Abstract
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.
Highlights
Lon protease is a multifunctional enzyme, and its functions include the degradation of damaged proteins and naturally short lived proteins, ATPase and chaperonelike activities, as well as DNA binding
The results showed that hydrophobic interactions may play important roles in the dimerization of Brevibacillus thermoruber Lon (Bt-Lon), and ionic interactions are mainly responsible for the assembly of hexamers
By aligning homologous regions of Bt-Lon, E. coli Lon, and Ms-Lon (Fig. 1C), we propose that the residues 290 –316 within the Bt-Lon N-terminal domain, called oligomerization domain, is involved in the oligomerization of BtLon
Summary
Enzymes and Chemicals—Restriction enzymes were purchased from New England Biolabs (London, UK). Peptidase assays contained 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 1.0 mM ATP, 0.3 mM fluorogenic peptide, Glt-AAF, and 5 g of proteins in a total volume of 200 l. Reaction mixtures consisted of 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 1.0 mM ATP, and 2–5 g of Bt-Lon in a total volume of 100 l and were incubated for 30 min at 50 °C or at indicated temperatures. Four g of Bt-Lon (1 M) was incubated in buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 10 mM MgCl2 in the absence and the presence of increasing concentrations of NaCl or SDS in a total volume of 50 l. The electrostatic potential of the modeled Bt-LonSSD was calculated by the program GRASP [43]
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