Fig 1.
Characterization and enzymatic activity of ClpP proteases from P. plecoglossicida.
(A) Schematic representation of a P. plecoglossicida gene cluster encoding Clp protein family components, numbers correspond to genomic loci of the proteins. (B) Phylogenetic tree of ClpP proteins constructed using neighbor-joining method based on multiple sequence alignment (S1A Fig). Bootstrap values (>50%) indicate branch support, with species and protein accessions labeled accordingly. (C) Representative multiple sequence alignment of selected ClpP homologs highlighting catalytic residues: Serine-S and Histidine-H marked with black asterisks, while Aspartic-D and Proline-P are indicated by red asterisks. Size-exclusion chromatography (SEC) elution profiles of recombinant PpClpP1 (D) and PpClpP2 (E). (F) Assessment of peptidase activity in P. plecoglossicida ClpP. (a) Comparative analysis of peptidase activity in PpClpP1 versus PpClpP2. (b) Peptidase activity of PpClpP1 catalytic triad mutants. (c) Peptidase activity of PpClpP2 catalytic triad mutants. Values indicated at the base of each bar denote the fold-change in fluorescence intensity relative to the DMSO-treated control. Bars represent the means of three independent experiments ± SD. All experiments were performed in triplicate. *p < 0.05, **p < 0.01.
Fig 2.
Structural architecture of PpClpP1.
(A) Surface representation of the tetradecameric PpClpP1 complex (PDB ID 9UXT) showing top and side views, with each monomer in the heptameric rings colored distinctly for clarity. (B) Ribbon diagram of an individual subunit highlighting the secondary structure elements, including six α-helices (denoted with α-letters) and ten β-strands (denoted with β-numbers), with the catalytic triad (S-H-P) prominently marked by yellow circles.
Fig 3.
Contrasting structural features of PpClpP1 compared to homologs.
(A) Domain architecture comparison showing sequence length and catalytic active sites between PpClpP1 (blue) and other structurally characterized ClpP homologs (gray). (B) a, Structural superposition of PpClpP1 monomer with representative ClpP structures from E. coli (EcClpP1, 3HLN), P. aeruginosa (PaClpP1/2, 7M1M/7M1L), S. aureus (SaClpP, 3V5E), M. Tuberculosis (MtbClpP1/P2, 6VGK), L. monocytogenes (LmClpP1/2, 4JCT/4JCQ), H. pylori (HpClpP2, 2ZL0), B. subtilis (BsClpP2, 7FEQ), and T. thermophilus (TtClpP2, 6HWM). b-c, Dashed boxes highlight the N-terminal α-helical regions (absent in PpClpP1) and catalytic triad sites, with each homolog colored distinctly. (C) Detailed views of catalytic triad configurations in various ClpP homologs (a-l) and their structural superposition (m), demonstrating the unique Ser-His-Pro arrangement in PpClpP1 compared to conventional Ser-His-Asp/Asn triads in other species, (n) Cryo-EM density for the catalytic triad of PpClpP1. The comparative analysis reveals PpClpP1’s distinctive structural adaptations at both N-terminal and active site regions.
Fig 4.
Distinct structural features and functional interplay between PpClpP1 and PpClpP2.
(A) Side views of electrostatic potential surfaces for the PpClpP1 tetradecamer (left) and the predicted PpClpP2 tetradecamer (right) generated by AlphaFold. (B) Top views of electrostatic potential surfaces for the PpClpP1 tetradecamer (left) and the predicted PpClpP2 tetradecamer (right). Inset images illustrate the axial pore diameter sizes of PpClpP1 (42.7 Å) and PpClpP2 (25.2 Å), respectively. (C) Structural alignment of PpClpP1 (Cryo-EM structure) with PpClpP2 (predicted model) highlighting domain organization (left) and catalytic triad configurations (right), including superposition with P. aeruginosa ClpP1 (PaClpP1). (D) Purification of the co-expressed PpClpP1-PpClpP2 complex. (a) Schematic of the initial Strep-tag affinity purification. Supernatant: supernatant after E. colicell lysis; Flow-through: flow-through fraction from the Streptactin resin; Wash: washing with buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8.0); Elution: elution with buffer containing 2.5 mM D-desthiobiotin, 150 mM NaCl, 100 mM Tris-HCl, pH 8.0. (b) Subsequent Ni-NTA affinity purification of the eluate from panel a. Flow-through: flow-through fraction from the Ni-NTA resin; Elution: elution with buffer containing 300 mM imidazole, 150 mM NaCl, 50 mM Tris-HCl, pH 8.0. (E) Comparative analysis of the peptidase activities among the PpClpP1P2 heterotetradecamer, the PpClpP1 homotetradecamer, and the PpClpP2 homotetradecamer. (F) The structure model of PpClpP1P2 heterotetradecamer predicted using AlphaFold. (G) Cartoon model depicting the interaction between a single PpClpP1 subunit and a single PpClpP2 subunit. (H) Analysis of the electrostatic potential at the homomeric (PpClpP1-PpClpP1) and heteromeric (PpClpP1-PpClpP2) interaction interfaces.
Fig 5.
Biochemical and functional characterization of PpClpX interactions with homomeric PpClpP1, PpClpP2, and the heteromeric PpClpP1P2 Complex.
(A) Purification and analytical ultracentrifugation (AUC) analysis of the truncated PpClpX (65-414 aa). The sedimentation profile indicates that PpClpX elutes as a stable hexamer. An SDS-PAGE analysis of the purified protein is shown on the right. (B-D) Pull-down assays assessing the interaction between PpClpX and different PpClpP isoforms. (B) His pull-down assay with PpClpX-His, PpClpP1-Strep can not co-elute with PpClpX-His. (C) Strep pull-down assay with PpClpP2-Strep, PpClpX-His was specifically pulled down by PpClpP2-Strep. (D) Strep pull-down assay with the PpClpP1P2 heterocomplex, PpClpX-His was pulled down by the PpClpP1P2 heterocomplex. (E-F) Degradation assays of fluorescent substrates by the respective active protease complexes. Degradation of GFP-ssrA (E) or FITC-casein (F). Significant protease activity is observed only in the presence of both PpClpX (full-length) and PpClpP2 or the PpClpP1P2 heterocomplex. Values indicated at the base of each bar denote the fold-change in fluorescence intensity relative to the BSA-treated control. Bars represent the means of three independent experiments ± SD. All experiments were performed in triplicate. **p < 0.01.
Fig 6.
Characterization of BTZ-mediated inhibition of PpClpP1 and its antibacterial effects.
(A) Determination of the growth curves of the wild-type P. plecoglossicida PQLYC4, ΔPpClpP1, ΔPpClpP2, and complemented strains (designated as cΔPpClpP1and cΔPpClpP2). (B) Differential scanning fluorimetry (DSF) analysis of PpClpP1 in the presence of small molecules (ADEP and BTZ). (C) Isothermal titration calorimetry (ITC) binding isotherm for BTZ (300 μM) titrated into PpClpP1 (30 μM), with derived binding parameters (Kd = 98 ± 3.41 μM). (D) Effect of BTZ on the peptidase activity of PpClpP1. (E) Effect of BTZ on the peptidase activity of PpClpP2. BTZ sensitivity assays of wild-type P. plecoglossicida PQLYC4 (F), ΔPpClpP1 (G), and ΔPpClpP2 mutant strains (H). Left, the bacterial cultures were adjusted to OD600 ≈ 0.4 with double distilled water, and 10-fold serial dilution was performed on TSB (with 1.25 μM BTZ), and plated on TSB agar to determine the number of surviving colony forming bacteria. Right, when freshly cultured bacteria grew to an OD600 value of 0.1, add BTZ small molecules at different concentrations (μM), spectrophotometry was used to measure the OD600 value at appropriate intervals. Bars represent the means of three independent experiments ± SD. All experiments were performed in triplicate.
Fig 7.
Elucidation of the BTZ binding mode within PpClpP1.
(A) 2D-molecular docking (left) and 3D-molecular docking (right) between BTZ and the PpClpP1 monomer. (B) The surface structure diagram of the PpClpP1 tetradecamer, with orange and cyan representing the PpClpP1 monomers in the two heptameric rings, respectively. The black dashed lines outline the removed two PpClpP1 monomers (left). Depiction of the hydrophobic binding pocket that BTZ binds in the context of the PpClpP1 monomer. Red sticks are the small molecule BTZ. (C) Substrate-binding pocket of PpClpP1, the residues involved in the binding to the BTZ are shown as sticks. (D) Enlarged view of the catalytic triad and BTZ in the PpClpP1 monomer. The black measurement lines represent the average distances between residues S73/H96 and the benzene ring on BTZ, which are 4.1 Å and 5.7 Å respectively. (E) Differential scanning fluorimetry (DSF) analysis of various PpClpP1 mutant proteins with BTZ. The ΔTm value represents the temperature difference between the melting temperature (Tm) of the PpClpP1 mutant + BTZ and that of the wild-type PpClpP1 + BTZ. (F) The binding affinity of BTZ to wild-type PpClpP1 and its mutants was quantified by isothermal titration calorimetry (ITC). The derived Kd values are shown within. Results are representative of those from three independent experiments. Data represent mean values ± SD.
Fig 8.
Comparative analysis of ligand binding modes across ClpP homologs.
(A) Cartoon representation of the PpClpP1 monomer in BTZ-bound (marked red) and (B) TtClpP BTZ-bound (marked gray) states. (C) Superposition of BTZ-bound PpClpP (cyan) and TtClpP (light blue) reveals distinct binding geometries (left), with electrostatic potential maps illustrating differential interaction patterns (right). Detailed views of binding interfaces demonstrate residue-specific contacts (red spheres) in PpClpP1 (D) versus TtClpP (E). (F) Comparative analysis includes ADEP-bound (orange) EcClpP dimer, showing inter-subunit binding (violet/light pink subunits) with surface and electrostatic potential representations (right) that contrast sharply with BTZ’s monomer-restricted binding mode.
Fig 9.
Assembly model of ClpP family proteins in P. plecoglossicida.
(A) The ClpP1 assembles in a homotetradecameric form and is capable of exhibiting low peptidase activity. However, BTZ can bind to ClpP1 and inhibit its peptidase activity. (B) The ClpP2 assembles in a homotetradecameric form and is capable of exhibiting high peptidase activity. (C) The formation of a P. plecoglossicida ClpP1P2 heterotetradecamer. (D) Potential assembly mode of the P. plecoglossicida ClpP protein complex with AAA+ unfoldases. The assembly mode of the ClpP2 homotetradecamer and AAA+ unfoldases complex (model 1), the assembly mode of the ClpP1P2 heterotetradecamer and AAA+ unfoldases complex (model 2).