UNDERSTANDING THE ACTIVATION OF BACTERIAL PROTEASE CLPP BY ACYLDEPSIPEPTIDE ANTIBIOTIC

Abstract

Acyldepsipeptide (ADEP1) is an antibiotic that binds to Escherichia coli ClpP, mimicking the interaction that the protease typically establishes with ClpA/ClpX ATPases in bacterial cells. Binding of ADEP1 causes the N-terminal end of the ClpP to adopt a structured β-hairpin and triggers opening of the axial gate in the tetradecameric ClpP. Open conformation of the axial gate causes translocation of the substrates into the catalytic chamber of ClpP and the resultant uncontrolled proteolysis renders cellular death making ADEP1 a potent antibiotic. Our current understanding about the ADEP1-induced open conformation of the axial gate is limited. Based on the existing X-ray structures, it is unclear whether the mechanism of ADEP1-mediated activation of ClpP is conserved in Gram-positive and Gram-negative bacteria. To understand the activation mechanism of ClpP by ADEP1, we obtained Bacillus subtilis ClpP variants with amino acid substitutions in the N-terminal region and tested the effect of these mutations on substrate translocation using fluorescence-based proteolytic assays and cryo-electron microscopy. We found that compromising the integrity of the β-hairpin adopted by the N-terminal region prevented translocation of the substrate into the catalytic chamber of B. subtilis ClpP. These results suggest that the structural requirements for a functional axial channel are conserved in Gram-positive and Gram-negative bacteria. This study defines the structural requirements for ADEP1-mediated activation of the ClpP protease and serves as a model for the functioning of ClpP in the context of the ClpAP and ClpXP complexes.