The antibiotic ADEP reprogrammes ClpP, switching it from a regulated to an uncontrolled protease

Abstract

A novel class of antibiotic acyldepsipeptides (designated ADEPs) exerts its unique antibacterial activity by targeting the peptidase caseinolytic protease P (ClpP). ClpP forms proteolytic complexes with heat shock proteins (Hsp100) that select and process substrate proteins for ClpP-mediated degradation. Here, we analyse the molecular mechanism of ADEP action and demonstrate that ADEPs abrogate ClpP interaction with cooperating Hsp100 adenosine triphosphatases (ATPases). Consequently, ADEP treated bacteria are affected in ClpP-dependent general and regulatory proteolysis. At the same time, ADEPs also activate ClpP by converting it from a tightly regulated peptidase, which can only degrade short peptides, into a proteolytic machinery that recognizes and degrades unfolded polypeptides. In vivo nascent polypeptide chains represent the putative primary target of ADEP-activated ClpP, providing a rationale for the antibacterial activity of the ADEPs. Thus, ADEPs cause a complete functional reprogramming of the Clp–protease complex.

Crystal structure of Mycobacterium tuberculosis ClpP1P2 suggests a model for peptidase activation by AAA+ partner binding and substrate delivery

“…The synergy of ADEPs and agonists in activating ClpP1P2 indicates that both molecules bind preferentially to functional ClpP1P2 and thus stabilize this species relative to any inactive conformations. There is substantial evidence that ADEP toxicity results from a gain-of-function mechanism in which the open axial pores of ADEP-bound ClpP allow entry and degradation of unfolded or misfolded proteins (14,15,20,21). Because we find that ADEPs both open the pores and activate M. tuberculosis ClpP1P2, it is plausible that rogue degradation of cellular proteins is also responsible for ADEP killing of M. tuberculosis (30).…”

“…ADEPs bind to many homomeric ClpP enzymes and activate cleavage of large peptides and unstructured proteins (20,21). They also inhibit M. tuberculosis growth in the presence of effluxpump inhibitors (30), strongly suggesting that ClpP1P2 also should be an ADEP target, with toxicity resulting either from activation of rogue degradation and/or from inhibition of interaction with a AAA+ partner.…”

“…Agonist and ADEP binding to ClpP1P2 were expected to mimic the binding of polypeptide substrates and AAA+ partners, respectively (14,15,20,21,23,29). For both activating ligands, however, surprises emerged.…”

“…Binding is mediated in part by tripeptide motifs [typically Ile-Gly-Phe or Leu-Gly-Phe (LGF)] in flexible loops in the AAA+ hexamer, which dock into hydrophobic pockets at subunit interfaces on each ClpP heptamer (16)(17)(18)(19). In a remarkable example of protein mimicry by a natural product, cyclic acyldepsipeptide (ADEP) antibiotics bind in the same hydrophobic pockets on ClpP and also open the axial pores, potentially leading to unregulated protein degradation and cell death (14,15,20,21).…”

Structure and Functional Properties of the Active Form of the Proteolytic Complex, ClpP1P2, from Mycobacterium tuberculosis

“…The latter crystals (unlike the crystallographic studies) were grown in the presence of an ADEP antibiotic, which was bound to the ClpC1/X docking site on the apical surface of Mtb ClpP heptamers. Binding of different ADEPs has been shown to activate degradation of unfolded proteins by ClpP (28). ADEPs allosterically induce a change in binding interactions between the N-terminal ␤-hairpin and residues on the surface of ClpP, causing a reorientation of the ␤-hairpins and widening of the axial channel, which presumably allows unfolded polypeptides to pass into the degradation chamber (26,39).…”