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. / Thesis / Master of Science (MSc)

<p>Caseinolytic protease P (ClpP) is a compartmentalized bacterial protease tightly regulated by AAA+ proteins such as ClpA and ClpX within <em>Escherichia coli</em>. It is known that the amino terminus is required to gate the axial entry pores of ClpP however the conformation adopted during activation by ClpA and ClpX has not been properly characterized. Recently it has been determined that binding of a group of antimicrobials termed acyldepsipeptides induces the open conformation of the axial channel independent of ClpA or ClpX to mediate the translocation of unfolded proteins. To determine the structural determinates required to stabilize the open conformation of the axial channel during acyldepsipeptide binding we generated amino terminal variants by site directed mutagenesis. It was found that the formation and anchoring of a β-hairpin element at the amino terminus was crucial for the effective translocation of protein substrates. These results describe the structural requirements that mediate substrate translocation during acyldepsipeptide induced activation and provide a model for the structural requirements of the ClpP amino terminus during formation of the ClpAP and ClpXP holocomplexes.</p> / Master of Science (MSc)