Antibacterial activity of and resistance to small molecule inhibitors of the ClpP peptidase.
ABSTRACT: There is rapidly mounting evidence that intracellular proteases in bacteria are compelling targets for antibacterial drugs. Multiple reports suggest that the human pathogen Mycobacterium tuberculosis and other actinobacteria may be particularly sensitive to small molecules that perturb the activities of self-compartmentalized peptidases, which catalyze intracellular protein turnover as components of ATP-dependent proteolytic machines. Here, we report chemical syntheses and evaluations of structurally diverse ?-lactones, which have a privileged structure for selective, suicide inhibition of the self-compartmentalized ClpP peptidase. ?-Lactones with certain substituents on the ?- and ?-carbons were found to be toxic to M. tuberculosis. Using an affinity-labeled analogue of a bioactive ?-lactone in a series of chemical proteomic experiments, we selectively captured the ClpP1P2 peptidase from live cultures of two different actinobacteria that are related to M. tuberculosis. Importantly, we found that the growth inhibitory ?-lactones also inactivate the M. tuberculosis ClpP1P2 peptidase in vitro via formation of a covalent adduct at the ClpP2 catalytic serine. Given the potent antibacterial activity of these compounds and their medicinal potential, we sought to identify innate mechanisms of resistance. Using a genome mining strategy, we identified a genetic determinant of ?-lactone resistance in Streptomyces coelicolor, a non-pathogenic relative of M. tuberculosis. Collectively, these findings validate the potential of ClpP inhibition as a strategy in antibacterial drug development and define a mechanism by which bacteria could resist the toxic effects of ClpP inhibitors.
Project description:Clp chaperone-proteases are cylindrical complexes built from ATP-dependent chaperonerings that stack onto a proteolytic ClpP double-ring core to carry out substrate protein degradation.Interaction of the ClpP particle with the chaperone is mediated by an N-terminal loop and a hydrophobic surface patch on the ClpP ring surface. In contrast to E. coli, Myco bacterium tuberculosis harbors not only one but two ClpP protease subunits, ClpP1 and ClpP2,and a homo-heptameric ring of each assembles to form the ClpP1P2 double-ring core. Consequently,this hetero double-ring presents two different potential binding surfaces for the interaction with the chaperones ClpX and ClpC1. To investigate whether ClpX or ClpC1 might preferentially interact with one or the other double-ring face, we mutated the hydrophobicchaperone-interaction patch on either ClpP1 or ClpP2, generating ClpP1P2 particles that are defective in one of the two binding patches and thereby in their ability to interact with their chaperone partners. Using chaperone-mediated degradation of ssrA-tagged model substrates, we show that both Mycobacterium tuberculosis Clp chaperones require the intact interaction face of ClpP2 to support degradation, resulting in an asymmetric complex where chaperones only bind to the ClpP2 side of the proteolytic core. This sets the Clpproteases of Mycobacterium tuberculosis, and probably other Actinobacteria, apart from the well-studied E. coli system, where chaperones bind to both sides of the protease core,and it frees the ClpP1 interaction interface for putative new binding partners [corrected].
Project description:Caseinolytic peptidase P (ClpP), a double-ring peptidase with 14 subunits, collaborates with ATPases associated with diverse activities (AAA+) partners to execute ATP-dependent protein degradation. Although many ClpP enzymes self-assemble into catalytically active homo-tetradecamers able to cleave small peptides, the Mycobacterium tuberculosis enzyme consists of discrete ClpP1 and ClpP2 heptamers that require a AAA+ partner and protein-substrate delivery or a peptide agonist to stabilize assembly of the active tetradecamer. Here, we show that cyclic acyldepsipeptides (ADEPs) and agonist peptides synergistically activate ClpP1P2 by mimicking AAA+ partners and substrates, respectively, and determine the structure of the activated complex. Our studies establish the basis of heteromeric ClpP1P2 assembly and function, reveal tight coupling between the conformations of each ring, show that ADEPs bind only to one ring but appear to open the axial pores of both rings, provide a foundation for rational drug development, and suggest strategies for studying the roles of individual ClpP1 and ClpP2 rings in Clp-family proteolysis.
Project description: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.
Project description:The ClpP protease complex and its regulatory ATPases, ClpC1 and ClpX, inMycobacterium tuberculosis(Mtb) are essential and, therefore, promising drug targets. TheMtbClpP protease consists of two heptameric rings, one composed of ClpP1 and the other of ClpP2 subunits. Formation of the enzymatically active ClpP1P2 complex requires binding of N-blocked dipeptide activators. We have found a new potent activator, benzoyl-leucine-leucine (Bz-LL), that binds with higher affinity and promotes 3-4-fold higher peptidase activity than previous activators. Bz-LL-activated ClpP1P2 specifically stimulates the ATPase activity ofMtbClpC1 and ClpX. The ClpC1P1P2 and ClpXP1P2 complexes exhibit 2-3-fold enhanced ATPase activity, peptide cleavage, and ATP-dependent protein degradation. The crystal structure of ClpP1P2 with bound Bz-LL was determined at a resolution of 3.07 Å and with benzyloxycarbonyl-Leu-Leu (Z-LL) bound at 2.9 Å. Bz-LL was present in all 14 active sites, whereas Z-LL density was not resolved. Surprisingly, Bz-LL adopts opposite orientations in ClpP1 and ClpP2. In ClpP1, Bz-LL binds with the C-terminal leucine side chain in the S1 pocket. One C-terminal oxygen is close to the catalytic serine, whereas the other contacts backbone amides in the oxyanion hole. In ClpP2, Bz-LL binds with the benzoyl group in the S1 pocket, and the peptide hydrogen bonded between parallel ?-strands. The ClpP2 axial loops are extended, forming an open axial channel as has been observed with bound ADEP antibiotics. Thus occupancy of the active sites of ClpP allosterically alters sites on the surfaces thereby affecting the association of ClpP1 and ClpP2 rings, interactions with regulatory ATPases, and entry of protein substrates.
Project description:Mycobacterial Clp-family proteases function via collaboration of the heteromeric ClpP1P2 peptidase with a AAA+ partner, ClpX or ClpC1. These enzymes are essential for M. tuberculosis viability and are validated antibacterial drug targets, but the requirements for assembly and regulation of functional proteolytic complexes are poorly understood. Here, we report the reconstitution of protein degradation by mycobacterial Clp proteases in vitro and describe novel features of these enzymes that distinguish them from orthologues in other bacteria. Both ClpX and ClpC1 catalyse ATP-dependent unfolding and degradation of native protein substrates in conjunction with ClpP1P2, but neither mediates protein degradation with just ClpP1 or ClpP2. ClpP1P2 alone has negligible peptidase activity, but is strongly stimulated by translocation of protein substrates into ClpP1P2 by either AAA+ partner. Interestingly, our results support a model in which both binding of a AAA+ partner and protein-substrate delivery are required to stabilize active ClpP1P2. Our model has implications for therapeutically targeting ClpP1P2 in dormant M. tuberculosis, and our reconstituted systems should facilitate identification of novel Clp protease inhibitors and activators.
Project description:The Clp protease complex in Mycobacterium tuberculosis is unusual in its composition, functional importance and activation mechanism. Whilst most bacterial species contain a single ClpP protein that is dispensable for normal growth, mycobacteria have two ClpPs, ClpP1 and ClpP2, which are essential for viability and together form the ClpP1P2 tetradecamer. Acyldepsipeptide antibiotics of the ADEP class inhibit the growth of Gram-positive firmicutes by activating ClpP and causing unregulated protein degradation. Here we show that, in contrast, mycobacteria are killed by ADEP through inhibition of ClpP function. Although ADEPs can stimulate purified M. tuberculosis ClpP1P2 to degrade larger peptides and unstructured proteins, this effect is weaker than for ClpP from other bacteria and depends on the presence of an additional activating factor (e.g. the dipeptide benzyloxycarbonyl-leucyl-leucine in vitro) to form the active ClpP1P2 tetradecamer. The cell division protein FtsZ, which is a particularly sensitive target for ADEP-activated ClpP in firmicutes, is not degraded in mycobacteria. Depletion of the ClpP1P2 level in a conditional Mycobacterium bovis BCG mutant enhanced killing by ADEP unlike in other bacteria. In summary, ADEPs kill mycobacteria by preventing interaction of ClpP1P2 with the regulatory ATPases, ClpX or ClpC1, thus inhibiting essential ATP-dependent protein degradation.
Project description:In most bacteria, Clp protease is a conserved, non-essential serine protease that regulates the response to various stresses. Mycobacteria, including Mycobacterium tuberculosis (Mtb) and Mycobacterium smegmatis, unlike most well studied prokaryotes, encode two ClpP homologs, ClpP1 and ClpP2, in a single operon. Here we demonstrate that the two proteins form a mixed complex (ClpP1P2) in mycobacteria. Using two different approaches, promoter replacement, and a novel system of inducible protein degradation, leading to inducible expression of clpP1 and clpP2, we demonstrate that both genes are essential for growth and that a marked depletion of either one results in rapid bacterial death. ClpP1P2 protease appears important in degrading missense and prematurely terminated peptides, as partial depletion of ClpP2 reduced growth specifically in the presence of antibiotics that increase errors in translation. We further show that the ClpP1P2 protease is required for the degradation of proteins tagged with the SsrA motif, a tag co-translationally added to incomplete protein products. Using active site mutants of ClpP1 and ClpP2, we show that the activity of each subunit is required for proteolysis, for normal growth of Mtb in vitro and during infection of mice. These observations suggest that the Clp protease plays an unusual and essential role in Mtb and may serve as an ideal target for antimycobacterial therapy.
Project description:BACKGROUND: Caseinolytic 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. RESULTS: We 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. CONCLUSION: Despite 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.
Project description:Caseinolytic protease P (ClpP) has emerged as a promising new target for antibacterial development. While ClpPs from single isoform expressing bacteria have been studied in detail, the function and regulation of systems with more than one ClpP homologue are still poorly understood. Herein, we present fundamental studies toward understanding the ClpP system in C. difficile, an anaerobic spore-forming pathogen that contains two chromosomally distant isoforms of ClpP. Examination of proteomic and genomic data suggest that ClpP1 is the primary isoform responsible for normal growth and virulence, but little is known about the function of ClpP2 or the context required for the formation of functional proteases. For the first time in a pathogenic bacterium, we demonstrate that both isoforms are capable of forming operative proteases. Interestingly, ClpP1 is the only homologue that possesses characteristic response to small molecule acyldepsipeptide activation. On the contrary, both ClpP1 and ClpP2 respond to cochaperone activation to degrade an ssrA-tagged substrate. These observations indicate that ClpP2 is less susceptible to acyldepsipeptide activation but retains the ability to interact with a known cochaperone. Homology models reveal no obvious characteristics that would allow one to predict less efficient acyldepsipeptide binding. The reported findings establish the uniqueness of the ClpP system in C. difficile, open new avenues of inquiry, and highlight the importance of more detailed structural, genetic, and biological characterization of the ClpP system in C. difficile.
Project description:Caseinolytic proteases (ClpPs) are large oligomeric protein complexes that contribute to cell homeostasis as well as virulence regulation in bacteria. Although most organisms possess a single ClpP protein, some organisms encode two or more ClpP isoforms. Here, we elucidated the crystal structures of ClpP1 and ClpP2 from pathogenic Listeria monocytogenes and observe an unprecedented regulation principle by the catalytic triad. Whereas L. monocytogenes (Lm)ClpP2 is both structurally and functionally similar to previously studied tetradecameric ClpP proteins from Escherichia coli and Staphylococcus aureus, heptameric LmClpP1 features an asparagine in its catalytic triad. Mutation of this asparagine to aspartate increased the reactivity of the active site and led to the assembly of a tetradecameric complex. We analyzed the heterooligomeric complex of LmClpP1 and LmClpP2 via coexpression and subsequent labeling studies with natural product-derived probes. Notably, the LmClpP1 peptidase activity is stimulated 75-fold in the complex providing insights into heterooligomerization as a regulatory mechanism. Collectively, our data point toward different preferences for substrates and inhibitors of the two ClpP enzymes and highlight their structural and functional characteristics.