Functional domains of Brevibacillus thermoruber Lon protease for oligomerization and DNA binding -: Role of N-terminal and sensor and substrate discrimination domains
Functional domains of Brevibacillus thermoruber Lon protease for oligomerization and DNA binding -: Role of N-terminal and sensor and substrate discrimination domains
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DOI：
10.1074/jbc.m403562200
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发表时间：
2004-08-13
影响因子：
4.8
通讯作者：
Wu, SH
中科院分区：
文献类型：
--
作者：
Lee, AYL;Hsu, CH;Wu, SH
Lon蛋白酶是一种多功能酶，其功能包括降解受损蛋白质和天然短寿命蛋白质、ATP酶和分子伴侣样活性，以及DNA结合。来自热橡胶短芽孢杆菌 WR-249 (Bt-Lon) 的热稳定性 Lon 蛋白酶已被克隆，并具有 N 端结构域、中央 ATP 酶结构域（包括传感器和底物辨别 (SSD) 结构域）和 C 端蛋白酶结构域。在这里，我们提出了 Bt-Lon 的详细结构-功能表征，不仅剖析了 Bt-Lon 结构域在寡聚化、催化活性、类伴侣活性和 DNA 结合活性中的各自作用，而且还描述了寡聚化的性质。设计、表达和纯化了 7 个 Bt-Lon 截短突变体。我们的结果表明 N 末端结构域对于寡聚化至关重要。 N端结构域的截短导致寡聚化失败，并导致蛋白水解、ATP酶和分子伴侣样活性失活，但保留了DNA结合活性，这表明Bt-Lon的寡聚化是其催化和分子伴侣样活性的先决条件。我们进一步发现 SSD 参与基于凝胶迁移率变化测定的 DNA 结合。另一方面，Bt-Lon 的寡聚化通过化学交联实验揭示的二聚体 7 四聚体 7 六聚体组装模型进行。结果还表明，疏水相互作用可能在Bt-Lon二聚化过程中发挥重要作用，而离子相互作用主要负责六聚体的组装。
Lon protease is a multifunctional enzyme, and its functions include the degradation of damaged proteins and naturally short lived proteins, ATPase and chaperone-like activities, as well as DNA binding. A thermostable Lon protease from Brevibacillus thermoruber WR-249 (Bt-Lon) has been cloned and characterized with an N-terminal domain, a central ATPase domain that includes a sensor and substrate discrimination (SSD) domain, and a C-terminal protease domain. Here we present a detailed structure-function characterization of Bt-Lon, not only dissecting the individual roles of Bt-Lon domains in oligomerization, catalytic activities, chaperone-like activity, and DNA binding activity but also describing the nature of oligomerization. Seven truncated mutants of Bt-Lon were designed, expressed, and purified. Our results show that the N-terminal domain is essential for oligomerization. The truncation of the N-terminal domain resulted in the failure of oligomerization and led to the inactivation of proteolytic, ATPase, and chaperone-like activities but retained the DNA binding activity, suggesting that oligomerization of Bt-Lon is a prerequisite for its catalytic and chaperone-like activities. We further found that the SSD is involved in DNA binding based on gel mobility shift assays. On the other hand, the oligomerization of Bt-Lon proceeds through a dimer 7 tetramer 7 hexamer assembly model revealed by chemical cross-linking experiments. The results also showed that hydrophobic interactions may play important roles in the dimerization of Bt-Lon, and ionic interactions are mainly responsible for the assembly of hexamers.