The active ClpP protease from M. tuberculosis is a complex composed of a heptameric ClpP1 and a ClpP2 ring

Abstract

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.

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).

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.

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).

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.

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 protease has the inhibitor sensitivity characteristic of serine proteases. Peptidase activity against Z-Gly-Gly-Leu-AMC (0.1 mM) was measured after 30 min preincubation of the enzyme (1.2 μg) with or without inhibitors at room temperature. The error ranges of the inhibition did not exceed 5%. (B) To learn whether the active-site charge relay system is functional in the absence of activator, 1.2 μg of wild-type or active-site Ser-Ala mutants of ClpP1, ClpP2, or a ClpP1/ClpP2 mixtures were incubated for 30 min with a biotinylated derivative of an active-site inhibitor fluoroethoxyphosphinyl. The binding of the inhibitor was determined by SDS–PAGE followed by western blot analysis with an anti-biotin antibody. (C) To determine how ClpP1 and ClpP2 contribute to the enzymatic activity of ClpP1P2, either ClpP1 or ClpP2 was inactivated separately by pretreatment with dichloroisocoumarin (0.1 mM) or by an active-site mutation (active site Ser to Ala). The inactivated ClpP1 or ClpP2 was then mixed with its normal (untreated WT) counterpart in the presence of activator Z-Leu-Leu, and hydrolysis of hydrophobic and acidic peptide substrates and casein was measured. FITC-casein was not used to measure proteinase activity of DCL-treated enzymes due to the overlapping of the fluorescence spectra of FITC and DCL. Figure source data can be found in Supplementary data.

Figure 6

ClpC1 activates degradation of casein, but not peptides, by ClpP1P2 only in the presence of activator and ATP. (A) ClpC1 activates casein but not peptide degradation by ClpP1P2. ClpP1P2 (2.5 μg) and ClpC1 (32 μg) were mixed in 100 μl of reaction buffer containing 2 mM ATP and 8 mM MgCl₂ (see Materials and methods) in the presence or absence of the peptide activator. Enzymatic activity was measured fluorometrically using Z-Gly-Gly-Leu-AMC or FITC-casein as substrates. The rate of degradation of ClpP1P2 in the presence of the activator was taken as 100%. (B) ClpC1 activates casein degradation by ClpP1P2 but not by ClpP1 or ClpP2 alone. Degradation of FITC-casein by ClpP1 (2.5 μg), ClpP2 (2.9 μg), or ClpP1P2 (2.7 μg) was measured as in (A). (C) The stimulation of FITC-casein degradation by ClpP1P2 requires ATP and the peptide activator. Degradation was measured as in (A) and (B) with different concentrations of ClpC1. (D) Casein stimulates ATPase activity of ClpC1. In all, 2 μg of ClpC1 was incubated with 5 μg FITC-casein, or with ClpP1P2+/− activator, or with the activator alone, in 20 μl of reaction buffer for 30 min and the ATPase activity was measured by Malachite Green method (see Materials and methods).

Figure 7

Mechanism of formation of active ClpP1P2. The proposed mechanism is based on the results in (1) Figure 4A showing that the optimal activity of ClpP1P2 is reached at the equal molar amounts of ClpP1 and ClpP2; (2) Figure 3A showing the dissociation of ClpP1 and ClpP2 tetradecamers into heptamers, and their re-association into mixed ClpP1P2 complex in the presence of the activator; (3) Figure 3B showing physical interaction between the rings; and (4) Figure 4B demonstrating crosslinking of only one type of subunits within the rings.