Department of Plant Sciences, Weizmann Institute of Science, Rehovot 76100, Israel.
J Biol Chem. 2010 Apr 30;285(18):13550-60. doi: 10.1074/jbc.M109.095075. Epub 2010 Feb 24.
In animals, protease inhibitors of the serpin family are associated with many physiological processes, including blood coagulation and innate immunity. Serpins feature a reactive center loop (RCL), which displays a protease target sequence as a bait. RCL cleavage results in an irreversible, covalent serpin-protease complex. AtSerpin1 is an Arabidopsis protease inhibitor that is expressed ubiquitously throughout the plant. The x-ray crystal structure of recombinant AtSerpin1 in its native stressed conformation was determined at 2.2 A. The electrostatic surface potential below the RCL was found to be highly positive, whereas the breach region critical for RCL insertion is an unusually open structure. AtSerpin1 accumulates in plants as a full-length and a cleaved form. Fractionation of seedling extracts by nonreducing SDS-PAGE revealed the presence of an additional slower migrating complex that was absent when leaves were treated with the specific cysteine protease inhibitor L-trans-epoxysuccinyl-L-leucylamido (4-guanidino)butane. Significantly, RESPONSIVE TO DESICCATION-21 (RD21) was the major protease labeled with the L-trans-epoxysuccinyl-L-leucylamido (4-guanidino)butane derivative DCG-04 in wild type extracts but not in extracts of mutant plants constitutively overexpressing AtSerpin1, indicating competition. Fractionation by nonreducing SDS-PAGE followed by immunoblotting with RD21-specific antibody revealed that the protease accumulated both as a free enzyme and in a complex with AtSerpin1. Importantly, both RD21 and AtSerpin1 knock-out mutants lacked the serpin-protease complex. The results establish that the major Arabidopsis plant serpin interacts with RD21. This is the first report of the structure and in vivo interaction of a plant serpin with its target protease.
在动物中，丝氨酸蛋白酶抑制剂家族与许多生理过程有关，包括血液凝固和先天免疫。丝氨酸蛋白酶抑制剂的特征是具有反应中心环（RCL），其显示蛋白酶靶序列作为诱饵。RCL 切割导致不可逆的、共价的丝氨酸蛋白酶抑制剂复合物。AtSerpin1 是一种拟南芥蛋白酶抑制剂，在植物中广泛表达。在其天然应激构象下，重组 AtSerpin1 的 X 射线晶体结构在 2.2Å 处确定。发现 RCL 下方的静电表面电势非常正，而对于 RCL 插入至关重要的缺口区域是一种异常开放的结构。AtSerpin1 在植物中积累为全长和切割形式。幼苗提取物的非还原 SDS-PAGE 级分显示存在另一个迁移速度较慢的复合物，当叶片用特异性半胱氨酸蛋白酶抑制剂 L-trans-epoxysuccinyl-L-leucylamido（4-胍基）丁烷处理时，该复合物不存在。重要的是，响应脱水 21（RD21）是用 L-trans-epoxysuccinyl-L-leucylamido（4-胍基）丁烷衍生物 DCG-04 在野生型提取物中标记的主要蛋白酶，但在持续过表达 AtSerpin1 的突变体植物提取物中没有标记，表明存在竞争。非还原 SDS-PAGE 级分后用 RD21 特异性抗体进行免疫印迹显示，蛋白酶既作为游离酶积累，也与 AtSerpin1 形成复合物。重要的是，RD21 和 AtSerpin1 敲除突变体都缺乏丝氨酸蛋白酶抑制剂复合物。结果表明，主要的拟南芥植物丝氨酸蛋白酶与 RD21 相互作用。这是首次报道植物丝氨酸蛋白酶与其靶蛋白酶的结构和体内相互作用。
