Abstract
The Clp protease complex degrades a multitude of substrates, which are engaged by a AAA+ chaperone such as ClpX and subsequently digested by the dynamic, barrel-shaped ClpP protease. Acyldepsipeptides (ADEPs) are natural product-derived antibiotics that activate ClpP for chaperone-independent protein digestion. Here we show that both protein and small-molecule activators of ClpP allosterically control the ClpP barrel conformation. We dissect the catalytic mechanism with chemical probes and show that ADEP in addition to opening the axial pore directly stimulates ClpP activity through cooperative binding. ClpP activation thus reaches beyond active site accessibility and also involves conformational control of the catalytic residues. Moreover, we demonstrate that substoichiometric amounts of ADEP potently prevent binding of ClpX to ClpP and, at the same time, partially inhibit ClpP through conformational perturbance. Collectively, our results establish the hydrophobic binding pocket as a major conformational regulatory site with implications for both ClpXP proteolysis and ADEP-based anti-bacterial activity.
Highlights
The Clp protease complex degrades a multitude of substrates, which are engaged by a AAA þ chaperone such as ClpX and subsequently digested by the dynamic, barrel-shaped ClpP protease
We started by screening a small compilation of ADEP derivatives[28], which we found to be potent in inducing SaClpP proteolysis (Supplementary Fig. 1a–c)
Isothermal titration calorimetry (ITC) experiments in which ADEP7 was added to a solution of SaClpP showed nonstandard behaviour[30] (Fig. 1d) with the reaction becoming more exothermic during the initial increase of the ADEP7:SaClpP ratio
Summary
The Clp protease complex degrades a multitude of substrates, which are engaged by a AAA þ chaperone such as ClpX and subsequently digested by the dynamic, barrel-shaped ClpP protease. Proteomic experiments showed that this uncontrolled proteolysis leads to the depletion of several essential proteins such as the bacterial cytoskeleton protein FtsZ, which in turn causes impaired cell division and cell death[26,27] Since this unique antibiotic mechanism exploits a cellular machinery and activates it for destruction, ADEPs were recently shown to be active against bacterial persister cells and established biofilms of pathogenic Staphylococcus aureus[27]. Our results reveal an additional layer of regulation in the ClpXP system: the link between ClpP barrel conformation and occupation of the allosteric sites This demonstrates that the allosteric binding pocket of ClpP functions as a conformational switch that controls the accessibility of the active sites and their activity
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