BACKGROUND
in all organisms, atp-dependent proteases play an essential role by removing short-lived regulatory proteins whose rapid elimination is critical for cell metabolism and growth  <cit> .
they also allow riddance of misfolded and damaged proteins that accumulate in a variety of circumstances, notably during environmental stress  <cit> .
simultaneous orchestrated action of key regulatory proteins as well as protein quality control mechanism assure successful survival and virulence of bacteria.
the clp  proteolytic system is one of the major atp-dependent proteolytic complexes in bacteria.
it consists of a barrel-shaped tetradecameric clpp peptidase organized in two-stacked heptameric rings.
the active sites for peptide bond cleavage are sequestered inside a proteolytic chamber accessible only through narrow axial pores and whose entry is restricted to unfolded polypeptides  <cit> .
in e.coli, where the biochemical properties of clpp have been extensively studied, the peptidase can on its own efficiently degrade small peptides of up to six amino acid residues  <cit> .
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  <cit> .
this atpase complex, a ring-shaped 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.
mycobacterium tuberculosis , the etiologic agent of tuberculosis in humans, is one of the deadliest pathogens on earth, killing nearly  <dig> million people each year  <cit> .
mtb has unique biological properties that enable it to persist in phagosomes for decades in a poorly understood latent form and from where it can be reactivated and cause active tuberculosis  <cit> .
the phagosome is a hostile environment which is acidic, nutrient and oxygen poor, and oxidative and nitrosative due to the production of reactive oxygen and nitrogen species by the host  <cit> .
consistent with a powerful adaptive capacity, microarray studies have shown that mtb survival in macrophages is accompanied by changes in expression of about  <dig> genes, many of them involved in starvation, nitrosative and oxidative stress responses  <cit> .
one of the factors potentially involved in the pathogenesis of mtb is the clpp proteolytic complex, and growing evidences have involved this protease in the virulence of numerous other pathogen bacteria including salmonella typhimurium  <cit> , listeria monocytogenes  <cit> , streptococcus pneumoniae  <cit> , staphylococcus aureus  <cit> , and helicobacter pylori  <cit> .
the complete genome sequencing of the best-characterized m. tuberculosis strain  has revealed the presence of two paralog genes encoding putative clpp peptidase, clpp <dig>  and clpp <dig>  , probably organized as an operon  <cit> .
presence of multiple clpp genes is common to actinomyces, whereas most other bacteria contain only one clpp gene.
clpp <dig> has been predicted as essential for growth of mtb by transposon mutagenesis  <cit> .
both genes have their expression up-regulated during reaeration of mtb cultures  <cit>  and are important for mtb to replicate in macrophages  <cit> .
despite their potential importance in survival and virulence of mtb, to our knowledge, functional studies of these peptidases have not yet been reported.
in this study, in order to gain insight into the mechanism of protein degradation by clpps from mtb, we have produced and purified recombinant clpp <dig> and clpp <dig> and tested their peptidase activities.
no conventional chymotryptic activity could be detected toward the model peptide suc-ly-amc  under conditions that normally favor clpp activity.
however, clpp <dig> was shown to have a proteolytic activity responsible for its own cleavage after the arg <dig> residue and cleavage of clpp <dig> after the ala <dig> residue.
we have investigated whether the cleavage of model peptides by clpp <dig> and clpp <dig> would require their proteolytic processing, deletion of the amino termini that could prevent peptide entry, or the presence of the atpases clpc and clpx.
we have also tested how the truncations in the amino-termini of the proteins would change their oligomeric assembly and found that they led to a higher order of assembly.
RESULTS
recombinant clpp <dig> and clpp <dig> do not cleave the suc-ly-amc peptide but can inhibit e. coli clpp activity
the rv2461c and rv2460c genes encode proteins of respectively  <dig> and  <dig> amino acids annotated as clpp <dig> and clpp <dig>  alignment of the primary sequence of those proteins with that of e. coli clpp shows a conservation of the ser, his, and asp catalytic triad residues , indicating that they both have a putative ser peptidase activity.
in order to study the mycobacterial clpp peptidase activities, clpp <dig>  clpp <dig> and a clpp1-clpp <dig> operon were expressed under the control of t <dig> promoter in e. coli bl <dig> cells.
when the corresponding proteins were purified by a single ni2+ affinity chromatography and left for several weeks at 4°c, all the preparations exhibited a peptidase activity toward the suc-ly-amc fluorogenic peptides .
however, replacing the active site serine residue of clpp <dig>  and clpp <dig>  by alanine residues did not abolish the observed peptidase activity .
when clpp <dig> and clpp <dig> were produced in e. coli cells lacking endogenous clpp , no peptidase activity toward the suc-ly-amc peptide was detected .
also, when an extract was prepared from empty vector-expressing bl <dig> cells and subjected to a ni2+ affinity chromatography, and incubated with the suc-ly-amc peptide, no peptidase activity was found .
therefore, e. coli clpp rather than mtb clpp <dig> and clpp <dig> was responsible for the observed peptidase activities and the co-purification of e. coli clpp depends on the presence of clpp <dig> or clpp <dig>
it is noteworthy that not all protein preparations had the same specific peptidase activity toward suc-ly-amc nor it could be detected after the same duration of incubation at 4°c.
the proteins purified when the clpp1-clpp <dig> operon was overexpressed showed the most rapidly detectable peptidase activity  <dig> were used whereas  <dig> μg of the other preparations were used), and the clpp <dig> preparation needed to be incubated a longer time at 4°c in order to detect e. coli clpp peptidase activity.
in order to eliminate the contaminating e. coli clpp peptidase activity, the proteins purified when the clpp1-clpp <dig> operon was overexpressed were subjected to a second chromatographic step.
the elution fraction of the ni2+ column contained mainly clpp <dig> and a small amount of clpp <dig> .
when subjected to a size exclusion chromatography , this protein mixture eluted as a main peak with an elution volume of  <dig>  ml, which corresponds to a species having an apparent molecular mass of about  <dig> kda .
active e. coli clpp peptidase assembles in a tetradecameric complex of about  <dig> kda.
therefore, this peak should not contain any active tetradecamer of e. coli clpp.
fractions corresponding to the mycobacterial clpp peak were pooled and stored at 4°c.
as seen in figure 1c, after two days no peptidase activity was detectable.
after  <dig> days of storage at 4°c, a peptidase activity was measurable and this activity kept increasing after  <dig> days of storage.
comparable peptidase activity was observed if the protein mixture was not subjected to a sec but no peptidase activity could be observed if the clpp1-clpp <dig> operon was overexpressed in the sg1146a strain and the proteins subjected to a sec purification step .
this suggests that the major peak eluting from the sec column at  <dig>  ml contained, in addition to mtb clpps, inactive subunits of e. coli clpp that could reassemble into an active complex upon storage at 4°c.
this would imply that e. coli clpp subunits could associate with mtb clpp <dig> and clpp <dig> to form complexes inactive toward the suc-ly-amc peptide.
an interaction between e. coli clpp and mtb clpp <dig> or clpp <dig> was demonstrated by co-producing clpp <dig> or clpp <dig> with untagged e. coli clpp and showing that the binding of clpp to a ni2+ resin depended on the mtb clpp <dig> or clpp <dig> .
to verify that clpp <dig> or clpp <dig>  when interacting with e. coli clpp, would inhibit clpp peptidase activity, we tested whether expression of clpp <dig> and clpp <dig> could decrease cleavage of suc-ly-amc peptide by e.coli clpp.
as seen in figure 1d, a peptidase activity toward suc-ly-amc peptide was detected in total extract of bl <dig> that could be attributed to e. coli clpp since it dramatically decreased when clpp was inactivated .
expressing clpp <dig>  clpp <dig>  or the clpp1-clpp <dig> operon led to a decrease in e. coli clpp peptidase activity, indicating that mtb clpps subunits may interact with e. coli clpp subunits and inhibit its activity.
such kind of property has never been described for any other recombinant clpps produced in e. coli and might reflect a difference in the assembly pathway of protomers into tetradecamers.
our finding that the purified proteins released the active e. coli clpp more rapidly when clpp <dig> and clpp <dig> are expressed from an operon than when mtb clpps are produced independently might indicate a stronger, perhaps physiological, interaction between clpp <dig> and clpp <dig>  the copurification of clpp <dig> with clpp <dig> makes such a hypothesis very likely.
those findings demonstrated the need to produce mtb clpp <dig> and clpp <dig> in a bacterial strain that does not contain any endogenous e. coli clpp.
since we could not detect any chymotryptic peptidase activity by mtb clpp <dig> and clpp <dig> toward the suc-ly-amc peptide, we also tested cleavage of other peptides.
clpp <dig> and clpp <dig>  whether produced independently or from the clpp1-clpp <dig> operon, did not cleave the bz-vgr-amc  or boc-lrr-amc  peptides , indicating that they do not exhibit trypsin-like activity toward those peptides in the conditions used in our assay.
recombinant mtb clpp <dig> and clpp <dig> do not assemble into tetradecamers in solution
one of the hypotheses to explain the absence of any peptidase activity observed in our assay is the absence of a tetradecameric assembly.
therefore, the presence of a correct tetradecameric assembly was examined on purified clpp <dig> and clpp <dig>  when subjected to sec, purified clpp <dig> eluted as a species having an apparent molecular mass of  <dig> kda, compatible with monomers and dimers of clpp <dig> .
purified clpp <dig> eluted as a molecular complex having an apparent molecular mass of about  <dig> kda .
these results indicated that neither clpp <dig> nor clpp <dig> assembled in tetradecamers in these conditions.
the possibility that both clpp <dig> and clpp <dig> were needed to promote tetradecamer assembly was tested.
as seen in figure  <dig> , coproducing clpp <dig> with clpp <dig> did not dramatically change the elution volume of purified clpp <dig> and similarly coproducing clpp <dig> with clpp <dig> did not influence the elution volume of clpp <dig> .
furthermore, mixing purified clpp <dig> and clpp <dig> did not change the elution volumes of individual clpp <dig> or clpp <dig> .
therefore, tetradecameric assembly was not mutually induced by the presence of either clpp <dig> or clpp <dig>
it is noteworthy that the presence of a his tag at the c-termini of clpp <dig> and clpp <dig> could not explain the absence of tetradecamer formation since producing and purifying untagged clpp <dig> and clpp <dig> led to the same oligomeric assembly as his tagged peptidases .
peptidase activity and assembly of processed clpp <dig> and clpp2
we then explored different procedures to stimulate the peptidase activity and to promote correct tetradecameric assembly of clpp <dig> and clpp <dig>
in e. coli, clpp matures as an active peptidase by the autoproteolytic removal of the first  <dig> amino acid residues  <cit> .
in the x-ray structure of mtb clpp <dig>  the first  <dig> residues are not visible and it was suggested that the n-terminus of the protein was disordered and might prevent the entry of small peptides into the central proteolytic cavity, explaining the lack of peptidase activity  <cit> .
we thus tested whether processed clpp <dig> and clpp <dig> would correctly assemble in tetradecamers and exhibit chymotryptic activity.
processed e. coli clpp has been shown to start with ala <dig>  <cit> .
based on alignment with e.coli clpp, clpp <dig> is not expected to be processed .
if aligned with the whole sequence of streptomyces clpp <dig> , it could be processed at met <dig>  however, it was shown that streptomyces clpp <dig> had another putative initiation codon according to the codon usage  <cit>  and if aligned with the streptomyces clpp <dig> sequence starting with this second putative initiation codon mtb clpp <dig> would be processed at the ser <dig> residue .
based on alignement with e.coli clpp, clpp <dig> is predicted to be processed at tyr <dig>  in streptomyces, mature clpp <dig> was shown to start at val <dig>  <cit> , which would predict that mature mtb clpp <dig> starts with the ile <dig> residue .
several truncated variants of clpp <dig> starting with met <dig>  or ser <dig> , and of clpp <dig> starting with arg <dig> , tyr <dig> , ile <dig> , or leu <dig>  residues were produced and purified in sg1146a cells , and analyzed for peptide cleavage and oligomeric assembly.
removing the putative prosequences of clpp <dig> did not change its elution during sec .
likewise, most of the processed clpp <dig> variants eluted with an elution volume comparable to that of wild-type clpp <dig> .
however, the clpp2r <dig> variant surprisingly eluted as three main species.
in order to determine precisely the nature of these different oligomeric species, full-length clpp <dig> and the clpp2r <dig> variant were subjected to size-exclusion chromatography coupled online to a triple detector array  and further analyzed by static light scattering measurements.
in sec-tda experiment, the analysis of full-length clpp <dig> peak indicated the presence of a range of assemblies with a weight average molecular mass of  <dig> kda compatible with hexamers or heptamers of clpp <dig> subunits .
analysis of the clpp2r <dig> variant by sec-tda indicated species with molecular masses of  <dig>  kda,  <dig>  kda and  <dig>  kda corresponding to 21-mer, heptamer, and dimer species respectively .
the 21-mer oligomeric species was also observed during the elution of the clpp2y <dig>  but in a lower amount .
this high molecular weight complex is reminiscent of that observed with e. coli clpp in the absence of salt  <cit>  and could correspond to three stacked heptameric rings.
therefore, removing the putative prosequence of clpp <dig> did promote the formation of higher order oligomers but not of the expected tetradecamer.
none of those clpp <dig> or clpp <dig> variants exhibited any activity toward suc-ly-amc or boc-lrr-amc peptides .
thus, removing putative prosequences was not sufficient for allowing clpp <dig> and clpp <dig> to assemble into tetradecamers with activity toward model peptides.
deleting the entry gate does not activate clpp <dig> and clpp2
since the n-terminal extremity of clpp <dig> influenced its oligomerisation state, we tested whether further deletions in their n-termini would favor assembly of clpp <dig> and clpp <dig> in tetradecamer.
recent studies have demonstrated a gating mechanism for e. coli clpp comparable to that of proteasomes, a self-compartmentalized protease with a similar barrel-shaped architecture.
in general, the first  <dig> residues of mature clpp  adopt a flexible conformation that can explain why they are not always visible in electron density map.
in the mature processed clpp, the n-terminal extremity can adopt different secondary structures and therefore modulate the size of the entry pore  <cit> .
in the "up" conformation, a portion of the n-terminus extends outwards the access pore whereas in the "down" conformation it is located within the axial pore.
for e. coli clpp, removing the residues involved in the gating mechanism allowed degradation of unfolded substrates in the absence of the atpase complex  <cit> .
for the eukaryotic 20s proteasomes, the entry gate precludes entry of even small size peptides  <cit> .
in mtb clpp <dig>  the residues ser15-glu <dig> fold as a helix that is longer than the corresponding helix in clpp orthologues and that protruded into the axial pore of the tetradecamer, making a gating mechanism likely  <cit> .
to see whether removing the putative gate in mtb clpp <dig> and clpp <dig> would have an effect on peptide cleavage, n-terminal gate deletion variants were constructed based on the alignment with e. coli clpp .
in e. coli, deletion of the first  <dig>   <dig>  and  <dig> first amino acids  activated degradation of unfolded substrates to a different extent, with the Δ 14clpp variant being the most active.
corresponding deletion variants were produced and their peptidase activity and tetradecameric assembly were tested.
none of them exhibited any significant peptidase activity toward the suc-ly-amc peptide , suggesting that the absence of chymotryptic activity toward this peptide in wt clpp <dig> and clpp <dig> was not due to any obstruction of the pore entrance by their n-termini.
when we examined their assembly by sec-tda, we found that when the first  <dig> residues of clpp <dig> were absent, an additional peak was detected.
indeed, full-length clpp <dig> exhibited the presence of a several assemblies with molecular masses ranging from  <dig> to  <dig>  kda compatible with complexes varying from monomers to trimers of clpp <dig> subunits .
the clpp1l <dig> variant eluted as a sharper peak corresponding to a species having a molecular mass complex of about  <dig> kda, compatible with clpp <dig> heptamers .
thus, removing the n-terminal extremity of clpp <dig> likely favors assembly of the clpp <dig> heptamericring.
deleting the amino terminal residues of clpp <dig> slightly decreased its elution volume during sec but did not promote higher order oligomeric assembly above heptameric form .
these finding indicated that further removing the n-terminal extremities of clpp <dig> and clpp <dig> could stabilize the heptamericring.
coproduction of the peptidases with the atpases clpc <dig> and clpx
most of clpps, when tested, are able to degrade small peptides in the absence of the atpase complex.
one exception is the human mitochondrial clpp that required the atpase clpx to assemble into stable tetradecamer with a high peptidase activity  <cit> .
in the mtb genome, three genes have been annotated as clp/hsp <dig> proteolysis associated atpase, clpc <dig> , clpc <dig> , and clpx  .
primary sequences of clpc <dig> and clpx contain the typical clp atpase features, i. e. one or two aaa  domains.
however, clpc <dig> protein does not exhibit any aaa or other atpase motif and the purified clpc <dig> did not have any atpase activity .
the presence of two clpn domains in the clpc <dig> sequence might have been at the origin of the mistaken annotation of this open reading frame as a clp atpase.
therefore, only clpc <dig> and clpx seem to be putative atpase partners for clpp <dig> and clpp <dig> peptidases.
to investigate whether mtb clpp <dig> and clpp <dig> require their atpase partners to hydrolyze peptides, clpp <dig> and clpp <dig> were produced with clpc or clpx  and peptide cleavage was tested in total extracts.
the presence of clpx or clpc did not result in suc-ly-amc hydrolysis .
therefore, the absence of suc-ly-amc cleavage by clpp <dig> and clpp <dig> was not due to a requirement for the atpase complex.
clpp <dig> is responsible for clpp <dig> and clpp <dig> processing
when produced in e. coli, clpp <dig> migrated as two bands on sds-page .
microsequencing revealed that the upper band contained a protein starting with sqvtdm, corresponding to a full-length clpp <dig> that had its initiating methionine residue removed.
the smaller protein in clpp <dig> preparation  started with snsqg, corresponding to clpp <dig> starting at the ser <dig> residue.
when the ser catalytic residue of clpp <dig> was replaced by an ala , this form of clpp <dig> was not visible .
therefore, proteolytic activity of clpp <dig> was responsible for its own cleavage.
clpp <dig> migrated mainly as a single band on sds-page .
microsequencing showed that it corresponded to the full-length clpp <dig> .
when clpp <dig> was coproduced with clpp <dig>  two bands were obtained .
the protein of the upper band started with mnsqns residues and corresponded to full-length clpp <dig>  the protein in the lower band  started with ryilp and corresponded to clpp <dig> starting at the arg <dig> residue.
therefore, coproducing clpp <dig> and clpp <dig> led to processing of clpp <dig>  in order to test which peptidase was responsible for clpp <dig> processing, we inactivated clpp <dig> and clpp <dig> by replacing their active site serine by an alanine residue  in the clpp1-clpp <dig> operon.
inactivating clpp <dig> only did not prevent its processing  whereas clpp <dig> processing was not observed when clpp <dig> was inactivated .
altogether, these results demonstrated that recombinant clpp <dig> possessed a proteolytic activity responsible for its own cleavage after the arg <dig> residue and for clpp <dig> cleavage after the ala <dig> residue.
it is noteworthy that the truncated clpp <dig> form produced by the proteolytic activity of clpp <dig> corresponded to the putative processed clpp <dig> as predicted by sequence alignment.
in clpp <dig>  the processing cleavage site catalyzed by clpp <dig> was in the vicinity of that predicted by sequence alignment.
in conclusion, even though no suc-ly-amc cleavage was observed by clpp <dig>  this peptidase exhibited the ability to hydrolyze a peptide bond.
discussion
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  <cit> .
in this study, we have shown that, in conditions that normally allow suc-ly-amc cleavage by e. coli clpp, mtb clpp <dig> and clpp <dig> do not hydrolyze this model peptide.
however, a proteolytic activity was uncovered for clpp <dig>  ruling out the possibility that, at least for clpp <dig>  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 clpp <dig> or clpp <dig> 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 clpp <dig> and clpp <dig>  predicted to be highly flexible, since removing these extremities did not result in peptide cleavage.
furthermore, coproducing the clpx and clpc atpase complexes with clpp <dig> and clpp <dig> 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  and a more compact state that likely corresponds to an inactive state   <cit> .
recombinant clpp <dig> and clpp <dig> 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. coli clpp.
indeed, the nature of the amino acid in the p <dig> 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 p <dig> position   <cit> .
however, this feature is not shared by all clpps since p. falciparum clpp exhibited a preference for an arg residue in p <dig> position  <cit> .
likewise, mtb clpp <dig> and clpp <dig> could require another amino acid residue at the p <dig> position.
consistent with such a hypothesis is our finding that clpp <dig> can cleave a peptide bond after ala and arg residues.
in the light of these findings, short peptides of those specificities  were tested but none of them were cleaved by clpp <dig> or clpp <dig>  determination of the peptide specificities of mtb clpps must await further studies.
in this study, we have also shown that recombinant mtb clpp <dig> and clpp <dig> could not assemble as tetradecamers in solution.
determining the x-ray structure of clpp <dig> has shown that clpp <dig> 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 clpp <dig> was mainly isolated as a heptamer in solution  <cit> .
our findings suggest that a weak interaction between heptamers could also apply for clpp <dig> assembly.
in our study, we showed that deleting the amino-terminal extremity of clpp <dig> and clpp <dig> 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.
<cit>  demonstrated that the first  <dig> residues in the mature e. coli clpp could adopt two different conformations.
in the x-ray structures of the unprocessed full length mtb clpp <dig>  the first  <dig> residues were not visible and the ser15-glu <dig> portion formed a α-helix longer than in other orthologous clpps that partially occupies the axial pore  <cit> .
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 clpp <dig> 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  <cit> .
CONCLUSIONS
in this study, we have uncovered an unconventional mechanism of oligomeric assembly and proteolytic activity for mtb clpp <dig> and clpp <dig> that are distinct from those of other clpps, especially the well-characterized e. coli clpp.
a better knowledge of such atp-dependent proteases which are potentially important for the survival and virulence of mtb could offer new hope in the need for developing new drug to treat tuberculosis.

 in all organisms, atp-dependent proteases play an essential role by removing short-lived regulatory proteins whose rapid elimination is critical for cell metabolism and growth. they also allow riddance of misfolded and damaged proteins that accumulate in a variety of circumstances, notably during environmental stress. simultaneous orchestrated action of key regulatory proteins as well as protein quality control mechanism assure successful survival and virulence of bacteria. the clp proteolytic system is one of the major atp-dependent proteolytic complexes in bacteria. it consists of a barrel-shaped tetradecameric clpp peptidase organized in two-stacked heptameric rings. the active sites for peptide bond cleavage are sequestered inside a proteolytic chamber accessible only through narrow axial pores and whose entry is restricted to unfolded polypeptides. in e.coli, where the biochemical properties of clpp have been extensively studied, the peptidase can on its own efficiently degrade small peptides of up to six amino acid residues. 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. this atpase complex, a ring-shaped 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. mycobacterium tuberculosis, the etiologic agent of tuberculosis in humans, is one of the deadliest pathogens on earth, killing nearly million people each year. mtb has unique biological properties that enable it to persist in phagosomes for decades in a poorly understood latent form and from where it can be reactivated and cause active tuberculosis. the phagosome is a hostile environment which is acidic, nutrient and oxygen poor, and oxidative and nitrosative due to the production of reactive oxygen and nitrogen species by the host. consistent with a powerful adaptive capacity, microarray studies have shown that mtb survival in macrophages is accompanied by changes in expression of about genes, many of them involved in starvation, nitrosative and oxidative stress responses. one of the factors potentially involved in the pathogenesis of mtb is the clpp proteolytic complex, and growing evidences have involved this protease in the virulence of numerous other pathogen bacteria including salmonella typhimurium, listeria monocytogenes, streptococcus pneumoniae, staphylococcus aureus, and helicobacter pylori. the complete genome sequencing of the best-characterized m. tuberculosis strain has revealed the presence of two paralog genes encoding putative clpp peptidase, clpp and clpp, probably organized as an operon. presence of multiple clpp genes is common to actinomyces, whereas most other bacteria contain only one clpp gene. clpp has been predicted as essential for growth of mtb by transposon mutagenesis. both genes have their expression up-regulated during reaeration of mtb cultures and are important for mtb to replicate in macrophages. despite their potential importance in survival and virulence of mtb, to our knowledge, functional studies of these peptidases have not yet been reported. in this study, in order to gain insight into the mechanism of protein degradation by clpps from mtb, we have produced and purified recombinant clpp and clpp and tested their peptidase activities. no conventional chymotryptic activity could be detected toward the model peptide suc-ly-amc under conditions that normally favor clpp activity. however, clpp was shown to have a proteolytic activity responsible for its own cleavage after the arg residue and cleavage of clpp after the ala residue. we have investigated whether the cleavage of model peptides by clpp and clpp would require their proteolytic processing, deletion of the amino termini that could prevent peptide entry, or the presence of the atpases clpc and clpx. we have also tested how the truncations in the amino-termini of the proteins would change their oligomeric assembly and found that they led to a higher order of assembly.  recombinant clpp and clpp do not cleave the suc-ly-amc peptide but can inhibit e. coli clpp activity the rv2461c and rv2460c genes encode proteins of respectively and amino acids annotated as clpp and clpp alignment of the primary sequence of those proteins with that of e. coli clpp shows a conservation of the ser, his, and asp catalytic triad residues, indicating that they both have a putative ser peptidase activity. in order to study the mycobacterial clpp peptidase activities, clpp clpp and a clpp1-clpp operon were expressed under the control of t promoter in e. coli bl cells. when the corresponding proteins were purified by a single ni2+ affinity chromatography and left for several weeks at 4°c, all the preparations exhibited a peptidase activity toward the suc-ly-amc fluorogenic peptides. however, replacing the active site serine residue of clpp and clpp by alanine residues did not abolish the observed peptidase activity. when clpp and clpp were produced in e. coli cells lacking endogenous clpp, no peptidase activity toward the suc-ly-amc peptide was detected. also, when an extract was prepared from empty vector-expressing bl cells and subjected to a ni2+ affinity chromatography, and incubated with the suc-ly-amc peptide, no peptidase activity was found. therefore, e. coli clpp rather than mtb clpp and clpp was responsible for the observed peptidase activities and the co-purification of e. coli clpp depends on the presence of clpp or clpp it is noteworthy that not all protein preparations had the same specific peptidase activity toward suc-ly-amc nor it could be detected after the same duration of incubation at 4°c. the proteins purified when the clpp1-clpp operon was overexpressed showed the most rapidly detectable peptidase activity were used whereas μg of the other preparations were used), and the clpp preparation needed to be incubated a longer time at 4°c in order to detect e. coli clpp peptidase activity. in order to eliminate the contaminating e. coli clpp peptidase activity, the proteins purified when the clpp1-clpp operon was overexpressed were subjected to a second chromatographic step. the elution fraction of the ni2+ column contained mainly clpp and a small amount of clpp. when subjected to a size exclusion chromatography, this protein mixture eluted as a main peak with an elution volume of ml, which corresponds to a species having an apparent molecular mass of about kda. active e. coli clpp peptidase assembles in a tetradecameric complex of about kda. therefore, this peak should not contain any active tetradecamer of e. coli clpp. fractions corresponding to the mycobacterial clpp peak were pooled and stored at 4°c. as seen in figure 1c, after two days no peptidase activity was detectable. after days of storage at 4°c, a peptidase activity was measurable and this activity kept increasing after days of storage. comparable peptidase activity was observed if the protein mixture was not subjected to a sec but no peptidase activity could be observed if the clpp1-clpp operon was overexpressed in the sg1146a strain and the proteins subjected to a sec purification step. this suggests that the major peak eluting from the sec column at ml contained, in addition to mtb clpps, inactive subunits of e. coli clpp that could reassemble into an active complex upon storage at 4°c. this would imply that e. coli clpp subunits could associate with mtb clpp and clpp to form complexes inactive toward the suc-ly-amc peptide. an interaction between e. coli clpp and mtb clpp or clpp was demonstrated by co-producing clpp or clpp with untagged e. coli clpp and showing that the binding of clpp to a ni2+ resin depended on the mtb clpp or clpp. to verify that clpp or clpp when interacting with e. coli clpp, would inhibit clpp peptidase activity, we tested whether expression of clpp and clpp could decrease cleavage of suc-ly-amc peptide by e.coli clpp. as seen in figure 1d, a peptidase activity toward suc-ly-amc peptide was detected in total extract of bl that could be attributed to e. coli clpp since it dramatically decreased when clpp was inactivated. expressing clpp clpp or the clpp1-clpp operon led to a decrease in e. coli clpp peptidase activity, indicating that mtb clpps subunits may interact with e. coli clpp subunits and inhibit its activity. such kind of property has never been described for any other recombinant clpps produced in e. coli and might reflect a difference in the assembly pathway of protomers into tetradecamers. our finding that the purified proteins released the active e. coli clpp more rapidly when clpp and clpp are expressed from an operon than when mtb clpps are produced independently might indicate a stronger, perhaps physiological, interaction between clpp and clpp the copurification of clpp with clpp makes such a hypothesis very likely. those findings demonstrated the need to produce mtb clpp and clpp in a bacterial strain that does not contain any endogenous e. coli clpp. since we could not detect any chymotryptic peptidase activity by mtb clpp and clpp toward the suc-ly-amc peptide, we also tested cleavage of other peptides. clpp and clpp whether produced independently or from the clpp1-clpp operon, did not cleave the bz-vgr-amc or boc-lrr-amc peptides, indicating that they do not exhibit trypsin-like activity toward those peptides in the conditions used in our assay. recombinant mtb clpp and clpp do not assemble into tetradecamers in solution one of the hypotheses to explain the absence of any peptidase activity observed in our assay is the absence of a tetradecameric assembly. therefore, the presence of a correct tetradecameric assembly was examined on purified clpp and clpp when subjected to sec, purified clpp eluted as a species having an apparent molecular mass of kda, compatible with monomers and dimers of clpp. purified clpp eluted as a molecular complex having an apparent molecular mass of about kda. these results indicated that neither clpp nor clpp assembled in tetradecamers in these conditions. the possibility that both clpp and clpp were needed to promote tetradecamer assembly was tested. as seen in figure, coproducing clpp with clpp did not dramatically change the elution volume of purified clpp and similarly coproducing clpp with clpp did not influence the elution volume of clpp. furthermore, mixing purified clpp and clpp did not change the elution volumes of individual clpp or clpp. therefore, tetradecameric assembly was not mutually induced by the presence of either clpp or clpp it is noteworthy that the presence of a his tag at the c-termini of clpp and clpp could not explain the absence of tetradecamer formation since producing and purifying untagged clpp and clpp led to the same oligomeric assembly as his tagged peptidases. peptidase activity and assembly of processed clpp and clpp2 we then explored different procedures to stimulate the peptidase activity and to promote correct tetradecameric assembly of clpp and clpp in e. coli, clpp matures as an active peptidase by the autoproteolytic removal of the first amino acid residues. in the x-ray structure of mtb clpp the first residues are not visible and it was suggested that the n-terminus of the protein was disordered and might prevent the entry of small peptides into the central proteolytic cavity, explaining the lack of peptidase activity. we thus tested whether processed clpp and clpp would correctly assemble in tetradecamers and exhibit chymotryptic activity. processed e. coli clpp has been shown to start with ala. based on alignment with e.coli clpp, clpp is not expected to be processed. if aligned with the whole sequence of streptomyces clpp, it could be processed at met however, it was shown that streptomyces clpp had another putative initiation codon according to the codon usage and if aligned with the streptomyces clpp sequence starting with this second putative initiation codon mtb clpp would be processed at the ser residue. based on alignement with e.coli clpp, clpp is predicted to be processed at tyr in streptomyces, mature clpp was shown to start at val, which would predict that mature mtb clpp starts with the ile residue. several truncated variants of clpp starting with met or ser, and of clpp starting with arg, tyr, ile, or leu residues were produced and purified in sg1146a cells, and analyzed for peptide cleavage and oligomeric assembly. removing the putative prosequences of clpp did not change its elution during sec. likewise, most of the processed clpp variants eluted with an elution volume comparable to that of wild-type clpp. however, the clpp2r variant surprisingly eluted as three main species. in order to determine precisely the nature of these different oligomeric species, full-length clpp and the clpp2r variant were subjected to size-exclusion chromatography coupled online to a triple detector array and further analyzed by static light scattering measurements. in sec-tda experiment, the analysis of full-length clpp peak indicated the presence of a range of assemblies with a weight average molecular mass of kda compatible with hexamers or heptamers of clpp subunits. analysis of the clpp2r variant by sec-tda indicated species with molecular masses of kda, kda and kda corresponding to 21-mer, heptamer, and dimer species respectively. the 21-mer oligomeric species was also observed during the elution of the clpp2y but in a lower amount. this high molecular weight complex is reminiscent of that observed with e. coli clpp in the absence of salt and could correspond to three stacked heptameric rings. therefore, removing the putative prosequence of clpp did promote the formation of higher order oligomers but not of the expected tetradecamer. none of those clpp or clpp variants exhibited any activity toward suc-ly-amc or boc-lrr-amc peptides. thus, removing putative prosequences was not sufficient for allowing clpp and clpp to assemble into tetradecamers with activity toward model peptides. deleting the entry gate does not activate clpp and clpp2 since the n-terminal extremity of clpp influenced its oligomerisation state, we tested whether further deletions in their n-termini would favor assembly of clpp and clpp in tetradecamer. recent studies have demonstrated a gating mechanism for e. coli clpp comparable to that of proteasomes, a self-compartmentalized protease with a similar barrel-shaped architecture. in general, the first residues of mature clpp adopt a flexible conformation that can explain why they are not always visible in electron density map. in the mature processed clpp, the n-terminal extremity can adopt different secondary structures and therefore modulate the size of the entry pore. in the "up" conformation, a portion of the n-terminus extends outwards the access pore whereas in the "down" conformation it is located within the axial pore. for e. coli clpp, removing the residues involved in the gating mechanism allowed degradation of unfolded substrates in the absence of the atpase complex. for the eukaryotic 20s proteasomes, the entry gate precludes entry of even small size peptides. in mtb clpp the residues ser15-glu fold as a helix that is longer than the corresponding helix in clpp orthologues and that protruded into the axial pore of the tetradecamer, making a gating mechanism likely. to see whether removing the putative gate in mtb clpp and clpp would have an effect on peptide cleavage, n-terminal gate deletion variants were constructed based on the alignment with e. coli clpp. in e. coli, deletion of the first and first amino acids activated degradation of unfolded substrates to a different extent, with the Δ 14clpp variant being the most active. corresponding deletion variants were produced and their peptidase activity and tetradecameric assembly were tested. none of them exhibited any significant peptidase activity toward the suc-ly-amc peptide, suggesting that the absence of chymotryptic activity toward this peptide in wt clpp and clpp was not due to any obstruction of the pore entrance by their n-termini. when we examined their assembly by sec-tda, we found that when the first residues of clpp were absent, an additional peak was detected. indeed, full-length clpp exhibited the presence of a several assemblies with molecular masses ranging from to kda compatible with complexes varying from monomers to trimers of clpp subunits. the clpp1l variant eluted as a sharper peak corresponding to a species having a molecular mass complex of about kda, compatible with clpp heptamers. thus, removing the n-terminal extremity of clpp likely favors assembly of the clpp heptamericring. deleting the amino terminal residues of clpp slightly decreased its elution volume during sec but did not promote higher order oligomeric assembly above heptameric form. these finding indicated that further removing the n-terminal extremities of clpp and clpp could stabilize the heptamericring. coproduction of the peptidases with the atpases clpc and clpx most of clpps, when tested, are able to degrade small peptides in the absence of the atpase complex. one exception is the human mitochondrial clpp that required the atpase clpx to assemble into stable tetradecamer with a high peptidase activity. in the mtb genome, three genes have been annotated as clp/hsp proteolysis associated atpase, clpc, clpc, and clpx. primary sequences of clpc and clpx contain the typical clp atpase features, i. e. one or two aaa domains. however, clpc protein does not exhibit any aaa or other atpase motif and the purified clpc did not have any atpase activity. the presence of two clpn domains in the clpc sequence might have been at the origin of the mistaken annotation of this open reading frame as a clp atpase. therefore, only clpc and clpx seem to be putative atpase partners for clpp and clpp peptidases. to investigate whether mtb clpp and clpp require their atpase partners to hydrolyze peptides, clpp and clpp were produced with clpc or clpx and peptide cleavage was tested in total extracts. the presence of clpx or clpc did not result in suc-ly-amc hydrolysis. therefore, the absence of suc-ly-amc cleavage by clpp and clpp was not due to a requirement for the atpase complex. clpp is responsible for clpp and clpp processing when produced in e. coli, clpp migrated as two bands on sds-page. microsequencing revealed that the upper band contained a protein starting with sqvtdm, corresponding to a full-length clpp that had its initiating methionine residue removed. the smaller protein in clpp preparation started with snsqg, corresponding to clpp starting at the ser residue. when the ser catalytic residue of clpp was replaced by an ala, this form of clpp was not visible. therefore, proteolytic activity of clpp was responsible for its own cleavage. clpp migrated mainly as a single band on sds-page. microsequencing showed that it corresponded to the full-length clpp. when clpp was coproduced with clpp two bands were obtained. the protein of the upper band started with mnsqns residues and corresponded to full-length clpp the protein in the lower band started with ryilp and corresponded to clpp starting at the arg residue. therefore, coproducing clpp and clpp led to processing of clpp in order to test which peptidase was responsible for clpp processing, we inactivated clpp and clpp by replacing their active site serine by an alanine residue in the clpp1-clpp operon. inactivating clpp only did not prevent its processing whereas clpp processing was not observed when clpp was inactivated. altogether, these results demonstrated that recombinant clpp possessed a proteolytic activity responsible for its own cleavage after the arg residue and for clpp cleavage after the ala residue. it is noteworthy that the truncated clpp form produced by the proteolytic activity of clpp corresponded to the putative processed clpp as predicted by sequence alignment. in clpp the processing cleavage site catalyzed by clpp was in the vicinity of that predicted by sequence alignment. in conclusion, even though no suc-ly-amc cleavage was observed by clpp this peptidase exhibited the ability to hydrolyze a peptide bond. discussion 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 clpp and clpp do not hydrolyze this model peptide. however, a proteolytic activity was uncovered for clpp ruling out the possibility that, at least for clpp 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 clpp or clpp 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 clpp and clpp predicted to be highly flexible, since removing these extremities did not result in peptide cleavage. furthermore, coproducing the clpx and clpc atpase complexes with clpp and clpp 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 and a more compact state that likely corresponds to an inactive state. recombinant clpp and clpp 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. coli clpp. indeed, the nature of the amino acid in the p 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 p position. however, this feature is not shared by all clpps since p. falciparum clpp exhibited a preference for an arg residue in p position. likewise, mtb clpp and clpp could require another amino acid residue at the p position. consistent with such a hypothesis is our finding that clpp can cleave a peptide bond after ala and arg residues. in the light of these findings, short peptides of those specificities were tested but none of them were cleaved by clpp or clpp determination of the peptide specificities of mtb clpps must await further studies. in this study, we have also shown that recombinant mtb clpp and clpp could not assemble as tetradecamers in solution. determining the x-ray structure of clpp has shown that clpp 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 clpp was mainly isolated as a heptamer in solution. our findings suggest that a weak interaction between heptamers could also apply for clpp assembly. in our study, we showed that deleting the amino-terminal extremity of clpp and clpp 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. demonstrated that the first residues in the mature e. coli clpp could adopt two different conformations. in the x-ray structures of the unprocessed full length mtb clpp the first residues were not visible and the ser15-glu portion formed a α-helix longer than in other orthologous clpps that partially occupies the axial pore. 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 clpp 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.  in this study, we have uncovered an unconventional mechanism of oligomeric assembly and proteolytic activity for mtb clpp and clpp that are distinct from those of other clpps, especially the well-characterized e. coli clpp. a better knowledge of such atp-dependent proteases which are potentially important for the survival and virulence of mtb could offer new hope in the need for developing new drug to treat tuberculosis. 
use of fluorogenic model peptides of diverse specificities failed to show peptidase activity with recombinant mycobacterial clpp or clpp however, we found that clpp had a proteolytic activity responsible for its own cleavage after the arg residue and cleavage of clpp after the ala 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 clpp and clpp did not result in cleavage of model peptides. deleting the amino-terminal extremity of clpp and clpp 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. despite the conservation of a ser protease catalytic triad in their primary sequences, mycobacterial clpp and clpp do not have conventional peptidase activity toward peptide models and display an unusual mechanism of self-assembly. the concomitant presence of both clpp and clpp did not result in tetradecameric assembly. caseinolytic proteases are barrel-shaped self-compartmentalized peptidases involved in eliminating damaged or short-lived regulatory proteins.
 caseinolytic proteases are barrel-shaped self-compartmentalized peptidases involved in eliminating damaged or short-lived regulatory proteins. the mycobacterium tuberculosis genome contains two genes coding for putative clpps, clpp and clpp respectively, that are likely to play a role in the virulence of the bacterium.  we report the first biochemical characterization of clpp and clpp 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 clpp or clpp however, we found that clpp had a proteolytic activity responsible for its own cleavage after the arg residue and cleavage of clpp after the ala 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 clpp and clpp did not result in cleavage of model peptides. we have also shown that recombinant clpp and clpp 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 clpp and clpp did not result in tetradecameric assembly. deleting the amino-terminal extremity of clpp and clpp 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.  despite the conservation of a ser protease catalytic triad in their primary sequences, mycobacterial clpp and clpp 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. 