Dynamic Oligomerization Processes of <i>Bacillus subtilis</i> ClpP Protease Induced by ADEP1 Studied with High-Speed Atomic Force Microscopy

Bacterial ClpPs are a highly conserved family of serine proteases that associate with members of the AAA+ ATPase (ATPase associated with diverse cellular activities) family to degrade protein substrates. The antibiotic A54556 factor (ADEP1) induces uncontrolled proteolysis by forming an ATPase-independent ClpP-ADEP complex. Cryo-EM analysis of <i>Bacillus subtilis</i> ClpP (<i>Bs</i>-ClpP) has demonstrated that ADEP1 binding shifts the protease to an active extended conformation and opens its axial entry pores. However, the dynamic oligomerization processes of <i>Bs</i>-ClpP induced by ADEP1 remain unclear. In this study, we used a combination of biochemical studies and high-speed atomic force microscopy (HS-AFM) to reveal how ADEP1 affects the oligomerization states and protease activity of <i>Bs</i>-ClpP, inducing the active extended state and protease activity of <i>Bs</i>-ClpP. HS-AFM observations demonstrated that the <i>Bs</i>-ClpP tetradecamer (2R state) forms via a progression from monomers to oligomers and then from oligomers to heptamers (R state) in the presence of ADEP1. Our results suggest that ADEP1 binding to monomeric <i>Bs</i>-ClpP triggers conformational changes that facilitate <i>Bs</i>-ClpP oligomerization (R and 2R states) and activation.