Function-Structural Studies on the Lon Protease from Function-Structural Studies on the Lon Protease from Brevibacillus thermoruber WR-249
Date Issued
2004
Date
2004
Author(s)
Lee, Alan Yueh-Luen
DOI
en-US
Abstract
Protein quality control plays key roles in prokaryotic and eukaryotic cells by depicting cellular surveillance systems of structural and functional integrity of proteins inside a cell. Oligomeric ATP-dependent proteases consisting of the chaperone and the protease machinery carry out such quality control. These energy-dependent proteases are especially important under stress conditions because increasing amounts of misfolding and damaged proteins may accumulate during various stresses.
A thermostable Lon protease from Brevibacillus thermoruber WR-249 (Bt-Lon) has been cloned and characterized. The Br. thermoruber Lon gene (Bt-lon) encodes an 88-kDa protein characterized with an N-terminal domain, a central ATPase domain including an SSD (sensor- and substrate- discrimination) domain, and a C-terminal protease domain. The Bt-lon is a heat-inducible gene and may be controlled under a putative Bacillus subtilis sA-dependent promoter but in the absence of CIRCE (controlling inverted repeat of chaperone expression). Bt-lon was expressed in E. coli and its protein product was purified. Bt-Lon is a multi-functional enzyme and its functions include the degradation of proteins, ATPase and chaperone-like activities, and DNA binding. The optimal temperature of ATPase activity for Bt-Lon is at 70oC, while the optimal temperature of peptidase and DNA-binding activities is 50oC. The peptidase activity of Bt-Lon increases substantially in the presence of ATP. Furthermore, the substrate specificity of Bt-Lon was found to be different from that of E. coli Lon by using fluorogenic peptides as substrates. Notably, this Bt-Lon protein shows the chaperone-like activity by preventing aggregation of denatured insulin B chain in a dose-dependent and ATP-independent manner. In the thermal denaturation experiments, Bt-Lon was found to display an indicator of thermostability value, Tm of 71.5 oC. Sequence comparison with a mesophilic Lon protease shows differences in the rigidity, electrostatic interactions or hydrogen bonding of Bt-Lon relevant to thermostability. Additionally, the native Br. thermoruber Lon protease (Bt-Lon) showed a hexameric structure as revealed by analytical gel filtration chromatography, and its nature of oligomerization was investigated. The chemical crosslinking experiments revealed that the oligomerization of Bt-Lon proceeds through a dimer « tetramer « hexamer assembly model. Our results also showed that hydrophobic interactions may play important roles in the dimerization of Bt-Lon and that ionic interactions are mainly responsible for hexamer assembly.
To identify the roles of individual domains in the oligomerization process and the functional activities, seven truncated mutants of Bt-Lon were designed, expressed and purified. We examined the Bt-Lon mutants using assays that reflect five different aspects of Bt-Lon activity: ATP-independent oligomerization, ATP-dependent proteolysis, ATPase activity, chaperone-like activity, and DNA-binding activity. Our results show that the N-terminal domain is essential for the oligomerization. The truncation of N-terminal domain resulted in the failure of oligomerization and led to the inactivation of proteolytic, ATPase and chaperone-like activities, suggesting that oligomerization of Bt-Lon is a prerequisite for its catalytic and chaperone-like activities. However, the N-terminal region of Bt-Lon is not involved in the interaction with DNA. We further found that the SSD domain with a previously uncertain function is involved in DNA-binding based on gel mobility shift assays (GMSA).
Subjects
DNA-結合蛋白質
熱穩定性
Lon 蛋白酶
chaperone-like activity
DNA-binding protein
thermostability
Lon protease
AAA+ protein
Type
other
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