The active ClpP protease fromM. tuberculosisis a complex composed of a heptameric ClpP1 and a ClpP2 ring

Abstract

Mycobacterium tuberculosis (Mtb) contains two clpP genes, both of which are essential for viability. We expressed and purified Mtb ClpP1 and ClpP2 separately. Although each formed a tetradecameric structure and was processed, they lacked proteolytic activity. We could, however, reconstitute an active, mixed ClpP1P2 complex after identifying N‐blocked dipeptides that stimulate dramatically (>1000‐fold) ClpP1P2 activity against certain peptides and proteins. These activators function cooperatively to induce the dissociation of ClpP1 and ClpP2 tetradecamers into heptameric rings, which then re‐associate to form the active ClpP1P2 2‐ring mixed complex. No analogous small molecule‐induced enzyme activation mechanism involving dissociation and re‐association of multimeric rings has been described. ClpP1P2 possesses chymotrypsin and caspase‐like activities, and ClpP1 and ClpP2 differ in cleavage preferences. The regulatory ATPase ClpC1 was purified and shown to increase hydrolysis of proteins by ClpP1P2, but not peptides. ClpC1 did not activate ClpP1 or ClpP2 homotetradecamers and stimulated ClpP1P2 only when both ATP and a dipeptide activator were present. ClpP1P2 activity, its unusual activation mechanism and ClpC1 ATPase represent attractive drug targets to combat tuberculosis.

Structural and functional insights into caseinolytic proteases reveal an unprecedented regulation principle of their catalytic triad

“…Pioneering work in mycobacteria revealed a heterooligomeric assembly of ClpP1 and ClpP2 in which the interaction of both heptameric rings stimulated activity and exhibited specialized substrate preferences (26). Here we elucidate the high-resolution X-ray structures of LmClpP1, LmClpP1-N172D, as well as LmClpP2 and evaluate their activity by customized probes as well as substrate turnover assays.…”

“…For a cyanobacterial system, heptameric rings of mixed composition have been reported that interact with different chaperones (22). In contrast, ClpP proteins from L. monocytogenes (LmClpP1 and LmClpP2) as well as from Mycobacterium tuberculosis have been found to assemble into heterooligomeric complexes composed of two homoheptamers (25,26). Inhibition of LmClpP2 with lactonebased inhibitors led to down-regulation of virulence without affecting viability (27).…”

“…Inhibition of LmClpP2 with lactonebased inhibitors led to down-regulation of virulence without affecting viability (27). In contrast, both mycobacterial ClpP subunits are essential for bacterial survival, emphasizing defined functional roles of ClpP proteins among species (26,28).…”

Crystal structure of Mycobacterium tuberculosis ClpP1P2 suggests a model for peptidase activation by AAA+ partner binding and substrate delivery

“…S6 A and B). Notably, all peptides known to function as agonists contain an N-terminal carboxybenzyl group (29). The "reverse" binding orientation also explains why agonist peptides are not cleaved, because no peptide bond is positioned for nucleophilic attack by the active-site serine (Fig.…”

“…The importance of Clp-family proteolysis in M. tuberculosis is highlighted by the facts that the clpP1, clpP2, clpX, and clpC1 genes are all essential and that mechanism-based ClpP inhibitors suppress growth (24,(26)(27)(28). Recent studies indicate that M. tuberculosis ClpP1 and ClpP2 form discrete heptameric rings that assemble into an active ClpP1P2 tetradecamer only in the presence of a ClpX or ClpC1 AAA+ partner and one additional factor, either protein substrates being actively translocated into the degradation chamber or N-blocked peptide agonists (23,29). Because M. tuberculosis resistance to conventional antibacterial drugs is a major health hazard, there is substantial interest in developing drugs that target ClpP1P2.…”