Generation of Lasso Peptide-Based ClpP Binders.

Area of Science:

• Bacterial Proteolysis
• Enzyme Engineering
• Molecular Biology

Background:

• The Clp protease system is crucial for bacterial protein homeostasis, comprising ClpP and Clp-ATPases.
• Clp-ATPases utilize specific loops to interact with ClpP's hydrophobic pockets, facilitating substrate degradation.
• Microcin J25 (MccJ25) is a lasso peptide with potential for bioengineering.

Purpose of the Study:

• To engineer MccJ25 by incorporating Clp-ATPase-like motifs into its structure.
• To investigate the binding affinity and activity modulation of engineered MccJ25 variants with ClpP.
• To compare the effects of engineered MccJ25 with known ClpP activators like ADEPs.

Main Methods:

• Genetic engineering of MccJ25 to introduce conserved tripeptide motifs.
• Assays to evaluate the binding of MccJ25 variants to ClpP from *Staphylococcus aureus* and *Bacillus subtilis*.
• Proteolytic activity assays to measure ClpP activation and substrate degradation.
• Analysis of interactions between engineered MccJ25 and acyldepsipeptide antibiotics (ADEPs).

Main Results:

• Nine MccJ25 variants were generated, with one variant (12IGF) showing significant ClpP activation.
• 12IGF stabilized ClpP tetradecamers and enhanced peptide degradation, unlike ADEPs which trigger unregulated degradation.
• Synergistic effects were observed between 12IGF and ADEPs, suggesting complex regulatory interactions.

Conclusions:

• Engineered MccJ25 variants can modulate bacterial Clp protease activity.
• The 12IGF variant represents a novel tool for studying ClpP function and regulation.
• The distinct mechanism of 12IGF activation offers new avenues for therapeutic development targeting bacterial proteolysis.