The active ClpP protease from M. tuberculosis is a complex composed of a heptameric ClpP1 and a ClpP2 ring
2012; Springer Nature; Volume: 31; Issue: 6 Linguagem: Inglês
10.1038/emboj.2012.5
ISSN1460-2075
AutoresTatos Akopian, Olga Kandror, Ravikiran M. Raju, Meera Unnikrishnan, Eric J. Rubin, Alfred L. Goldberg,
Tópico(s)Peptidase Inhibition and Analysis
ResumoArticle27 January 2012free access The active ClpP protease from M. tuberculosis is a complex composed of a heptameric ClpP1 and a ClpP2 ring Tatos Akopian Tatos Akopian Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Olga Kandror Olga Kandror Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Ravikiran M Raju Ravikiran M Raju Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA, USA Search for more papers by this author Meera UnniKrishnan Meera UnniKrishnan Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA, USA Search for more papers by this author Eric J Rubin Eric J Rubin Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA, USA Search for more papers by this author Alfred L Goldberg Corresponding Author Alfred L Goldberg Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Tatos Akopian Tatos Akopian Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Olga Kandror Olga Kandror Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Ravikiran M Raju Ravikiran M Raju Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA, USA Search for more papers by this author Meera UnniKrishnan Meera UnniKrishnan Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA, USA Search for more papers by this author Eric J Rubin Eric J Rubin Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA, USA Search for more papers by this author Alfred L Goldberg Corresponding Author Alfred L Goldberg Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Author Information Tatos Akopian1,‡, Olga Kandror1,‡, Ravikiran M Raju2, Meera UnniKrishnan2, Eric J Rubin2 and Alfred L Goldberg 1 1Department of Cell Biology, Harvard Medical School, Boston, MA, USA 2Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA, USA ‡These authors contributed equally to this work *Corresponding author. Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA. Tel.: +1 617 432 1854; Fax: +1 617 232 0173; E-mail: [email protected] The EMBO Journal (2012)31:1529-1541https://doi.org/10.1038/emboj.2012.5 There is an Article (March 2012) associated with this Have you seen?. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction Tuberculosis is a devastating disease that affects worldwide about 100 million people and causes nearly 2 million deaths annually. It has been estimated that one third of all humans is infected with latent Mycobacterium tuberculosis (Mtb). Moreover, Mtb has become increasingly resistant to available antibiotics. Consequently, it is important to identify and characterize new therapeutic targets in Mtb and to synthesize selective inhibitors. Ideal targets for drug development should be enzymes essential for bacterial viability that differ in physicochemical properties and specificity from those present in humans. ClpP1 has been recently validated to be essential in Mtb (Ollinger et al, 2012) and in related studies, we established that the proteases encoded by clpP1 and by clpP2 are required both for the growth of Mtb and for its virulence during murine infection (Sassetti et al, 2003; Raju et al, 2011). Therefore, ClpP1 and ClpP2 are highly attractive drug targets, especially since they are not present in the cytosol of mammalian cells (where protein breakdown occurs primarily by the ubiquitin proteasome pathway or by lysosomes), and these enzymes differ markedly from the mitochondrial Clp complex. ClpP is a highly conserved, multimeric serine protease originally discovered (Hwang et al, 1987; Katayama-Fujimura et al, 1987) and extensively characterized in E. coli (Maurizi et al, 1990b, 1994, 1998; Yu and Houry, 2007). ClpP homologues exist in a wide range of bacteria, as well as in mitochondria and chloroplasts in eukaryotes (Porankiewicz et al, 1999). The clpP gene in E. coli encodes a polypeptide of 207 amino acids, of which 14 residues are autolytically cleaved to yield the mature protein of 21.5 kDa (Maurizi et al, 1990a). The active enzyme is a tetradecameric structure composed of two heptameric rings that form a hollow cylinder with 14 proteolytic sites compartmentalized within its central chamber (Flanagan et al, 1995; Shin et al, 1996; Wang et al, 1997). Substrates enter into the chamber through two axial openings with a diameter of about 10 Å, which limits the size of polypeptides degraded. By itself, E. coli ClpP is able to rapidly hydrolyse only oligopeptides, but not large globular proteins. The degradation of large proteins requires the presence of an AAA ATPase complex, such as ClpA or ClpX in E. coli or ClpC in other species (Kress et al, 2009). These hexameric structures associate with both ends of ClpP to form the active 4-ring ATP-dependent protease (Maurizi, 1991; Maurizi et al, 1998; Kim et al, 2001). These ATPases bind selectively certain protein substrates, unfold them, and translocate the linearized polypeptides into the ClpP proteolytic chamber for degradation (Hoskins et al, 1998; Ortega et al, 2000; Ishikawa et al, 2001; Reid et al, 2001). In addition to substrate recognition, the human mitochondrial ClpX complex promotes the assembly of the ClpP complex into an active form (Kang et al, 2005). Most organisms possess a single clpP gene, while some microorganisms (e.g., Streptomyces, Actinomycetes, and Cyanobacteria) and plants (e.g., Arabidopsis thaliana) have two or more clpPs (Porankiewicz et al, 1999; Viala et al, 2000; Peltier et al, 2001, 2004; Schelin et al, 2002; Viala and Mazodier, 2002; Butler et al, 2006; Sjogren et al, 2006; Stanne et al, 2007; Andersson et al, 2009). The functional significance of these multiple species is unclear. Mtb contains two clpP genes, clpP1 and clpP2, both of which are essential for viability (Sassetti et al, 2003) and infectivity, as shown in Raju et al (2011). Although both appear to encode serine proteases, prior attempts (Ingvarsson et al, 2007; Benaroudj et al, 2011) to express and characterize Mtb ClpP1 and ClpP2 in E. coli yielded complexes that lacked proteolytic activity, as did our initial attempts to express ClpP1 and ClpP2 in E. coli. We hypothesized that those attempts failed because they were based on the assumption that ClpP1 and ClpP2 are distinct enzymes, while in fact, the active enzyme in vivo is a mixed complex. Here, we demonstrate that ClpP1 and ClpP2, when overproduced independently, form tetradecameric complexes that lack any proteolytic activity. However, when these complexes are mixed together in the presence of certain small activating molecules, which we accidentally discovered, these tetradecamers dissociate into heptameric rings, which then re-associate into a mixed tetradecameric complex that is capable of degrading model peptides as well as some unstructured proteins. These low molecular weight activators clearly represent a novel form of enzyme regulation and stimulate ClpP1P2 activity in a very different manner from the regulatory ATPase complex, ClpC1, which we show enhances specifically the degradation of proteins. Thus, ClpP1P2 differs markedly from other members of the ClpP family and has a number of highly unusual structural, enzymatic, and regulatory properties. These unique qualities of ClpP1P2, taken together with its essential role during infection, make it an attractive target for drug development. Results Isolation of processed ClpP1 and ClpP2 Mtb clpP1 or clpP2 genes were expressed as C-terminal fusions with 6 × His and/or Myc tags under the control of a tetracycline-inducible promoter. Since previous efforts and our initial attempts to produce active ClpP1 and ClpP2 in E. coli were unsuccessful (Ingvarsson et al, 2007; Benaroudj et al, 2011), we attempted to separately express ClpP1 and ClpP2 under conditions resembling those in Mtb by using the closely related non-pathogenic species M. smegmatis. Purification on an Ni-NTA agarose column yielded large amounts of nearly pure proteins, each with an apparent molecular weight of ∼22 kDa (Figure 1A). When ClpP1 and ClpP2 were subjected to gel filtration on an S-300 Sephacryl column, both were eluted as single homogenous peaks with a molecular mass of about 300 kDa (Figure 3A, top panel). Thus, both ClpP1 and ClpP2 had the same elution profile as E. coli ClpP and appeared to be 14-subunit 2-ring complexes. Figure 1.Purification of processed but inactive ClpP1 and ClpP2 and reconstitution of the active ClpP1P2 complex. (A) Coomassie staining of ClpP1 (2.3 μg) and ClpP2 (2.7 μg) after purification. (B) Sequences of ClpP1 and ClpP2 proteins expressed in M. smegmatis. Arrows indicate the sites of proteolytic processing determined by Mass Spectrometry and N-terminal sequencing of ClpP1 and ClpP2 purified as in (A). (C) ClpP1P2 (2.1 μg) possesses peptidase activity but only in the presence of activating dipeptide Z-Leu-Leu. ClpP1 (1.8 μg) or ClpP2 (2.4 μg) alone did not show any activity with or without the activator. Enzymatic activity was measured fluorometrically using Z-Gly-Gly-Leu-AMC as a substrate. (D) Activator also stimulates degradation of longer peptides (Mca-KKPTPIQLN-Dpa(Dnp)-amide) and proteins (FITC-casein) by ClpP1P2 (0.36 and 2.9 μg, respectively). Download figure Download PowerPoint The ClpP1 and ClpP2 bands from the SDS–PAGE were digested by trypsin and chymotrypsin and analysed by MS/MS. Eighty-three peptides were identified for ClpP1 (92% coverage by amino acids) and 70 peptides for ClpP2 (94% coverage). Although mass spectrometry thus demonstrated nearly all the expected peptides, N-terminal sequencing indicated that ∼70% of both proteins were N-terminally processed with major cleavage sites at Asp6-Met7 for ClpP1 and Ala12-Arg13 for ClpP2 (Figure 1B). (In addition, minor cleavages were also detected at Thr5-Asp6 and Met7-Arg8 for ClpP1 and Arg13-Tyr14 for ClpP2.) It is noteworthy that the extent of this processing varied in different preparations and correlated with their ability to support enzymatic activity. Thus, N-terminal processing of both gene products appears important for the formation of the active enzyme. Moreover, when full-length mutant forms of ClpP1 and ClpP2, which lacked enzymatic activity (see below), were expressed in M. smegmatis, a much smaller fraction of N-terminally processed forms could be detected. Therefore, it is likely that the proteolytic processing of mycobacterial ClpPs occurs primarily through an autocatalytic mechanism (possibly involving collaboration with the M. smegmatis enzymes). Accordingly, ClpP1 is cleaved after Asp (Figure 1B), which as shown below, is one of the preferred sites for Mtb ClpP (see below, Table I). Table 1. Mtb ClpP1P2 preferentially hydrolyses peptides with hydrophobic and acidic residues in P1 position Peptide substrate Relative rates of hydrolysis (%) Hydrophobic P1 residue Z-Gly-Gly-Leu-amc 100.0 Suc-Leu-Leu-Val-Tyr-amc 0.8 Suc-Leu-Tyr-amc 11.5 Z-Leu-Leu-Leu-amc 0.15 Z-Leu-Leu-amc 4.7 Z-Ala-Ala-Ala-amc 3.6 Suc-Ala-Leu-Pro-Phe-amc 0.12 Suc-Ala-Ala-Pro-Ala-amc 0.08 Ala-Ala-Phe-amc 87.0 Suc-Ala-Ala-Phe-amc 42.0 Acidic P1 residue Ac-Nle-Pro-Nle-Asp-amc 36.0 Z-Leu-Leu-Glu-amc 0.35 Basic P1 residue Z-Leu-Leu-Arg-amc 0.25 Z-Gly-Gly-Arg-amc 0.55 Z-Phe-Val-Arg-amc 0.18 Aminopeptidase substrates Ala-amc 0.78 Leu-amc 0.36 Phe-amc 0.26 Asp-amc 0.1 Peptidase activity of ClpP1P2 (1.8 μg) was measured with a variety of substrates (0.1 mM) and compared with activity obtained with Z-Gly-Gly-Leu-AMC (100%). In subsequent studies, we therefore expressed the constructs corresponding to the processed versions directly and obtained more homogenous preparations with higher activities. It is noteworthy that these shorter forms, which do not require N-terminal processing, could also be efficiently produced in E. coli. ClpP1 and ClpP2 form a mixed ClpP1P2 protease that requires certain short peptides for activation Neither ClpP1 nor ClpP2 alone had peptidase activity (Figure 1C), although both formed tetradecameric structures characteristic of the ClpP family. Because both genes are essential (Sassetti et al, 2003; Ollinger et al, 2012; Raju et al, 2011), we hypothesized that ClpP1 and ClpP2 are not two distinct enzymes, but instead associate to form a novel, mixed proteolytic complex. To test this possibility, we first attempted to co-express Mtb ClpP1 and ClpP2 in M. smegmatis. The two proteins associated in vivo since they could be co-immunoprecipitated from the cell extract (Raju et al, 2011). However, due to wide variations in the levels of ClpP1 and ClpP2 expression, the ratios between the co-purified ClpP1 and ClpP2 varied markedly in different preparations, and this heterogeneity prevented rigorous study of the active complex. Therefore, we expressed them separately and attempted to reconstitute such a mixed complex from pure components. In fact, mixing pure ClpP1 and ClpP2 together in high concentrations (up to 0.5 mg/ml) resulted in the appearance of very low peptidase activity against the fluorogenic substrate of E. coli ClpP, Suc-Leu-Tyr-AMC. During attempts to identify transition state-specific inhibitors of this low activity, we accidentally made the surprising, but very valuable discovery that a group of N-blocked peptide aldehydes that were substrate analogues not only did not inhibit, but actually stimulated this activity over 1000-fold. A similar dramatic activation was even found with certain related blocked peptides. For example, as shown in Figure 1C, a mixture of ClpP1 and ClpP2 was inactive in hydrolysing the Z-Gly-Gly-Leu-AMC or the quenched fluorescent substrate Mca-GHQQYKMK-Dpa(Dnp)-amide, but in the presence of the activating peptide Z-Leu-Leu, both substrates and the unfolded protein, casein, were efficiently cleaved (Figure 1C and D). The activating peptides and peptide aldehydes only induced peptidase activity if both ClpP1 and ClpP2 were present together. It is noteworthy that at 37°C (under our standard assay conditions) the activation occurred without any noticeable delay after the addition of the peptide activator. Also, the activator had to be continually present for enzymatic activity. When the activator was removed by gel filtration or if its concentration was reduced by dilution, activity was lost, but it could be regained fully upon restoration of activator to its prior concentration (Figure 2C). Figure 2.Certain short peptides and peptide aldehydes dramatically activate ClpP1P2 by binding to multiple sites. (A) Effects of various short peptide aldehydes and related peptides or peptide alcohols on activity of ClpP1P2 (1.3 μg). Peptidase activity was measured with Z-Gly-Gly-Leu-AMC. The specific activity with Z-Leu-norleucinal was 4.25 μmole/mg/min, which was taken as 100%. (B) Determination of Hill coefficient of Z-Leu-Leu and Z-Leu-leucinal in the hydrolysis of Z-Gly-Gly-Leu-AMC (0.1 mM) and Ac-Nle-Pro-Nle-Asp-amc (0.1 mM) by ClpP1P2 (2.4 μg). (C) Activation of ClpP1P2 by Z-Leu-Leu is readily reversible. Enzyme (1.8 μg) was incubated in the presence of activator Z-Leu-Leu (5 mM) and then diluted 200-fold in the buffer with and without the activator. Enzymatic activity was assayed with Z-Gly-Leu-Leu-AMC (0.1 mM). Re-addition of the activator restored the ClpP1P2 activity completely. Download figure Download PowerPoint The strongest stimulation against Z-Gly-Gly-Leu-AMC, as well as other substrates, was found with Z-Leu-leucinal (Figure 2), but the longer aldehyde Z-Leu-Leu-leucinal was significantly less active. Several other hydrophobic dipeptide aldehydes (e.g., Z-Val-phenylalaninal), acidic peptide aldehydes (e.g., Z-Pro-Nle-aspartal) and alkyl aldehydes did not show any stimulatory capacity. The effective peptide aldehydes presumably should bind to at least some of the enzymes' 14 active sites. However, the related peptide Z-Leu-Leu and its alcohol derivative Z-Leu-leucinol (which presumably should not bind strongly to the active sites) could also activate ClpP1P2, although only at much higher concentrations than the corresponding aldehydes. A much smaller stimulation was observed with blocked peptides Z-Leu, Z-Gly-Leu and Z-Gly-Leu-Leu (Figure 2A). The concentration dependence for activation, by Z-Leu-leucinal (Kd=0.24 mM) and Z-Leu-Leu (Kd=2.2 mM), revealed a highly cooperative mechanism with a Hill coefficient of 5–7 (Figure 2B). Thus, multiple molecules probably bind to ClpP1P2 to stimulate its activity. Though substrate analogues, these activators are not cleaved, since upon incubation with ClpP1P2, no new amino groups could be detected using the sensitive fluorescamine assay. It is noteworthy that although the aldehyde had a higher affinity, at high concentrations, Z-Leu-Leu caused a greater activation than Z-Leu-leucinal (Figure 2B). Also because peptides are more stable and much less expensive than the corresponding aldehydes, in subsequent studies, we routinely induce Mtb ClpP1P2 activity using Z-Leu-Leu (subsequently referred to as the 'activator'). Activation involves dissociation of ClpP1 and ClpP2 tetradecamers and formation of 2-ring ClpP1P2 complex Because the activators stimulate only ClpP1 and ClpP2 together (but not pure ClpP1 or ClpP2; Figure 1C and D), they probably activate by promoting the formation of a new mixed ClpP1P2 complex. We therefore examined how the presence of an activator affects the sizes of these different complexes. Upon size-exclusion chromatography, a mixture of ClpP1 and ClpP2 behaved as tetradecamers exactly like pure Mtb ClpP1 or ClpP2 and E. coli ClpP (Figure 3A, upper panel). However, when the activator Z-Leu-Leu was present (Figure 3A, lower panel), both ClpP1 and ClpP2 peaks were eluted as a single lower molecular weight peak, resembling γ-globulin (150 kDa) in size. Thus, the tetradecameric (presumably 2-ring) complexes composed of a single subunit type dissociated into heptamers. However, in the presence of the activator, the ClpP1/ClpP2 mixture was eluted as a 300-kDa peak that coincided with the peptidase activity and corresponded in size to ClpP tetradecamers (Figure 3A, lower panel). The ClpP1P2 complexes were isolated from the peak using Ni-NTA (by His-tagged ClpP2) or anti-Myc (by Myc-tagged ClpP1) columns, and the presence of both proteins in resin-bound material was confirmed by MS (see Materials and methods). Figure 3.With the activator present, ClpP1 and ClpP2 tetradecamers dissociate into heptamers but ClpP1/ClpP2 mixture forms an enzymatically active tetradecamer. (A) Size-exclusion chromatography of ClpP1 (0.28 mg), ClpP2 (0.34 mg), and ClpP1/ClpP2 mixture (0.31 mg) was carried out using Sephacryl S300 column in the absence (upper panel) and presence (lower panel) of activator Z-Leu-Leu, which was also present in the running buffer. Samples (0.2 ml) were applied to the column and 0.5 ml fractions were collected and assayed for protein content and activity with Z-Gly-Gly-Leu-AMC. The column was calibrated with γ-thyroglobulin (670 K), γ-globulin (158 K), ovalbumin (44 K), and E. coli ClpP (300 K). (B) The change in fluorescence emission spectrum of ClpP1/ClpP2 mixture (12.8 μg in 100 μl) upon addition of activator indicates a conformational change due to complex formation between ClpP1 and ClpP2. The addition of activator to ClpP1/ClpP2 mixture shifted the peak of ClpP1 Trp174 emission from 345 to 338 nm (lower panel), while no change was observed with ClpP1 alone (upper panel). Download figure Download PowerPoint Thus, the activating peptide causes the dissociation of ClpP1 and ClpP2 tetradecamers into heptamers and favours their subsequent association to form the active tetradecameric ClpP1P2 complex. By contrast, no changes in elution pattern were observed when E. coli ClpP was incubated with this activator. Conformational changes accompanying formation of ClpP1P2 complex The dissociation and re-association of multimeric ClpP1 and ClpP2 rings must involve activation-induced major changes in subunit conformation. Because ClpP1 (but not ClpP2) contains a Trp residue, we can use it to monitor conformational changes that may accompany the formation of an active ClpP1P2 complex from inactive ClpP1 and ClpP2 ones. Although no spectral changes were observed with dissociation of the ClpP1 tetradecamer upon addition of the activator, the formation of the active ClpP1P2 complex appears to involve changes in ClpP1's conformation, because the fluorescence of Trp174 in ClpP1 shifted its maximal fluorescence from 345 in pure ClpP1 to 338 nm. (Figure 3B). Thus, the interaction between ClpP1 and ClpP2 subunits leading to activation is associated with changes in the subunits' conformation. It is noteworthy that similar changes in Trp174 fluorescence occurred when active-site mutants of ClpP1 and ClpP2 that lack enzymatic activity (see below) were mixed in the presence of the activator. Thus, enzymatic activity of both ClpPs is not necessary for their dissociation—re-association and the major structural changes associated with this activation process. To confirm that such a mixed ClpP1P2 complex actually exists in vivo, we tested whether endogenous ClpP1 and ClpP2 associate in wild-type M. smegmatis. As described in the related manuscript (Raju et al, 2011), we employed mycobacterial recombineering to add a C-myc tag to the C-terminus of genomic ClpP2. The C-myc-tagged ClpP2 was isolated together with associated proteins using an anti-myc resin, and the material eluted with the Myc peptide was resolved by SDS–PAGE. Bands corresponding by size to ClpP2 and ClpP1 were analysed by Mass Spectrometry, and the presence of both subunits was confirmed, indicating that mixed ClpP1P2 complexes are present in mycobacteria. Mtb ClpP1P2 is composed of one ClpP1 and one ClpP2 heptameric ring To determine the subunit composition of this ClpP1P2 complex, we varied the relative concentrations of ClpP1 and ClpP2 in the presence of an activator (Figure 4A). Upon increasing the amount of ClpP1 with a constant amount of ClpP2, peptidase activity gradually increased and reached its maximum when these components were present in close to equimolar amounts. Conversely, when ClpP1 content was held constant and the amount of ClpP2 increased, maximal activity was also obtained with equimolar concentrations. In different experiments using different ClpP1 and ClpP2 preparations, the optimal ClpP1/ClpP2 molar ratio ranged from 0.82 to 1.15. Thus, the active complex contains equal numbers of ClpP1 and ClpP2 subunits. Figure 4.ClpP1P2 has maximal activity when equimolar amounts of ClpP1 and ClpP2 are present (A) and is composed of heptameric rings containing only ClpP1 or ClpP2 subunits (B). (A) Activity of ClpP1P2 at different ClpP1:ClpP2 ratios. Constant amounts of ClpP2 (0.85 μg) were mixed with increasing amounts of ClpP1, and Z-Gly-Gly-Leu-AMC hydrolysis was measured in the presence of activator. (B) Crosslinking of ClpP1P2 subunits by glutaraldehyde. After 0.5 and 20 h incubation at room temperature of ClpP1P2 (12 μg) with 0.125% glutaraldehyde, the reaction mixture was analysed by SDS–PAGE, followed by Mass Spectrometry. High molecular weight bands corresponding to seven crosslinked subunits were found to contain exclusively ClpP1 or ClpP2 peptides, indicating that each ring contains seven identical ClpP1 or ClpP2 subunits. In the insert, one tenth of the crosslinked material used in Figure 4B was resolved by SDS–PAGE and stained by Coomassie Blue. The gel was scanned, and the image enlarged using Photoshop. Download figure Download PowerPoint These findings and the rapidity of activation together strongly suggest that the active enzyme is composed of one ClpP1 and one ClpP2 ring. However, it is also possible that each heptameric ring contains a mixture of ClpP1 and ClpP2 subunits, as has been found for the cyanobacterium Synechococcus ClpP complexes (Stanne et al, 2007; Andersson et al, 2009). To determine the composition of the rings, we crosslinked the neighbouring subunits in the active ClpP1P2 tetradecamer with glutaraldehyde in the presence of the activator (Figure 4B). After a 0.5 h of incubation, seven distinct crosslinked bands were evident on SDS–PAGE corresponding to 1-, 2-, 3- 4-, 5-, 6-, and 7-mers. As expected, the larger crosslinked structures were the least abundant. After an overnight incubation, when crosslinking went to completion, all seven subunits, presumably comprising the rings, were crosslinked together, but still no band was observed with a molecular mass higher than that of a 7-mer. Thus, apparently, no crosslinking occurred between the two rings (which presumably requires a crosslinker with a longer spacer arm than glutaraldehyde). Analysis by mass spectrometry indicated that the crosslinked heptamers were composed only of ClpP1 or ClpP2 subunits, respectively, and no peptides corresponding to ClpP1–ClpP2 crosslinked were found. Thus, each heptameric ring in the Mtb ClpP1P2 protease is homogenous in composition. Cleavage specificity of Mtb ClpP1P2 To define the substrate preference of the ClpP1P2 active sites, we tested a variety of synthetic fluorescent peptides with hydrophobic, acidic, or basic residues in the P1 position (Table I). The best substrate was Z-Gly-Gly-Leu-AMC, while Suc-Ala-Ala-Phe-AMC and Ala-Ala-Phe-AMC also were readily cleaved. The failure of ClpP1P2 to degrade rapidly the widely used proteasome substrate Suc-Leu-Val-Tyr-AMC indicates major differences from enzymes in the mammalian cytosol. It is noteworthy that Z-Leu-Leu-AMC, the fluorescent peptide corresponding to the peptide activator employed routinely Z-Leu-Leu, was a poor substrate for the enzyme (Table I), and conversely the peptides corresponding to the best substrates, Z-Gly-Gly-Leu or Z-Gly-Leu, were poor as activators (Figure 2A). In addition to hydrophobic peptides, ClpP1P2 also efficiently hydrolyses a peptide with acidic residues in the P1 position, Ac-Nle-Pro-Nle-Asp-AMC. (We also found that this substrate is degraded by E. coli ClpP, which had been reported to cleave after aspartate residues in model polypeptides; Thompson and Maurizi, 1994). However, Mtb ClpP1P2 did not hydrolyse Z-Leu-Leu-Glu-AMC or peptides with basic P1 residue and was also inactive against a variety of unblocked amino acid-AMC substrates used to assay aminopeptidases (Table I). ClpP1P2 also could cleave a variety of longer quenched fluorescent peptides (e.g., Mca-GNTQFKRR-Dpa(Dnp)-amide, Mca-GHQQYAMK-Dpa(Dnp)-amide, Mca-GNQQYKMK-Dpa(Dnp)-amide and Mca-KKPTPIQLN-Dpa(Dnp)-amide), and could degrade slowly the largely unstructured protein FITC-casein, provided an activator was present (Figure 1D). Though ClpP1 or ClpP2 alone lack enzymatic activity, their catalytic triads are formed The sequences of both ClpP1 and ClpP2 appear to contain a Ser/His/Asp catalytic triad characteristic of serine proteases (Figure 1B). Accordingly, Mtb ClpP1P2 was sensitive to most standard inhibitors of serine proteases (Figure 5A), including agents that react with the active-site serine (dichloroisocoumarin, Powers and Kam, 1994 and biotinylated derivative of fluoroethoxyphosphinyl (FP-biotin), Liu et al, 1999), and peptide chloromethyl ketones (Szyk and Maurizi, 2006), which modify the catalytic histidine. By contrast, standard inhibitors of metalloproteases and cysteine proteases had no effect. Interestingly, the hydrolysis of both hydrophobic and acidic substrates was inhibited to similar extents by the peptide chloromethyl ketones, Z-LY-CMK or AAF-CMK. Figure 5.Both ClpP1 and ClpP2 contain functional active sites, but ClpP1's active sites are more important than ClpP2's in ClpP1P2 activity. (A) ClpP1P2
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