Cleavage Specificity of Mycobacterium tuberculosis ClpP1P2 Protease and Identification of Novel Peptide Substrates and Boronate Inhibitors with Anti-bacterial Activity

Abstract

The ClpP1P2 protease complex is essential for viability in Mycobacteria tuberculosis and is an attractive drug target. Using a fluorogenic tripeptide library (Ac-X3X2X1-aminomethylcoumarin) and by determining specificity constants (kcat/Km), we show that ClpP1P2 prefers Met ≫ Leu > Phe > Ala in the X1 position, basic residues or Trp in the X2 position, and Pro ≫ Ala > Trp in the X3 position. We identified peptide substrates that are hydrolyzed up to 1000 times faster than the standard ClpP substrate. These positional preferences were consistent with cleavage sites in the protein GFPssrA by ClpXP1P2. Studies of ClpP1P2 with inactive ClpP1 or ClpP2 indicated that ClpP1 was responsible for nearly all the peptidase activity, whereas both ClpP1 and ClpP2 contributed to protein degradation. Substrate-based peptide boronates were synthesized that inhibit ClpP1P2 peptidase activity in the submicromolar range. Some of them inhibited the growth of Mtb cells in the low micromolar range indicating that cleavage specificity of Mtb ClpP1P2 can be used to design novel anti-bacterial agents.

Keywords: Drug Development; Enzyme Inhibitor; Enzyme Mechanism; Infectious Disease; Peptide Chemical Synthesis; Protein Degradation.

FIGURE 1.

Mtb ClpP1P2 cleaves peptide-amc substrates mainly after Leu, Phe, Ala, and especially Met. A, to determine cleavage preferences for the X₁ position, ClpP1P2 activity was measured continuously using Ac-X₃X₂X₁-amc fluorogenic peptides library (at 10 μm) in buffer A containing 30 nm ClpP1P2. The ClpP1P2 activity against the best substrates with a given amino acid in X₁ position is reflected in the graph. B, average rate of hydrolysis of substrates with a given amino acid in the X₁ position. The activity was measured as in Fig. A. RFU, relative fluorescence units.

FIGURE 2.

Mtb ClpP1P2 cleavage preferences for the X₂ and X₃ positions with fixed Met, Leu, Phe or Ala at the X₁ position. ClpP1P2 activity was measured using Ac-X₃X₂X₁-amc fluorogenic peptides library as in Fig. 1A. The size of the dots represent the ClpP1P2 activity against the given peptide and is calculated as percent of the maximal value for each panel. Maximal activity (relative fluorescence units) for the libraries with fixed Met in X₁ position was 2300, with Leu 660, with Phe 490, or with Ala 160.

FIGURE 3.

Isolation of WT or mutant forms of ClpP1P2 complexes in native gel and determining their enzymatic activity. ClpP1 and ClpP2 (3 μg of each) were mixed together in buffer A to form WT ClpP1P2 or mutant ClpP1P2(S-A) or ClpP1(S-A)P2 complexes and isolated using the native PAGE in the presence or absence of the peptide activator Z-LL. The peptidase (with Ac-PKM-amc) and proteinase (with GFPssrA) activities of the enzyme were then determined directly in the gel as described under “Experimental Procedures.”

FIGURE 4.

Cleavage preferences of Mtb ClpP1P2 are different from those of human proteasomes. Specificity constants k_cat/K_m were determined using 70 nm of ClpP1P2 or 8.5 nm concentrations of human proteasomes at peptide-amc substrate concentration of 10 μm.

FIGURE 5.

Size distribution of peptide products and cleavage preferences for ClpP1P2. A, size distribution of individual peptides generated after cleavage of GFPssrA. Protein substrate was digested by ClpP1P2 in buffer A containing 1 μm GFPssrA, 75–100 nm ClpP1P2, 300–400 nm ClpX, and 2 mm Mg-ATP. The produced peptides were separated by centrifugal filter (10 kDa) and analyzed using Liquid LS-MS/MS. B and C, cleavage preferences for X₁ and X₁′ positions during GFPssrA degradation. The number of cleavages after and before a given amino acid is presented as a percent of the total number of that amino acid in the protein sequence. Peptides were generated and analyzed as in A.

FIGURE 6.

ClpP1 accounts for nearly all of the peptidase activity, although both ClpP1 and ClpP2 subunits contribute to protein breakdown. A, degradation of peptide-amc substrates by WT ClpP1P2 and mutant ClpP1P2(S-A) and ClpP1(S-A)P2 mutant complexes. To determine the contribution of each type of active site in peptide degradation, the peptidase activity of ClpP1P2 complexes was measured against the best peptide substrates as in Fig. 1A. The activity of the WT ClpP1P2 against each substrate was taken as 100%. B, degradation of protein substrates by WT ClpP1P2, ClpP1P2(S-A), and ClpP1(S-A)P2 mutant complexes. To determine the ClpP1 and ClpP2 contributions to protein degradation, the cleavage of fluorescent protein substrates, GFPssrA with ClpX present and FITC-casein with ClpC1 present, were measured, as described under “Experimental Procedures.” The rates of degradation by the WT ClpP1P2 are taken as 100%.

FIGURE 7.

A, peptide boronates inhibit the ClpP1 activity more strongly than the ClpP2 activity. Effects of peptide boronate inhibitor (N-(1H-benzo(b)thiophene-7-carbonyl)-Lys-boro-Met) on degradation of peptide (Ac-Pro-Lys-Met-amc) and protein (GFPssrA and FITC-casein) substrates by WT ClpP1P2, ClpP1P2(S-A), and ClpP1(S-A)P2 complexes are presented as IC₅₀ values. Enzymatic activity of ClpP1P2 complexes was measured as described under “Experimental Procedures.” B, substrate-based boronate derivatives inhibit protein degradation by ClpXP1P2. N-(2-(3,5-difluorophenyl)acetyl-Trp-boroMet (20 μm) prevents the degradation of GFPssrA by Mtb ClpXP1P2 (the degradation assay was carried out as in Fig. 5A). RFU, relative fluorescence units.

FIGURE 8.

ClpP1P2 is the target for boronate inhibitors in mycobacteria. A, GFPssrA substrate accumulates in Mtb in the presence of ClpP1P2 inhibitor. H37Rv strain expressing PMV261::GFP-ssrA was incubated with and without 10 μm concentrations of inhibitor (N-(3-phenyl)propanoyl)-Lys-boroMet). Fluorescence measurements were normalized by cell density. Fluorescence measurements were taken every 2.75 h for 42 h. Error bars represent 95% confidence intervals of triplicate experiment. B, growth curves for Mtb in the presence and absence of the inhibitor. C, ClpP1P2 is the target for boronate inhibitors in M. smegmatis. M. smegmatis constitutively expressing GFP-ssrA was grown to stationary phase then diluted to an A₆₀₀ = 0.003 and dispensed into a sterile flat-bottom 96-well plate. Peptide boronates (Ala(1-naphtyl)-Lys-boroLeu (Table 3, #4) or N-(2-(3,5-difluorophenyl)acetyl-Trp-boroMet) (Table 3, #12) at different concentrations were added to wells. The fluorescence of GFP was measured after 6 h at 37 °C and normalized to cell density. RFU, relative fluorescence units.