The functional ClpXP protease of Chlamydia trachomatis requires distinct clpP genes from separate genetic loci

Abstract

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, ...

The ClpX and ClpP2 Orthologs of Chlamydia trachomatis Perform Discrete and Essential Functions in Organism Growth and Development

“…Given the predicted binding of the ClpX inhibitors on the structure of ClpX Ctr , we sought to determine whether these drugs effectively blocked ClpX function in vivo. We utilized a chlamydial SsrA-tagged green fluorescent protein (GFP) variant [GFP(VAA)], which was previously shown to be a substrate of ClpX Ctr in vitro (46), or GFP tagged with a mutated SsrA tag [GFP(VDD)]. When mutating VAA to VDD, GFP is rendered resistant to ClpX recognition (47).…”

“…also harbor the transfer-messenger RNA (tmRNA)/SsrA tagging system for ribosomal rescue ( 18 , 52 – 55 ), which fits a model where ClpX Ctr may play an integral role in turnover of tagged partially translated peptides. A recent article, using an in vitro assay for SsrA-tagged GFP degradation, suggests this function of chlamydial ClpX may be conserved ( 46 ). Our in vivo data exhibiting SsrA-tagged GFP protection with the addition of a ClpX inhibitor also suggests ClpX Ctr can target SsrA-tagged substrates, but whether this tagging is for ribosomal rescue or more specific purposes ( 56 , 57 ) remains to be determined and is currently under investigation by our research group.…”

The ClpX and ClpP2 Orthologs of Chlamydia trachomatis Perform Discrete and Essential Functions in Organism Growth and Development

“…Whether ClpX Ctr can actually target SsrA-tagged substrates, and whether this tagging is for ribosomal rescue or more specific purposes (53, 54), remains to be determined and is currently under investigation by our research group. A recent article, using an SsrA-tagged GFP, suggests this function of chlamydial ClpX may be conserved (55).…”

“…Transformants were screened for inserts using colony PCR with Fermentas Master Mix (Thermo Scientific) and positive clones were grown for plasmid isolation (GeneJet Plasmid Miniprep Kit, Thermo Scientific). Sequence verified plasmids were then transformed into BL21(DE3) Δ clpPAX E. coli (55) for subsequent protein purification.…”

An allosteric switch regulatesMycobacterium tuberculosisClpP1P2 protease function as established by cryo-EM and methyl-TROSY NMR

“…Unlike for other ClpPs, the addition of ADEP alone does not activate MtClpP1P2 (32,49). The requirement for activators appears to be a unique feature of actinobacteria, as other species that contain two ClpP proteoforms, such as Listeria monocytogenes (50,51) and Chlamydia trachomatis (52), are functional even in their absence. X-ray structures of MtClpP1P2 crystalized in the presence of Bz-LL show that the overall architecture and fold of each ClpP protomer is indistinguishable from what is found in other bacterial ClpPs that retain activity in the apo form (53).…”