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.
Highlights
ATP-dependent proteases represent a family of molecular machines responsible for the regulated turnover of misfolded, aggregated, or degradation-tagged cellular proteins [1,2,3,4]
To determine whether mycobacterial ClpS is a functional adaptor of ClpC1, we performed stopped-flow fluorescence experiments using a method that reports on ClpC1 catalyzed unfolding of an SsrA-tagged protein
Association of S. aureus ClpC with MecA induces a structural rearrangement that transitions the complex from an inactive resting state to a fully functional chaperone [15]
Summary
ATP-dependent proteases represent a family of molecular machines responsible for the regulated turnover of misfolded, aggregated, or degradation-tagged cellular proteins [1,2,3,4]. The regulated removal of protein substrates in the cytoplasm is mediated by at least four different proteolytic complexes that are broadly divided into two groups that include the bacterial-like proteases (FtsH, Lon, ClpXP and ClpC1P) and the eukaryotic-like proteasome [5,6]. These proteases share a common architecture in which a ring-shaped AAA+ (ATPases Associated with various cellular Activities) ATPase associates with one or both ends of a barrel-shaped peptidase that contains active sites inaccessible to bulk solvent [3,5]. Saturation of ClpS binding to ClpA hexamers as indicated by a 6:1 ratio of ClpS:ClpA6 is not necessary for significant control of ClpA function but an intact N-terminal domain (NTD) is required for ClpS-dependent inhibition of ClpAP catalyzed degradation of SsrA-tagged protein substrates
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