title: Functional domains of Brevibacillus thermoruber lon protease for oligomerization and DNA binding: role of N-terminal and sensor and substrate discrimination domains.
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.
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei 115.
authors.forename: Alan Yueh-Luen
authors.initials: AY
authors.lastname: Lee
authors.forename: Chun-Hua
authors.initials: CH
authors.lastname: Hsu
authors.forename: Shih-Hsiung
authors.initials: SH
authors.lastname: Wu
mesh_terms: D049069:ATP-Dependent Proteases; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000595:Amino Acid Sequence; D001419:Bacteria / Q000378:metabolism*; D015153:Blotting, Western; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006360:Heat-Shock Proteins / Q000737:chemistry*; D007477:Ions; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010447:Peptide Hydrolases / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry*; D013997:Time Factors
chemical_list: D003432:Cross-Linking Reagents; D006360:Heat-Shock Proteins; D007477:Ions; D004247:DNA; D010447:Peptide Hydrolases; D049069:ATP-Dependent Proteases; D012697:Serine Endopeptidases; D000251:Adenosine Triphosphatases