Functional Domains of Brevibacillus thermoruber Lon Protease for Oligomerization and DNA Binding
Functional Domains of Brevibacillus thermoruber Lon Protease for Oligomerization and DNA Binding
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热橡胶短芽孢杆菌 Lon 蛋白酶寡聚化和 DNA 结合的功能域
DOI：
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发表时间：
2004
影响因子：
4.8
通讯作者：
Shih
中科院分区：
文献类型：
--
作者：
A. Lee;Chun‐Hua Hsu;Shih
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酶和伴侣样活性，以及DNA结合。已克隆了来自热红短芽孢杆菌WR-249（Bt-Lon）的热稳定Lon蛋白酶，并且其特征在于具有N-末端结构域、包括传感器和底物识别（SSD）结构域的中央ATP酶结构域和C-末端蛋白酶结构域。在这里，我们提出了一个详细的结构-功能表征的Bt-Lon，不仅解剖的Bt-Lon结构域在寡聚化，催化活性，伴侣样活性，和DNA结合活性的个人角色，但也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。我们的研究结果表明，N-末端结构域是必不可少的寡聚化。N端结构域的截短导致寡聚化的失败，并导致蛋白水解、ATP酶和分子伴侣样活性的失活，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DNA结合的凝胶迁移率变动分析的基础上。另一方面，Bt-Lon的寡聚化通过化学交联实验揭示的二聚体Particytetramer Partichexamer组装模型进行。结果还表明，疏水相互作用可能在Bt-Lon的二聚化过程中起重要作用，而离子相互作用则是Bt-Lon六聚体组装的主要原因。
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 ↔ tetramer ↔ 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.
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影响因子：
7
作者：
A. F. Neuwald;L. Aravind;J. Spouge;E. Koonin
通讯作者：
A. F. Neuwald;L. Aravind;J. Spouge;E. Koonin
DOI：
10.1016/s0021-9258(17)42340-4
发表时间：
1994-01
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
L. van Dyck;D. Pearce;F. Sherman
通讯作者：
L. van Dyck;D. Pearce;F. Sherman
DOI：
10.1016/s0021-9258(17)41804-7
发表时间：
1994-02
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
D. Parsell;A. Kowal;S. Lindquist
通讯作者：
D. Parsell;A. Kowal;S. Lindquist
影响因子：
2.9
作者：
TWINING, SS
通讯作者：
TWINING, SS
Mg2 连接寡聚化调节耻垢分枝杆菌 Lon (La) 蛋白酶的催化活性。
影响因子：
2.9
作者：
Rudyak,SG;Brenowitz,M;Shrader,TE
通讯作者：
Shrader,TE