Structure and activity of ClpB from Escherichia coli. Role of the amino-and -carboxyl-terminal domains.
ClpB is a member of a protein-disaggregating multi-chaperone system in Escherichia coli. The mechanism of protein-folding reactions mediated by ClpB is currently unknown, and the functional role of different sequence regions in ClpB is under discussion. We have expressed and purified the full-length ClpB and three truncated variants with the N-terminal, C-terminal, and a double N- and C-terminal deletion. We studied the protein concentration-dependent and ATP-induced oligomerization of ClpB, casein-induced activation of ClpB ATPase, and ClpB-assisted reactivation of denatured firefly luciferase. We found that both the N- and C-terminal truncation of ClpB strongly inhibited its chaperone activity. The reasons for such inhibition were different, however, for the N- and C-terminal truncation. Deletion of the C-terminal domain inhibited the self-association of ClpB, which led to decreased affinity for ATP and to decreased ATPase and chaperone activity of the C-terminally truncated variants. In contrast, deletion of the N-terminal domain did not inhibit the self-association of ClpB and its basal ATPase activity but decreased the ability of casein to activate ClpB ATPase. These results indicate that the N-terminal region of ClpB may contain a functionally significant protein-binding site, whereas the main role of the C-terminal region is to support oligomerization of ClpB.
Barnett
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ME
Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506, USA.
Zolkiewska
A
A
Zolkiewski
M
M
D000251:Adenosine Triphosphatases / Q000378:metabolism; D002942:Circular Dichroism; D049071:Endopeptidase Clp; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism*; D018832:Molecular Chaperones / Q000378:metabolism; D013329:Structure-Activity Relationship
D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C069775:ClpB protein, E coli