Abstract
BackgroundCaseinolytic proteases (ClpPs) are barrel-shaped self-compartmentalized peptidases involved in eliminating damaged or short-lived regulatory proteins. The Mycobacterium tuberculosis (MTB) genome contains two genes coding for putative ClpPs, ClpP1 and ClpP2 respectively, that are likely to play a role in the virulence of the bacterium.ResultsWe report the first biochemical characterization of ClpP1 and ClpP2 peptidases from MTB. Both proteins were produced and purified in Escherichia coli. Use of fluorogenic model peptides of diverse specificities failed to show peptidase activity with recombinant mycobacterial ClpP1 or ClpP2. However, we found that ClpP1 had a proteolytic activity responsible for its own cleavage after the Arg8 residue and cleavage of ClpP2 after the Ala12 residue. In addition, we showed that the absence of any peptidase activity toward model peptides was not due to an obstruction of the entry pore by the N-terminal flexible extremity of the proteins, nor to an absolute requirement for the ClpX or ClpC ATPase complex. Finally, we also found that removing the putative propeptides of ClpP1 and ClpP2 did not result in cleavage of model peptides.We have also shown that recombinant ClpP1 and ClpP2 do not assemble in the conventional functional tetradecameric form but in lower order oligomeric species ranging from monomers to heptamers. The concomitant presence of both ClpP1 and ClpP2 did not result in tetradecameric assembly. Deleting the amino-terminal extremity of ClpP1 and ClpP2 (the putative propeptide or entry gate) promoted the assembly in higher order oligomeric species, suggesting that the flexible N-terminal extremity of mycobacterial ClpPs participated in the destabilization of interaction between heptamers.ConclusionDespite the conservation of a Ser protease catalytic triad in their primary sequences, mycobacterial ClpP1 and ClpP2 do not have conventional peptidase activity toward peptide models and display an unusual mechanism of self-assembly. Therefore, the mechanism underlying their peptidase and proteolytic activities might differ from that of other ClpP proteolytic complexes.
Highlights
Caseinolytic proteases (ClpPs) are barrel-shaped self-compartmentalized peptidases involved in eliminating damaged or short-lived regulatory proteins
Recombinant ClpP1 and ClpP2 do not cleave the Suc-LYAmc peptide but can inhibit E. coli ClpP activity The Rv2461c and Rv2460c genes encode proteins of respectively 200 and 214 amino acids annotated as ClpP1 and ClpP2
When ClpP1 and ClpP2 were produced in E. coli cells lacking endogenous ClpP (SG1146a strain), no peptidase activity toward the Suc-LY-AMC peptide was detected (Figure 1A)
Summary
Caseinolytic proteases (ClpPs) are barrel-shaped self-compartmentalized peptidases involved in eliminating damaged or short-lived regulatory proteins. ATP-dependent proteases play an essential role by removing short-lived regulatory proteins whose rapid elimination is critical for cell metabolism and growth [1] They allow riddance of misfolded and damaged proteins that accumulate in a variety of circumstances, notably during environmental stress [2]. Degradation of longer peptides and proteins required the interaction of ClpP with an ATPase complex, namely ClpA or ClpX in most Gram negative bacteria and ClpX, ClpC, and possibly ClpE and ClpL in Gram positive bacteria [6] This ATPase complex, a ringshaped hexamer that aligns coaxially with the ClpP peptidase tetradecamer, allows recognition of polypeptide substrates and, through ATP hydrolysis, provides the energy required for protein unfolding and translocation inside the proteolytic chamber where the polypeptide chain is degraded to small peptides
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