The caseinolytic protease (ClpP) is an emerging antibacterial target. Pseudomonas plecoglossicida ( Pp ), a pathogen causing visceral white spot disease in Larimichthys crocea , encodes two ClpP paralogs, Pp ClpP1 and Pp ClpP2. This study characterizes their distinct structural and functional properties. Phylogenetic and biochemical analysis revealed that Pp ClpP2 functions as a canonical serine protease with high peptidase activity, while Pp ClpP1 is evolutionarily divergent, exhibiting low inherent activity due to an unconventional Ser-His-Pro catalytic triad and a truncated N-terminal domain. Cryo-EM structure determination of Pp ClpP1 confirmed a homotetradecameric assembly with a dilated axial pore and a non-canonical catalytic geometry. In contrast, AlphaFold-predicted Pp ClpP2 displayed a compact structure with a canonical Ser-His-Asp triad. The subunits formed a stable heterotetradecamer ( Pp ClpP1P2) with enhanced proteolytic activity compared to individual homotetradecameric. Pull-down assays demonstrated that Pp ClpP2, but not Pp ClpP1, specifically interacts with the unfoldase Pp ClpX, and the Pp ClpP1P2 heterotetradecamer further augmented Pp ClpX-mediated degradation of model substrates. Notably, the proteasome inhibitor bortezomib (BTZ) selectively inhibited Pp ClpP1 by binding to a unique pocket near the active site without engaging the catalytic serine, thereby suppressing bacterial growth in a Pp ClpP1-dependent manner. This study elucidates the structural basis of functional divergence between Pp ClpP paralogs, highlights their synergistic interplay in proteolysis, and identifies Pp ClpP1 as a druggable target for antibacterial development.
Chen et al. (Thu,) studied this question.