Chemical Biology Approaches Reveal Conserved Features of a C-Terminal Processing PDZ Protease

Several proteases like the high temperature requirement A (HtrA) protein family containing internal or C‐terminal PDZ domains play key roles in protein quality control in the cell envelope of Gram‐negative bacteria. While several HtrA proteases have been extensively characterized, many features of C‐terminal processing proteases such as tail‐specific protease (Tsp) are still unknown. To fully understand these cellular control systems, individual domains need to be targeted by specific peptides acting as activators or inhibitors. Here, we describe the identification and design of potent inhibitors and activators of Tsp. Suitable synthetic substrates of Tsp were identified and served as a basis for the generation of boronic acid‐based peptide inhibitors. In addition, a proteomic screen of E. coli cell envelope proteins using a synthetic peptide library was performed to identify peptides capable of amplifying Tsp's proteolytic activity. The implications of these findings for the regulation of PDZ proteases and for future mechanistic studies are discussed. Turning Tsp ON: Chemical biology approaches have revealed that the quality control protease Tsp is triggered into an active state via interactions with various activating clues.