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 Esch erichia coli, The mechanism of protein-folding reactions mediated by ClpB i s 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 d ouble N- and C-terminal deletion. We studied the protein concentration-depe ndent and ATP-induced oligomerization of ClpB, casein-induced activation of ClpB ATPase, and ClpB-assisted reactivation of denatured firefly luciferas e, We found that both the N- and C-terminal truncation of ClpB strongly inh ibited its chaperone activity. The reasons for such inhibition were differe nt, however, for the N- and C-terminal truncation. Deletion of the C-termin al domain inhibited the self-association of ClpB, which led to decreased af finity for ATP and to decreased ATPase and chaperone activity of the C term inally truncated variants. In contrast, deletion of the N-terminal domain d id not inhibit the self-association of ClpB and its basal ATPase activity b ut decreased the ability of casein to activate ClpB ATPase, These results i ndicate that the N-terminal region of ClpB may contain a functionally signi ficant protein-binding site, whereas the main role of the C-terminal region is to support oligomerization of ClpB.