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Mechanism of the allosteric activation of the ClpP protease machinery by substrates and active-site inhibitors

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Science Advances  04 Sep 2019:
Vol. 5, no. 9, eaaw3818
DOI: 10.1126/sciadv.aaw3818
  • Fig. 1 Bortezomib activates TtClpP for peptide and intrinsically disordered protein degradation.

    (A) TtClpP (1 μM complex) peptidase activity was measured with the substrate PKMamc (100 μM) in the presence of bortezomib at the indicated concentrations. As the peptide is cleaved, 7-amino-4-methylcoumarin is released, resulting in an increase in the measured fluorescence. Initial rates are plotted as a function of the bortezomib concentration. RFU, relative fluorescence units. (B) The activating effect of bortezomib is compared with the one observed with Bz-LL, a previously described activator of MtbClpP1P2. (C) The degradation of the unfolded protein substrate FITC-casein by TtClpP (0.1 μM complex) was measured in the presence of the indicated bortezomib concentrations. Initial degradation rates following temperature equilibration were plotted as a function of the bortezomib concentration. (D) TtClpP (1 μM complex) can degrade the intrinsically disordered E. coli FtsZ. The degradation of FtsZ by TtClpP was monitored by SDS-gel electrophoresis. While no degradation was observed with apoTtClpP, degradation of FtsZ was observed in the presence of 200 μM and 2 mM bortezomib.

  • Fig. 2 TtClpX activates TtClpP peptidase activity.

    (A) TtClpP (0.1 μM complex) peptidase activity was measured in the presence of TtClpX at the indicated ClpX6/ClpP14 ratios. (B) The activation of TtClpP peptidase activity in the absence (circles) or presence (triangles) of bortezomib (10 μM) is plotted as a function of the molar ClpX6/ClpP14 ratio. Half-maximal peptidase activity was obtained at 6.5 ± 0.5 and 2.4 ± 0.3 ClpX6/ClpP14 ratio for the apo and bortezomib-loaded TtClpP, respectively. (C) Activation of the TtClpP active site by bortezomib (100 μM) and TtClpX (1 μM), monitored by labeling with TAMRA-FP. TAMRA-FP (5 μM) was incubated with TtClpP (1 mg/ml), and aliquots of the reaction mixture were removed at the indicated time points. Labeling of the active-site serines is reflected by the increase in fluorescence observed.

  • Fig. 3 Bortezomib binding to TtClpP investigated by MAS NMR and solution NMR.

    (A) Secondary structures (loop, black; α-helix, red; β-strand, blue) as a function of sequence number obtained from the resonance assignments obtained by MAS NMR and analysis with TALOS-N (24) (tall bars). For residues for which no assignment has been obtained, the small bars are obtained by TALOS-N from a database approach. The bottom panel shows the comparison with the secondary structure obtained from the crystal structure (see Fig. 5). (B) Zooms onto 2D 1H-15N excerpts from 3D hCANH MAS NMR correlation spectra of TtClpP sedimented in the presence of 10 mM bortezomib (red) or without bortezomib (black). Chemical shift changes are indicated with arrows. Note that the CSPs are relatively small, presumably due to the difficulty of saturating the binding site in a sedimented sample, given the high Kd value, resulting in incompletely occupied binding sites. ppm, parts per million. (C) Combined CSPs upon addition of bortezomib from the added peak shifts in 1H, 13Cα, and 15N dimensions obtained from the spectra shown in (B). (D) Location of residues with substantial CSPs mapped onto the x-ray structure of the TtClpP:bortezomib complex obtained from the data shown in (C). (E) Solution-state NMR spectra of wild-type (left) and S97A (right) TtClpP in the absence and presence of bortezomib, showing large spectra changes for the former due to binding, but no effect on the mutant.

  • Fig. 4 Structure and dynamics of TtClpP from x-ray crystallography, MAS NMR, and MD simulations.

    (A) Side and top views of the TtClpP 14-mer. One TtClpP monomer per heptameric ring (light and dark gray) is highlighted in light and dark blue, respectively. (B) Cartoon representation of the TtClpP monomer in peptide-bound (left) and bortezomib-bound (right) states. Helices are named by letters, and strands are indicated by numbers. A zoom of the ligands present in the active sites (dashed boxes) is shown in (C). (C) Substrate-binding pocket of TtClpP. The residues involved in the binding to the model peptide (inset 1) and bortezomib (inset 2) are shown as sticks. (D and E) Residue-wise MAS NMR amide 15N R relaxation rate constants. High R rate constants point to enhanced nanosecond-to-millisecond motions and are found primarily in loop regions and in helix αE, as shown in (C). (F and G) MD-derived root mean square fluctuations (RMSFs) over the 1-μs-long MD trajectory of the assembled 14-mer ClpP.

  • Fig. 5 Cooperative bortezomib binding detected by ITC experiments.

    (A) Calorimetric titrations for the interaction of TtClpP (10 μM in the calorimetric cell) with bortezomib (1.4 mM in the injecting syringe) in 50 mM Hepes (pH 7.6) and 50 mM NaCl. Experiments were performed at three different temperatures: 25°, 35°, and 45°C. Thermograms (thermal power as a function of time) are displayed in the upper plots, and binding isotherms (ligand-normalized heat effects per injection as a function of the molar ratio, [L]T/[P]T) are displayed in the lower plots. The binding isotherms were analyzed with the MWC model for TtClpP, consisting of 14 identical subunits, each one containing a single ligand-binding site. Nonlinear least squares regression analysis allows the determination of the following binding parameters (see Table 1): association constants for the R and T states (KR, KT), binding enthalpies to the R and T states (ΔHR, ΔHT), conformational equilibrium constant and conformational enthalpy change between states R and T (γ, ΔHγ), and fraction of active protein (N). (B) MWC model for a 14-mer oligomeric protein. The protein can populate only two conformational states in equilibrium (with equilibrium constant γ): all subunits in R (relaxed, ellipsoidal shape) conformation or all subunits in T (tense, spherical shape) conformation. Subunits have a single ligand-binding site, exhibiting the R conformation a higher binding affinity (KR > KT). Ligand binding occurs through an independent (noncooperative) fashion within an oligomer (ligand-free subunits in light blue and ligand-bound subunits in dark blue). T conformation is favored at low ligand concentration; the higher ligand-binding affinity for R conformation promotes a highly cooperative compulsory concerted conformational change driven by ligand binding and involving all subunits within an oligomer, displacing the equilibrium toward the R conformation. (C) Molar fraction of the different protein species (total, ligand-free, and ligand-bound R and T conformations) as a function of ligand concentration: total fraction of subunits in R conformation (continuous black line), total fraction of subunits in T conformation (continuous red line), fraction of subunits in ligand-bound R conformation (dashed black line), fraction of subunits in ligand-bound T conformation (dashed red line), fraction of ligand-free subunits in R conformation (dotted black line, highlighted with an arrow), and fraction of ligand-free subunits in T conformation (dotted red line). In addition, the fraction of ligand-bound subunits in either R or T conformation is shown (dashed green line). It is obvious that the contribution of subunits in T conformation to the ligand binding is very small (dashed red line). At very low ligand concentration, 77% of the protein subunits are in T conformation and 23% are in R conformation, according to the value of the equilibrium constant γ equal to 3.3. At low ligand concentration, the total fraction of subunits in R conformation increases, while the total fraction of subunits in T conformation decreases due to the T→R conversion. However, both the fractions of ligand-bound subunits in R and T conformation increase due to ligand binding (although the increment in ligand-bound subunits in T conformation is negligible). At high ligand concentration, the total fraction of subunits in R conformation further increases, while that of total subunits in T conformation decreases, and the fraction of ligand-bound subunits in R conformation further increases due to ligand binding, but the fraction of ligand-bound subunits in T conformation decreases due to the T→R conversion. The fraction of ligand-free subunits in R conformation dominates this region, with a maximal population of 60%. This is due to the concerted conversion of subunits within a given protein oligomer (all subunits within an oligomer undergo the conformational change T→R, although not all of the subunits bind a ligand). At very high ligand concentration, the fraction of ligand-bound subunits in R conformation dominates the conformational ensemble, reaching a maximal population of 100%. The R↔T equilibrium shows a switchover or crossover point (R and T are equally populated) at around 2 μM free bortezomib concentration. (D) TtClpP (1 μM complex) peptidase activity was measured as a function of PKMamc concentration in the presence (15 μM) and absence of bortezomib.

  • Fig. 6 In silico pulling experiment of TtClpP from the extended state to the compressed state.

    (A and B) Profile of the force (A) and work (B) required for compressing the two rings toward each other as a function of the distance ζ between the rings (shown in the inset, with exaggerated distance for better visibility). Three independent 200-ns simulations were performed for apo TtClpP or TtClpP, in which 2 or 3 of the 14 binding sites were occupied with trialanine peptide (see Materials and Methods). (C and D) Conformations of TtClpP extended (C) and SaClpP compressed (PDB: 3QWD) (D) states observed in crystal structures. (E) Starting conformation of TtClpP in the steered MD simulations (shown as sphere representation). (F) Final conformation of the compressed state at the conclusion of the steered MD. Note that the peptides are still bound and that the structure is in good overall agreement with the compressed state from x-ray diffraction shown in (D). (G) DLS analysis of TtClpP (3 μM) supplemented without or with 5 mM bortezomib. The upper graph shows one representative curve of three replicates measured at 25°C for TtClpP without (red curve) or with 5 mM bortezomib (green curve), with the relative intensity (in %) plotted versus particle size [hydrodynamic radius, Rhyd (in nm)]. The average of the three replicates is indicated using a dotted line. The lower graph depicts average hydrodynamic radii (Rhyd) calculated from three independent experiments measured at 25° and 45°C (0 mM bortezomib, 25°C: 5.946 ± 0.036 nm; 0 mM bortezomib, 45°C: 5.940 ± 0.102 nm; 5 mM bortezomib, 25°C: 6.673 ± 0.192 nm; 5 mM bortezomib, 45°C: 6.702 ± 0.176 nm). Theoretical calculated hydrodynamic radii of compressed TtClpP (6.05 nm) and the extended TtClpP:bortezomib complex (6.47 nm) are indicated using a dotted black line (see also Materials and Methods).

  • Table 1 Thermodynamic parameters of bortezomib binding from ITC and fitting to the MWC model.

    KR and KT refer to the bortezomib association constants for the relaxed and tense state. ΔHR, ΔHT, and ΔHγ refer to the binding enthalpy of the R state, T state, and conformational enthalpy between R and T conformations. γ is the equilibrium constant between R and T conformations in the absence of the ligand. N is the fraction of active or binding-competent protein. Relative errors in equilibrium constants are 30%. Absolute error in enthalpies is 0.3 kcal/mol.

    T (°C)NKR
    (M−1)
    ΔHR
    (kcal/mol)
    KT
    (M−1)
    ΔHT
    (kcal/mol)
    γΔHγ
    (kcal/mol)
    25°C0.974.5 × 104−0.5No apparent cooperativity
    35°C0.978.0 × 104−0.92.8 × 104−0.13.3−0.3
    45°C1.006.5 × 104−1.52.5 × 104−0.33.2−0.2

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/9/eaaw3818/DC1

    Fig. S1. Biochemical characterization of TtClpP.

    Fig. S2. Characterization of GFPssrA degradation by TtClpXP.

    Fig. S3. Characterization of TtClpP peptidase activity in the absence and presence of bortezomib.

    Fig. S4. Characterization of FITC-casein degradation by TtClpP in the absence and presence of bortezomib and/or TtClpX.

    Fig. S5. Ligand validation by 2mFo-DFc, omit, and polder maps.

    Fig. S6. Distance RMSD of the 14 alanine tripeptide ligands with respect to their initial bound state in TtClpP for three independent, 1-μs-long MD simulations.

    Fig. S7. Analysis of structural differences between TtClpP and EcClpP.

    Table S1. Crystallographic data collection and refinement statistics.

    Reference (65)

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Biochemical characterization of TtClpP.
    • Fig. S2. Characterization of GFPssrA degradation by TtClpXP.
    • Fig. S3. Characterization of TtClpP peptidase activity in the absence and presence of bortezomib.
    • Fig. S4. Characterization of FITC-casein degradation by TtClpP in the absence and presence of bortezomib and/or TtClpX.
    • Fig. S5. Ligand validation by 2mFo-DFc, omit, and polder maps.
    • Fig. S6. Distance RMSD of the 14 alanine tripeptide ligands with respect to their initial bound state in TtClpP for three independent, 1-μs-long MD simulations.
    • Fig. S7. Analysis of structural differences between TtClpP and EcClpP.
    • Table S1. Crystallographic data collection and refinement statistics.
    • Reference (65)

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