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

## TL;DR

This study explores two protease variants in a fish pathogen, revealing their unique roles and how inhibiting one can stop bacterial growth.

## Contribution

The study provides structural and functional insights into PpClpP1 and PpClpP2, identifying PpClpP1 as a novel antimicrobial target.

## Key findings

- PpClpP1 has low activity due to an unconventional catalytic triad and truncated structure.
- PpClpP1 and PpClpP2 form a hybrid complex with enhanced proteolytic activity.
- Bortezomib selectively inhibits PpClpP1, suppressing bacterial growth.

## Abstract

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, PpClpP1 and PpClpP2. This study characterizes their distinct structural and functional properties. Phylogenetic and biochemical analysis revealed that PpClpP2 functions as a canonical serine protease with high peptidase activity, while PpClpP1 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 PpClpP1 confirmed a homotetradecameric assembly with a dilated axial pore and a non-canonical catalytic geometry. In contrast, AlphaFold-predicted PpClpP2 displayed a compact structure with a canonical Ser-His-Asp triad. The subunits formed a stable heterotetradecamer (PpClpP1P2) with enhanced proteolytic activity compared to individual homotetradecameric. Pull-down assays demonstrated that PpClpP2, but not PpClpP1, specifically interacts with the unfoldase PpClpX, and the PpClpP1P2 heterotetradecamer further augmented PpClpX-mediated degradation of model substrates. Notably, the proteasome inhibitor bortezomib (BTZ) selectively inhibited PpClpP1 by binding to a unique pocket near the active site without engaging the catalytic serine, thereby suppressing bacterial growth in a PpClpP1-dependent manner. This study elucidates the structural basis of functional divergence between PpClpP paralogs, highlights their synergistic interplay in proteolysis, and identifies PpClpP1 as a druggable target for antibacterial development.

Pseudomonas plecoglossicida is a bacterial pathogen that causes devastating visceral white spot disease in the economically important large yellow croaker (Larimichthys crocea). The bacterium encodes two variants of ClpP, a key protease that regulates protein turnover and bacterial fitness. Here, we show that these two variants, PpClpP1 and PpClpP2, have distinct structural and functional roles. While PpClpP2 acts as a typical, highly active protease, PpClpP1 has low intrinsic activity due to an unusual catalytic triad and a truncated structure. Strikingly, when combined, they form a hybrid complex with enhanced degradation capacity. We further find that only PpClpP2 interacts with the unfoldase PpClpX, which helps deliver substrates for breakdown. Importantly, the bortezomib selectively blocks PpClpP1—but not PpClpP2—by binding near its active site, and this inhibition suppresses bacterial growth. Our work reveals how these two protease variants cooperate in P. plecoglossicida and identifies PpClpP1 as a promising target for developing specific antibiotics against this aquaculture pathogen.