Abstract
Intramembrane proteases (IMPs) are a unique class of proteases that catalyze the proteolysis within the membrane and regulate diverse cellular processes in various organisms. RseP, an Escherichia coli site-2 protease (S2P) family IMP, is involved in the regulation of an extracytoplasmic stress response through the cleavage of membrane-spanning anti-stress-response transcription factor (anti-σE) protein RseA. Extracytoplasmic stresses trigger a sequential cleavage of RseA, in which first DegS cleaves off its periplasmic domain, and RseP catalyzes the second cleavage of RseA. The two tandem-arranged periplasmic PDZ (PDZ tandem) domains of RseP serve as a size-exclusion filter which prevents the access of an intact RseA into the active site of RseP IMP domain. However, RseP's substrate recognition mechanism is not fully understood. Here, we found that a periplasmic region of RseP, located downstream of the PDZ tandem, contains a segment (named H1) predicted to form an amphiphilic helix. Bacterial S2P homologs with various numbers of PDZ domains have a similar amphiphilic helix in the corresponding region. We demonstrated that the H1 segment forms a partially membrane-embedded amphiphilic helix on the periplasmic surface of the membrane. Systematic and random mutagenesis analyses revealed that the H1 helix is important for the stability and proteolytic function of RseP and that mutations in the H1 segment can affect the PDZ-mediated substrate discrimination. Cross-linking experiments suggested that H1 directly interacts with the DegS-cleaved form of RseA. We propose that H1 acts as an adaptor required for proper arrangement of the PDZ tandem domain to perform its filter function and for substrate positioning for its efficient cleavage.