Mycobacterium tuberculosis ClpC1 N-Terminal Domain Is Dispensable for Adaptor Protein-Dependent Allosteric Regulation

Abstract

ClpC1 hexamers couple the energy of ATP hydrolysis to unfold and, subsequently, translocate specific protein substrates into the associated ClpP protease. Substrate recognition by ATPases associated with various cellular activities (AAA+) proteases is driven by the ATPase component, which selectively determines protein substrates to be degraded. The specificity of these unfoldases for protein substrates is often controlled by an adaptor protein with examples that include MecA regulation of Bacillus subtilis ClpC or ClpS-mediated control of Escherichia coli ClpA. No adaptor protein-mediated control has been reported for mycobacterial ClpC1. Using pulldown and stopped-flow fluorescence methods, we report data demonstrating that Mycobacterium tuberculosis ClpC1 catalyzed unfolding of an SsrA-tagged protein is negatively impacted by association with the ClpS adaptor protein. Our data indicate that ClpS-dependent inhibition of ClpC1 catalyzed SsrA-dependent protein unfolding does not require the ClpC1 N-terminal domain but instead requires the presence of an interaction surface located in the ClpC1 Middle Domain. Taken together, our results demonstrate for the first time that mycobacterial ClpC1 is subject to adaptor protein-mediated regulation in vitro.

Genome‐wide interaction screen for Mycobacterium tuberculosis ClpCP protease reveals toxin–antitoxin systems as a major substrate class

“…To date, only one adaptor protein has been suggested for the Mtb Clp system based on sequence similarity to ClpS from other bacteria, yet unfortunately, its ORF ( rv1331 ; clps ) was not contained in the BACTH library. However, the one‐to‐one BACTH experiment with ClpS and the ClpC1 N‐terminal domain demonstrates that Mtb ClpS binds to the ClpC1 N‐domain, and, using further in vitro characterization, we observed that ClpS not only inhibits the degradation of ssrA‐tagged substrates [41] and ClpC1, but also promotes the degradation of N‐end rule substrates in Mtb. ClpCP in the absence of ClpS appears to have a similar affinity for GFPssrA as it does for FR‐li‐GFP in the presence of ClpS, unlike in E. coli , where affinity of ClpAP for GFPssrA is at least 20‐fold lower than the affinity of ClpAPS for FR‐li‐GFP [48].…”

“…It is possible that such an adapter might be found among the number of hits of unknown function, but without sequence similarity to known adapters would be difficult to identify. However, there exists one candidate adaptor protein of ClpC1 in Mtb that was identified based on its similarity to the ClpA adaptor protein ClpS of other bacteria [41]. In E. coli, ClpS switches the specificity of the chaperone ClpA from ssrA-tagged substrates to N-end rule substrates [42,43].…”

The unfoldase ClpC1 of Mycobacterium tuberculosis regulates the expression of a distinct subset of proteins having intrinsically disordered termini

“…It is highly up-regulated transcriptionally via various two component regulators under different stress conditions (26). To understand the underlying mechanism of regulation of Hsp20 protein by ClpC1P1P2, first we sought to determine whether the accessory proteins such as ClpS, which acts as a ClpC1 adapter (27), or SmpB, which is involved in ssrA tagging, are directly involved. Expression of clpS and smpB was depleted in cells using CRISPRi approach, and the level of Hsp20 was determined by anti-Hsp20 immunoblotting in the control, clpS(2) and smpB(2) strains.…”

Progression from remodeling to hibernation of ribosomes in zinc-starved mycobacteria

“…However, attempts to test the role of ClpC1 by CRISPRi were not successful due to partial depletion of corresponding transcripts. Because mycobacterial ClpC1 appears to interact with ClpS ( 37 ), we probed the role of ClpS in Mrf degradation. An isogenic Δ clpS deletion (YL15) accumulated significantly more Mrf than the parent strain (YL2) at an early growth stage ( Fig.…”