中科院分区：
文献类型：
--
作者：
Pirneskoski, A;Klappa, P;Ruddock, LW
真核生物内质网中二硫键的形成由广泛表达的酶蛋白二硫键异构酶（PDI）催化。PDI作为折叠多肽中天然二硫键形成的催化剂的有效性取决于催化二硫键-二硫醇交换、结合非天然蛋白质以及触发结合底物中的构象变化的能力，从而允许接近掩埋的半胱氨酸残基。已知PDI的B'结构域提供PDI的主要肽结合位点，并且该结构域对于催化蛋白质底物中的异构化反应而不是氧化反应是关键的。在这里，我们使用同源性建模来更精确地定义B'结构域的边界，并显示在B'和a'结构域之间存在结构域内接头。我们已经表达了如此定义的重组B'结构域;重组产物的稳定性和构象性质证实了结构域边界的有效性。我们已经模拟了B'结构域的三级结构，并确定了其中的主要底物结合位点。该位点内的突变，在分离的结构域和全长PDI中表达，大大降低了对小肽底物的结合亲和力，其中影响最大的是I272 W，该突变似乎没有结构影响。
Disulfide bond formation in the endoplasmic reticulum of eukaryotes is catalyzed by the ubiquitously expressed enzyme protein disulfide isomerase (PDI). The effectiveness of PDI as a catalyst of native disulfide bond formation in folding polypeptides depends on the ability to catalyze disulfide-dithiol exchange, to bind non-native proteins, and to trigger conformational changes in the bound substrate, allowing access to buried cysteine residues. It is known that the b' domain of PDI provides the principal peptide binding site of PDI and that this domain is critical for catalysis of isomerization but not oxidation reactions in protein substrates. Here we use homology modeling to define more precisely the boundaries of the b' domain and show the existence of an intradomain linker between the b' and a' domains. We have expressed the recombinant b' domain thus defined; the stability and conformational properties of the recombinant product confirm the validity of the domain boundaries. We have modeled the tertiary structure of the b' domain and identified the primary substrate binding site within it. Mutations within this site, expressed both in the isolated domain and in full-length PDI, greatly reduce the binding affinity for small peptide substrates, with the greatest effect being I272W, a mutation that appears to have no structural effect.