Principal Investigator Robert Sauer
PDZ proteases serve important roles in stress responses and virulence in bacteria. Human orthologs play roles in disease prevention. In general, these enzymes function as trimers or higher oligomers. Each subunit consists of a protease domain and one or two PDZ domains, which typically bind C-terminal peptides and function as allosteric regulators of activity. Despite the widespread biological importance of PDZ proteases, the mechanisms that control the activities of these enzymes are just beginning to be understood.
We have been studying DegS, a PDZ protease anchored to the periplasmic side of the E. coli inner membrane. When protein folding in the periplasm is compromised, DegS cleaves the membrane-spanning protein RseA at a single site. This event initiates a cascade of additional cleavages by other proteases that destroy the cytoplasmic domain of RseA and liberate the sE transcription factor, which enhances expression of genes that encode periplasmic chaperones, proteases, and biosynthetic enzymes. Hence, a signal linked to protein misfolding in the periplasm is transduced across the inner membrane via a proteolytic cascade, leading to adaptive changes in gene expression. Several years ago, we found that peptides ending with the C-terminal sequences of outer-membrane porins (OMPs) bound to the DegS PDZ domain and activated cleavage of RseA. These C-terminal peptides are inaccessible in properly assembled OMPs, suggesting that DegS senses envelope stress by binding sequences that become accessible only when folding and/or assembly is compromised.
We found that DegS behaves like a classical allosteric enzyme, with the tense state being inactive and the relaxed state being active, and have identified interactions that stabilize both conformational states using crystallography, mutagenesis, and enzymology. OMP peptides bind to both states and, depending upon the peptide sequence, shift the equilibrium towards the active state to different degrees. This mechanism allows very fast cellular responses to stress and also allows the system to reset quickly as the response proceeds. We are currently trying to cocrystalize an active-site mutant of DegS with RseA to understand how recognition occurs and are investigating how bound OMP peptides modulate the DegS allosteric equilibrium and trigger the requisite conformational changes. DegS cleavage of RseA is also inhibited by RseB, a protein which we have shown binds tightly to RseA. OMP peptides do not relieve RseB inhibition and thus there must be a second cellular signal produced during stress that relieves RseB inhibition. We are currently trying to identify this factor. We are also investigating a related stress-response system in P. aeruginosa that controls alginate production and is frequently mutated in strains isolated from the lungs of cystic-fibrosis patients. Finally, we are beginning to characterize other PDZ proteases to determine their biological roles and mechanisms.