Centre de Biophysique Moléculaire, UPR 4301 CNRS conventionnée avec l'Université d'Orléans, Orléans Cedex 2, France.
FEBS J. 2012 Dec;279(24):4466-78. doi: 10.1111/febs.12033. Epub 2012 Nov 12.
Greglin is an 83-residue serine protease inhibitor purified from the ovaries of the locust Schistocerca gregaria. Greglin is a strong inhibitor of subtilisin and human neutrophil elastase, acting at sub-nanomolar and nanomolar concentrations, respectively; it also inhibits neutrophil cathepsin G, α-chymotrypsin and porcine pancreatic elastase, but to a lesser extent. In the present study, we show that greglin resists denaturation at high temperature (95 °C) and after exposure to acetonitrile and acidic or basic pH. Greglin is composed of two domains consisting of residues 1-20 and 21-83. Mass spectrometry indicates that the N-terminal domain (1-20) is post-translationally modified by phosphorylations at three sites and probably contains a glycosylation site. The crystal structure of the region of greglin comprising residues 21-78 in complex with subtilisin was determined at 1.75 Å resolution. Greglin represents a novel member of the non-classical Kazal inhibitors, as it has a unique additional C-terminal region (70-83) connected to the core of the molecule via a supplementary disulfide bond. The stability of greglin was compared with that of an ovomucoid inhibitor. The thermostability and inhibitory specificity of greglin are discussed in light of its structure. In particular, we propose that the C-terminal region is responsible for non-favourable interactions with the autolysis loop (140-loop) of serine proteases of the chymotrypsin family, and thus governs specificity.
The atomic coordinates and structure factors for the greglin-subtilisin complex have been deposited with the RCSB Protein Data Bank under accession number 4GI3.
Greglin and Subtilisin Carlsberg bind by X-ray crystallography (View interaction).
Greglin 是一种从蝗虫 Schistocerca gregaria 的卵巢中纯化得到的 83 残基丝氨酸蛋白酶抑制剂。Greglin 是枯草杆菌蛋白酶和人中性粒细胞弹性蛋白酶的强抑制剂，分别在亚纳摩尔和纳摩尔浓度下发挥作用；它还抑制中性粒细胞组织蛋白酶 G、α-糜蛋白酶和猪胰弹性蛋白酶，但抑制作用较小。在本研究中，我们表明 greglin 能够抵抗高温（95°C）变性以及暴露于乙腈和酸性或碱性 pH 值的变性。Greglin 由两个结构域组成，包含残基 1-20 和 21-83。质谱分析表明，N 端结构域（1-20）通过三个位点的磷酸化进行翻译后修饰，可能含有一个糖基化位点。Greglin 与枯草杆菌蛋白酶复合物的 21-78 个残基区域的晶体结构在 1.75Å分辨率下确定。Greglin 代表一种新的非经典 Kazal 抑制剂成员，因为它具有独特的额外 C 端区域（70-83），通过补充的二硫键与分子的核心相连。比较了 greglin 的稳定性与卵白蛋白抑制剂的稳定性。根据其结构讨论了 greglin 的热稳定性和抑制特异性。特别是，我们提出 C 端区域负责与糜蛋白酶家族的丝氨酸蛋白酶的自溶环（140 环）产生不利相互作用，从而控制特异性。
Greglin-枯草杆菌蛋白酶复合物的原子坐标和结构因子已被提交给 RCSB 蛋白质数据银行，登录号为 4GI3。
通过 X 射线晶体学（查看相互作用）确定了 Greglin 和枯草杆菌蛋白酶的结合。
