[Dynamic Oligomerization Processes and Functional Characterization of Bacterial ClpP Protease].

ClpP protease is a highly conserved serine protease that plays a crucial role in bacterial protein quality control alongside its partner AAA+ ATPases. ClpP assembles into a barrel-shaped tetradecamer that degrades unfolded or misfolded proteins translocated by ATP-driven unfoldases, such as ClpC, ClpX, or ClpA. Acyldepsipeptide (ADEP) antibiotics bind to the hydrophobic pockets of ClpP, mimicking the natural interaction with ATPases, thus activating ClpP in an ATP-independent manner. ADEP binding induces major conformational changes that open the axial pores, enabling ClpP to degrade large protein substrates such as the cell division protein FtsZ, ultimately causing cell death. Our recent studies revealed that in Bacillus subtilis the ClpP proteolytic system regulates the intracellular levels of nonribosomal peptide synthetases SrfAA, SrfAB, and SrfAC, which are responsible for surfactin biosynthesis. Moreover, ADEP1-activated ClpP directly degraded SrfAA and SrfAB both in cells and in vitro, identifying new physiological substrates of the ADEP1-ClpP complex. High-speed atomic force microscopy (HS-AFM) analysis visualized the stepwise oligomerization of B. subtilis ClpP from monomers to heptamers and then to tetradecamers upon ADEP1 binding, revealing dynamic assembly processes underlying its activation. These findings enhance our understanding of bacterial protein degradation mechanisms and provide a molecular basis for the rational design of ClpP-targeting antibiotics with novel modes of action.