CONFORMATION OF THE REACTIVE SITE LOOP OF ALPHA-1-PROTEINASE INHIBITOR PROBED BY LIMITED PROTEOLYSIS
CONFORMATION OF THE REACTIVE SITE LOOP OF ALPHA-1-PROTEINASE INHIBITOR PROBED BY LIMITED PROTEOLYSIS
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DOI：
10.1021/bi00125a012
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发表时间：
1992-03-17
期刊：
影响因子：
2.9
通讯作者：
SALVESEN, G
中科院分区：
文献类型：
--
作者：
MAST, AE;ENGHILD, JJ;SALVESEN, G
阐明丝氨酸蛋白酶抑制剂的反应位点环（RSL）结构对于理解其抑制机制至关重要。 RSL 结构的维持可能取决于其与二级结构的主导单元（称为 A-sheet）的相互作用。我们通过使用几种酶对 α-1-蛋白酶抑制剂进行有限的蛋白水解来研究这些相互作用。 RSL 的 P1-P10 区域对蛋白水解极其敏感，表明残基 P3'-P13 暴露在原始抑制剂中。在切割反应位点上游的八个或九个残基后，蛋白质发生非共价聚合，有时形成环状。聚合是将一个分子的 P1-P8 或 P1-P9 区域插入相邻的蛋白水解修饰分子的 A 片层中而产生的。 RSL 内的切割位点对蛋白质的构象稳定性具有明显影响，因此随着更多氨基酸插入 A 片层，稳定性会增加。我们得出的结论是，原始 α-1-蛋白酶抑制剂的 A 片层与卵清蛋白的 A 片层相似，只是它包含一个插入了两个或三个 RSL 残基的凸起。 通过 RSL 的蛋白水解切割允许插入七个或八个 RSL 残基，导致片层的扩展。 α-1-蛋白酶抑制剂和几种丝氨酸蛋白酶抑制剂的 RSL 可能表现出比丝氨酸蛋白酶的其他蛋白质抑制剂中常见的显着更高的流动性。
Elucidation of the reactive site loop (RSL) structure of serpins is essential for understanding their inhibitory mechanism. Maintenance of the RSL structure is likely to depend on its interactions with a dominant unit of secondary structure known as the A-sheet. We investigated these interactions by subjecting alpha-1-proteinase inhibitor to limited proteolysis using several enzymes. The P1-P10 region of the RSL was extremely sensitive to proteolysis, indicating that residues P3'-P13 are exposed in the virgin inhibitor. Following cleavage eight or nine residues upstream from the reactive site, the protein noncovalently polymerized, sometimes forming circles. Polymerization resulted from insertion of the P1-P8 or P1-P9 region of one molecule into the A-sheet of an adjacent proteolytically modified molecule. The site of cleavage within the RSL had a distinct effect on the conformational stability of the protein, such that stability increased as more amino acids insert into the A-sheet. We conclude that the A-sheet of virgin alpha-1-proteinase inhibitor resembles that of ovalbumin, except that it contains a bulge where two or three RSL residues are inserted. Insertion of seven or eight RSL residues, allowed by proteolytic cleavage of the RSL, causes expansion of the sheet. It is likely that the RSL of alpha-1-proteinase inhibitor and several serpins exhibits significantly more mobility than is common among other protein inhibitors of serine proteinases.