Abstract
General and regulatory proteolysis in bacteria is executed by a set of ATP-dependent proteases composed of hexameric ring-forming AAA+ proteins and associated peptidase barrels (e.g., ClpP). These AAA+ proteases play crucial roles in stress protection and bacterial virulence. Here, we provide the first biochemical characterization of the potential drug target ClpE-ClpP from Enterococcus faecalis. We show that ClpE-ClpP forms an autonomous and efficient protease, which degrades misfolded and aggregated model substrates and the stress-responsive transcriptional regulator CtsR. This qualifies ClpE-ClpP as a central component of bacterial protein quality control systems and explains formerly reported stress-sensitive phenotypes of clpE mutants. ClpE substrate specificity is mediated by its N-terminal domain, which is crucial for targeting misfolded and aggregated proteins. ClpE assembles into a tetrahedral structure formed by four hexamers that interact via their coiled-coil M-domains. ClpP binding to ClpE tetrahedrons triggers the formation of large clusters of proteolytic complexes in vitro and in vivo. Such assembly in principle can allow for spatially confined proteolysis, separating the proteolytic activity of ClpE-ClpP complexes from other cellular processes. Indeed, ClpE M-domain mutants, which are deficient in cluster formation, exhibit increased toxicity in vivo.