Structural elements of PC2 required for interaction with its helper protein 7B2.
Structural elements of PC2 required for interaction with its helper protein 7B2.
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PC2 与其辅助蛋白 7B2 相互作用所需的结构元件。
DOI：
10.1074/jbc.273.2.1158
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Lindberg,I
中科院分区：
文献类型：
--
作者：
Zhu,X;Muller,L;Mains,RE;Lindberg,I
真核枯草杆菌蛋白酶家族成员的结构可分为如下四个不同的结构域：前区、催化结构域、P结构域和羧基末端区域。尽管这些酶在进化上相关，但只有激素原转化酶 2 (PC2) 需要 7B2 才能激活。为了检查 PC2 的每个结构域对 PC2-7B2 相互作用的潜在贡献，我们进行了顺序删除、定点诱变和结构域交换，以用 pro-PC1 的相应片段/氨基酸替换 pro-PC2 的各个结构域或特定氨基酸。然后，这些嵌合体和突变酶分子在 AtT-20 细胞中表达，并分析 7B2 结合、成熟能力和酶活性。结果表明：1）PC2前区是必需的，但不足以赋予7B2结合； 2) P结构域是PC2结构稳定所必需的，不能与PC1的P结构域互换； 3) 羧基末端结构域不参与 7B2 结合。 pro-PC2 的定点诱变进一步表明，催化结构域 Tyr-194 → Asp 中的单个残基替换可阻止 pro-PC2 结合 7B2 并阻止激活。该残基存在于富含芳香族氨基酸的环内，从枯草杆菌蛋白酶的晶体结构推断，该残基似乎位于分子的表面上。该环可能代表催化结构域内 7B2 的主要识别位点。
The structures of the eukaryotic subtilisin protease family members can be divided into four distinct domains as follows: the proregion, the catalytic domain, the P domain, and the carboxyl-terminal region. Although these enzymes are evolutionarily related, only prohormone convertase 2 (PC2) requires 7B2 for activation. To examine the potential contribution of each domain of PC2 to PC2–7B2 interactions, we performed sequential deletions, site-directed mutagenesis, and domain swapping to replace individual domains or particular amino acids of pro-PC2 with the corresponding segments/amino acids of pro-PC1. These chimeras and mutant enzyme molecules were then expressed in AtT-20 cells and analyzed for 7B2 binding, maturation ability, and enzymatic activity. The results revealed that 1) the PC2 proregion is required but is not sufficient to confer 7B2 binding; 2) the P domain is required for the stabilization of PC2 structure and is not exchangeable with the P domain of PC1; and 3) the carboxyl-terminal domain is not involved in 7B2 binding. Site-directed mutagenesis of pro-PC2 further showed that a single residue replacement in the catalytic domain, Tyr-194 → Asp, prevented pro-PC2 from binding 7B2 and blocked activation. This residue is present within a loop rich in aromatic amino acids which appears to be on the surface of the molecule as extrapolated from the crystal structure of subtilisin. This loop may represent the primary recognition site for 7B2 within the catalytic domain.