DegS - Stress sensor of the bacterial unfolded protein response

Unfolded Protein Response in bacteria. DegS and RseP comrise the site-1 and site-2 proteases in the specialized RIP pathway.

The envelope stress response of Gram-negative bacteria uses the mechanism of “regulated intramembrane proteolysis” (RIP) to transmit the periplasmic stress signal across the inner membrane into the cytosol. Ultimately, the response is carried out by the alternative sigma factor sE, which orchestrates the bacterial unfolded stress response (UPR). Under non-stress conditions, sE is captured in an inactive state by the cytoplasmic domain of the transmembrane protein RseA. Proteotoxic stress causes mislocalization of outer membrane proteins (OMPs) to the periplasm, thereby triggering a proteolytic cascade that leads to the degradation of RseA. First, the site-1 protease DegS becomes activated by the C-termini of unassembled OMPs and introduces the priming cut in RseA. This cut removes the bulk of the periplasmic domain of RseA, thereby abolishing inhibition of the site-2 membrane protease RseP. Degradation of RseA by RseP and cytosolic Clp proteases liberates sE to interact with RNA polymerase and activate UPR promoters.

The PDZ-protease DegS plays a key role within the bacterial UPR as it couples the sensing of the stress signal with the triggering of the response pathway. We are thus interested in understanding the molecular mechanism of how DegS perceives the signal "proteotoxic stress" and collaborates with the other proteases in the UPR pathway.

Our findings: 2004 (stress-sensing mechanism), 2008 (integration of different stress signals)

2008 Integration of different stress signals by DegS

DegS is capable to funnel different stress signals into the bacterial UPR.

The UPR of Escherichia coli is triggered by the accumulation of unassembled OMPs in the cellular envelope. The PDZ-protease DegS recognizes these mislocalized OMPs and initiates a proteolytic cascade leading ultimately to the expression of a variety of heat-shock proteins. To this end, the general features of how OMPs activate the protease function of DegS have not been systematically addressed. Furthermore, it is unknown how the PDZ domain keeps the protease inactive in the resting state, which is crucial for the functioning of the UPR pathway. In this work, we showed in atomic detail how DegS is able to integrate the information of distinct stress signals that originate from different OMPs containing a phi-X-Phe C-terminal motif. A dedicated loop of the protease domain, loop L3, serves as a versatile sensor for allosteric ligands. L3 is capable of interacting differently with ligands but reorients in a conserved manner to activate DegS. Our data also indicate that the PDZ domain directly inhibits protease function in the absence of stress signals by wedging loop L3 in a conformation that ultimately disrupts the proteolytic site. Thus, the PDZ domain and loop L3 of DegS define a novel molecular switch allowing strict regulation of the bacterial UPR.


2004 Structural basis of how DegS perceives stress signals

Overall structure of the DegS trimer
Signal transduction from PDZ domain to the proteolytic site

For DegS, it was shown that the OmpC C-terminus serves as stress-signaling peptide that binds to the PDZ domain and induces protease function. Our structural and biochemical data revealed the precise molecular mechanism of the stress-sensing mechanism. The crystal structure of DegS in complex with an OmpC-like peptide directly showed that the PDZ-bound activator contacts loop L3 of the protease domain. Reoriented by this interaction, loop L3 then induces the remodeling of the activation domain into its active conformation yielding the functional protease. Strikingly, the activation of DegS bears some similarities to the classical trypsin activation. In trypsin, transition from the inactive to an active state is achieved by reorientation of the very same activation domain formed by loops L1, L2, and LD. Moreover, activation of both DegS and trypsin proteases is connected with a disorder-to-order transition. The important difference to canonical trypsin-like proteases is that activation of DegS is reversible and tightly coupled to binding the OMP C-terminus. This coupling guarantees an immediate and flexible cellular response toward folding stress.

Collectively, these results point to a novel mechanism of stress sensing and proteolytic activation, in which the PDZ domain acts as the central regulatory motif. It detects unfolded proteins via specific C-terminal sequences that become accessible once the folding capacity of periplasmic chaperones is exceeded. Therefore, in DegS, the PDZ domain is not a simple protein-binding domain. It attains a novel regulatory function being directly involved in intra- and intermolecular signaling and controlling the activity of the attached protease unit.