Structural and mechanistic insights into caseinolytic protease inhibition for antimicrobial development against Pseudomonas plecoglossicida
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.