Most of our knowledge on the mechanism of protein degradation by ClpP protease is based on studies of E. coli
ClpP. This peptidase is able alone to hydrolyze the model dipeptide Suc-LY-Amc in vitro
]. In this study, we have shown that, in conditions that normally allow Suc-LY-Amc cleavage by E. coli
ClpP, MTB ClpP1 and ClpP2 do not hydrolyze this model peptide. However, a proteolytic activity was uncovered for ClpP1, ruling out the possibility that, at least for ClpP1, those peptidases were produced in E. coli
as totally inactive.
An absence of peptidase activity was not due to the presence of a His tag at the C-termini of ClpP1 or ClpP2 since purifying those peptidases without any tag did not allow hydrolysis of Suc-LY-Amc or of other model peptides, nor it promoted correct tetradecameric assembly.
A failure to detect model peptide cleavage was not due to an obstruction of the entry pore by the N-terminal extremities of ClpP1 and ClpP2, predicted to be highly flexible, since removing these extremities did not result in peptide cleavage. Furthermore, coproducing the ClpX and ClpC ATPase complexes with ClpP1 and ClpP2 did not allow Suc-LY-Amc cleavage, showing that an absence of peptide model hydrolysis by mycobacterial ClpPs was not due to an obligatory requirement for the ATPases.
One plausible explanation for an absence of Suc-LY-Amc cleavage would be that mycobacterial ClpPs require different physico-chemical conditions to hydrolyze this peptide than those needed by E. coli
ClpP. In fact, based on crystal structure determination, the tetradecamer of ClpPs from different organisms could be grouped into two structural states: an extended state fully active toward the Suc-LY-Amc model peptide (seen with E. coli
, Homo sapiens
, H. pylori
ClpPs) and a more compact state that likely corresponds to an inactive state (seen with M. tuberculosis
, S. pneumoniae
, Plasmodium falciparum
]. Recombinant ClpP1 and ClpP2 might be isolated in the compact state and require specific physico-chemical conditions to switch to the extended fully active conformation toward model peptides.
Also, the specificities of peptide bond hydrolysis could be different than those of E
ClpP. Indeed, the nature of the amino acid in the P1 position relative to the scissile peptide is important in controlling the hydrolysis rate. For instance, E. coli
ClpP has been shown to exhibit the greatest degradation rate when a large aromatic amino acid residue is in the P1 position (as in the Suc-LY-Amc peptide) [28
However, this feature is not shared by all ClpPs since P. falciparum
ClpP exhibited a preference for an Arg residue in P1 position [29
]. Likewise, MTB ClpP1 and ClpP2 could require another amino acid residue at the P1 position. Consistent with such a hypothesis is our finding that ClpP1 can cleave a peptide bond after Ala and Arg residues. In the light of these findings, short peptides of those specificities (H-A-Amc, Suc-AAA-Amc, H-GR-Amc, Boc-LRR-Amc, Bz-VGR-Amc) were tested but none of them were cleaved by ClpP1 or ClpP2. Determination of the peptide specificities of MTB ClpPs must await further studies.
In this study, we have also shown that recombinant MTB ClpP1 and ClpP2 could not assemble as tetradecamers in solution. Determining the X-ray structure of ClpP1 has shown that ClpP1 could assemble in a tetradecamer under the crystal conditions but the interactions between the two heptamers that stabilize the tetradecamer were weaker than those in other ClpPs; and ClpP1 was mainly isolated as a heptamer in solution [21
]. Our findings suggest that a weak interaction between heptamers could also apply for ClpP2 assembly. In our study, we showed that deleting the amino-terminal extremity of ClpP1 and ClpP2 favored a higher order assembly. In most of the deposited X-ray structures, the amino-terminal extremity of mature ClpP is unmodelled because non interpretable in the electron density. This suggested a high flexibility in this portion of the protein. Indeed, Bewley et al. [23
] demonstrated that the first 20 residues in the mature E. coli
ClpP could adopt two different conformations. In the X-ray structures of the unprocessed full length MTB ClpP1, the first 15 residues were not visible and the Ser15-Glu27 portion formed a α-helix longer than in other orthologous ClpPs that partially occupies the axial pore [21
]. The flexibility of this might hinder a correct assembly of mycobacterial ClpPs and a deviation in its conformation form other orthologous ClpPs might explain a difference in the stabilization of the tetradecamer. Whether an interaction of mycobacterial ClpPs with the ATPase ClpX and ClpC could stabilize a tetradecameric assembly remains to be tested.
Despite that recombinant ClpP1 did not assemble in a tetradecamer, it exhibited a proteolytic activity that was reminiscent of the autocatalytic processing of ClpP proteases. Autocatalytic processing in ClpP might not require the tetradecameric functional assembly required for the processive proteolytic activity of ClpP. Indeed, the minimal functional structure for the hydrolytic activity of ClpP was found to be a single heptameric ring [30