title: Site-directed mutagenesis of conserved charged amino acid residues in ClpB from Escherichia coli.
abstract: ClpB is a member of a multichaperone system in Escherichia coli (with DnaK, DnaJ, and GrpE) that reactivates strongly aggregated proteins. The sequence of ClpB contains two ATP-binding domains, each containing Walker consensus motifs. The N- and C-terminal sequence regions of ClpB do not contain known functional motifs. In this study, we performed site-directed mutagenesis of selected charged residues within the Walker A motifs (Lys212 and Lys611) and the C-terminal region of ClpB (Asp797, Arg815, Arg819, and Glu826). We found that the mutations K212T, K611T, D797A, R815A, R819A, and E826A did not significantly affect the secondary structure of ClpB. The mutation of the N-terminal ATP-binding site (K212T), but not of the C-terminal ATP-binding site (K611T), and two mutations within the C-terminal domain (R815A and R819A) inhibited the self-association of ClpB in the absence of nucleotides. The defects in self-association of these mutants were also observed in the presence of ATP and ADP. The four mutants K212T, K611T, R815A, and R819A showed an inhibition of chaperone activity, which correlated with their low ATPase activity in the presence of casein. Our results indicate that positively charged amino acids that are located along the intersubunit interface (this includes Lys212 in the Walker A motif of the N-terminal ATP-binding domain as well as Arg815 and Arg819 in the C-terminal domain) participate in intersubunit salt bridges and stabilize the ClpB oligomer. Interestingly, we have identified a conserved residue within the C-terminal domain (Arg819) which does not participate directly in nucleotide binding but is essential for the chaperone activity of ClpB.
authors.affiliation: Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506, USA.
authors.forename: Micheal E
authors.initials: ME
authors.lastname: Barnett
authors.forename: Michal
authors.initials: M
authors.lastname: Zolkiewski
mesh_terms: D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D024342:Amino Acids, Acidic / Q000235:genetics*; D024361:Amino Acids, Basic / Q000235:genetics*; D001120:Arginine / Q000235:genetics; D017124:Conserved Sequence; D049071:Endopeptidase Clp; D029968:Escherichia coli Proteins / Q000235:genetics*; D018698:Glutamic Acid / Q000235:genetics; D006360:Heat-Shock Proteins / Q000235:genetics*; D008239:Lysine / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013912:Threonine / Q000235:genetics
chemical_list: D024342:Amino Acids, Acidic; D024361:Amino Acids, Basic; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D013912:Threonine; D018698:Glutamic Acid; D001120:Arginine; D049071:Endopeptidase Clp; C069775:ClpB protein, E coli; D008239:Lysine; D000409:Alanine