Antibiotic acyldepsipeptides stimulate the Streptomyces Clp-ATPase/ClpP complex for accelerated proteolysis

Clp proteases consist of a proteolytic, tetradecameric core ClpP and AAA+ Clp-ATPases. Streptomycetes, producers of a plethora of secondary metabolites, encode up to five different ClpP homologs and the composition of their unusually complex Clp protease machinery has remained unsolved. Here, we report on the composition of the house-keeping Clp protease in Streptomyces , consisting of a hetero-tetradecameric core built of ClpP1, ClpP2 and the cognate Clp-ATPases ClpX, ClpC1 or ClpC2, all interacting with ClpP2 only. ADEP antibiotics dysregulate the Clp protease for unregulated proteolysis. We observed that ADEP binds Streptomyces ClpP1, but not ClpP2, thereby not only triggering the degradation of non-native protein substrates but also accelerating Clp-ATPase-dependent proteolysis. The explanation is the concomitant binding of ADEP and Clp-ATPases to opposite sides of the ClpP1P2 barrel, hence revealing a third, so far unknown mechanism of ADEP action, i.e., the accelerated proteolysis of native protein substrates by the Clp protease. Significance Clp proteases are antibiotic and anti-cancer drug targets. Composed of the proteolytic core ClpP and a regulatory Clp-ATPase, the protease machinery is important for protein homeostasis and regulatory proteolysis. The acyldepsipeptide antibiotic ADEP targets ClpP and has shown promise for treating multi-resistant and persistent bacterial infections. The molecular mechanism of ADEP is multi-layered. Here, we present a new way how ADEP can deregulate the Clp protease system. Clp-ATPases and ADEP bind to opposite sides of Streptomyces ClpP, accelerating the degradation of natural Clp protease substrates. We also demonstrate the composition of the major Streptomyces Clp protease complex, a heteromeric ClpP1P2 core with the Clp-ATPases ClpX, ClpC1 or ClpC2 exclusively bound to ClpP2, and the killing mechanism of ADEP in Streptomyces . ### Competing Interest Statement The authors have declared no competing interest.