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. 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.

Summary The Clp protease complex in Mycobacterium tuberculosis is unusual in its composition, functional importance, and activation mechanism. While most bacterial species contain a single ClpP protein that is dispensable for normal growth, mycobacteria have two ClpPs, ClpP1 and ClpP2, which are essential for viability and together form the ClpP1P2 tetradecamer. Acyldepsipeptide antibiotics of the ADEP class inhibit the growth of Gram-positive firmicutes by activating ClpP and causing unregulated protein degradation. Here we show that, in contrast, mycobacteria are killed by ADEP through inhibition of ClpP function. Although ADEPs can stimulate purified M. tuberculosis ClpP1P2 to degrade larger peptides and unstructured proteins, this effect is weaker than for ClpP from other bacteria and depends on the presence of an additional activating factor (e.g. the dipeptide benzyloxycarbonyl-leucyl-leucine in vitro) to form the active ClpP1P2 tetradecamer. The cell division protein FtsZ, which is a particularly sensitive target for ADEP-activated ClpP in firmicutes, is not degraded in mycobacteria. Depletion of the ClpP1P2 level in a conditional Mycobacterium bovis BCG mutant enhanced killing by ADEP unlike in other bacteria. In summary, ADEPs kill mycobacteria by preventing interaction of ClpP1P2 with the regulatory ATPases, ClpX or ClpC1, thus inhibiting essential ATP-dependent protein degradation.

clp proteases play a central role in bacterial physiology and, for some bacterial species, are even essential for survival. Also due to their conservation among bacteria including important human pathogens, clp proteases have recently attracted considerable attention as antibiotic targets. Here, we functionally reconstituted and characterized the clpXp protease of Chlamydia trachomatis (ctclpXp), an obligate intracellular pathogen and the causative agent of widespread sexually transmitted diseases in humans. our in vitro data show that ctclpXp is formed by a hetero-tetradecameric proteolytic core, composed of two distinct homologs of ClpP (ctClpP1 and ctClpP2), that associates with the unfoldase ctClpX via ctClpP2 for regulated protein degradation. Antibiotics of the ADEP class interfere with protease functions by both preventing the interaction of ctClpX with ctClpP1P2 and activating the otherwise dormant proteolytic core for unregulated proteolysis. thus, our results reveal molecular insight into ctclpXp function, validating this protease as an antibacterial target. Bacterial Clp proteases constitute compartmentalized macromolecular machines. On the molecular level, Clp proteases form large complexes that can be separated into two major components: a proteolytic core formed by a barrel-shaped tetradecamer of ClpP subunits 1 that has to associate with regulatory AAA+ Clp-ATPases (e.g. ClpX and ClpA in Escherichia coli, or ClpX and ClpC in Staphylococcus aureus) to allow for substrate recognition and proteolytic activity 2. In most bacteria including E. coli, S. aureus and Bacillus subtilis, 14 ClpP monomers arrange as two homo-heptameric rings, which stack vis-à-vis to form a cylindrical structure of about 90 Å in both diameter and height. Inside of the compartmentalized ClpP barrel, a spacious degradation chamber of approx. 50 Å width secludes the active sites of the protease located close to the equatorial plane of the ClpP barrel, which comprise 14 catalytic triads with the canonical residues typical for serine proteases (Ser, His, Asp). The compartmentalized structure of the ClpP tetradecamer effectively shields the active sites from potential protein substrates in the cytoplasmic environment, which can only be accessed by small peptides through narrow entry pores at the apical and distal surfaces of the ClpP barrel. ClpP itself is almost free of substrate specificity and is unable to degrade proteins on its own under natural conditions due to restricted substrate access to the inner proteolytic chamber of the ClpP barrel. Only small peptides that readily diffuse through the entrance pores are degraded 3. As such, the ClpP tetradecamer by itself should be considered as a peptidase but constitutes the dormant core of the larger proteolytic Clp complex. For proteolytic activity, the ClpP barrel has to associate with designated Clp-ATPases, hexameric unfoldases, which bind via distinct loops to the buried hydrophobic pockets at the apical sides of the ClpP barrel. The Clp-ATPases select natural Clp substrates, ...

Covering: up to 2017The bacterial Clp protease is a highly conserved and structurally versatile machine. It has gained a lot of recognition during the last decade as a novel antibacterial drug target with an unprecedented mechanism of action. Due to its complexity, there are distinct means of interfering with its natural functions and several compounds targeting this machine have been identified. In this review, we summarize the current state of knowledge about natural products deregulating Clp proteolysis, a crucial and delicate process within the cell. Among those, acyldepsipeptide antibiotics of the ADEP class (ADEPs) are characterized best. The molecular mechanism of ADEP-mediated deregulation sheds light on the inner workings of the Clp protease.