The Clp protease system fulfills a plethora of important functions in bacteria. It consists
of a tetradecameric ClpP barrel holding the proteolytic centers and two hexameric Clp-ATPase
rings, which recognize, unfold, and then feed substrate proteins into the ClpP barrel for proteolytic
degradation. Flexible loops carrying conserved tripeptide motifs protrude from the Clp-ATPases
and bind into hydrophobic pockets (H-pockets) on ClpP. Here, we set out to engineer microcin J25
(MccJ25), a ribosomally synthesized and post-translationally modified peptide (RiPP) of the lasso
peptide subfamily, by introducing the conserved tripeptide motifs into the lasso peptide loop region
to mimic the Clp-ATPase loops. We studied the capacity of the resulting lasso peptide variants to
bind to ClpP and affect its activity. From the nine variants generated, one in particular (12IGF) was
able to activate ClpP from Staphylococcus aureus and Bacillus subtilis. While 12IGF conferred stability
to ClpP tetradecamers and stimulated peptide degradation, it did not trigger unregulated protein
degradation, in contrast to the H-pocket-binding acyldepsipeptide antibiotics (ADEPs). Interestingly,
synergistic interactions between 12IGF and ADEP were observed