PDZ domains of RseP are not essential for sequential cleavage of RseA or stress‐induced σE activation in vivo

The Escherichia coli σ^E extracytoplasmic stress response monitors and responds to folding stress in the cell envelope. A protease cascade directed at RseA, a membrane‐spanning anti‐σ that inhibits σ^E activity, controls this critical signal‐transduction system. Stress cues activate DegS to cleave RseA; a second cleavage by RseP releases RseA from the membrane, enabling its rapid degradation. Stress control of proteolysis requires that RseP cleavage is dependent on DegS cleavage. Recent in vitro and structural studies found that RseP cleavage requires binding of RseP PDZ‐C to the newly exposed C‐terminal residue (Val148) of RseA, generated by DegS cleavage, explaining dependence. We tested this mechanism in vivo. Neither mutation in the putative PDZ ligand‐binding regions nor even deletion of entire RseP PDZ domains had significant effects on RseA cleavage in vivo, and the C‐terminal residue of DegS‐processed RseA also little affected RseA cleavage. Indeed, strains with a chromosomal rseP gene deleted for either PDZ domain and strains with a chromosomal rseA V148 mutation grew normally and exhibited almost normal σ^E activation in response to stress signals. We conclude that recognition of the cleaved amino acid by the RseP PDZ domain is not essential for sequential cleavage of RseA and σ^E stress response in vivo.     

Regulation of the Escherichia coli sigma-dependent envelope stress response

The Escherichia colisigma(E)-dependent stress response pathway controls the expression of genes encoding periplasmic folding catalysts, proteases, biosynthesis enzymes for lipid A (a component of lipopolysaccharide or LPS) and other proteins known or predicted to function in or produce components of the envelope. When E. coli is subjected to heat or other stresses that generate unfolded envelope proteins, sigma(E) activity is induced. Four key players in this signal transduction pathway have been identified: RseA, an inner membrane sigma(E) antisigma factor; RseB, a periplasmic protein that binds to the periplasmic face of RseA; and the DegS and YaeL proteases. The major point of regulation, the interaction between sigma(E) and RseA, is primarily controlled by the stability of RseA. Envelope stress promotes RseA degradation, which occurs by a proteolytic cascade initiated by DegS. There is evidence that one sigma(E)-inducing stress (OmpC overexpression) directly activates DegS to cleave RseA. Secondarily, envelope stress may relieve RseB-mediated enhancement of RseA activity. Additional levels of control upon sigma(E) activity may become evident upon further study of this stress response pathway.     

Analyzing the Interaction of RseA and RseB, the Two Negative Regulators of the ��E Envelope Stress Response, Using a Combined Bioinformatic and Experimental Strategy

The Escherichia coli envelope stress response is controlled by the alternative sigma factor, σ^E, and is induced when unfolded outer membrane proteins accumulate in the periplasm. The response is initiated by sequential cleavage of the membrane-spanning antisigma factor, RseA. RseB is an important negative regulator of envelope stress response that exerts its negative effects onσ^E activity through its binding to RseA. In this study, we analyze the interaction between RseA and RseB. We found that tight binding of RseB to RseA required intact RseB. Using programs that performed global and local sequence alignment of RseB and RseA, we found regions of high similarity and performed alanine substitution mutagenesis to test the hypothesis that these regions were functionally important. This protocol is based on the hypothesis that functionally dependent regions of two proteins co-evolve and therefore are likely to be sequentially conserved. This procedure allowed us to identify both an N-terminal and C-terminal region in RseB important for binding to RseA. We extensively analyzed the C-terminal region, which aligns with a region of RseA coincident with the major RseB binding determinant in RseA. Both allele-specific suppression analysis and cysteine-mediated disulfide bond formation indicated that this C-terminal region of similarity of RseA and RseB identifies a contact site between the two proteins. We suggest a similar protocol can be successfully applied to pairs of non-homologous but functionally linked proteins to find specific regions of the protein sequences that are important for establishing functional linkage.The Escherichia coli σ^E-mediated envelope stress response is the major pathway to ensure homeostasis in the envelope compartment of the cell (1-3). σ^E regulon members encode periplasmic chaperones and proteases, the machinery for inserting β-barrel proteins into the outer membrane and components controlling the synthesis and assembly of LPS (4-6). This pathway is highly conserved among γ-proteobacteria (6).The σ^E response is initiated when periplasmic protein folding and assembly is compromised (7-9). During steady state growth, σ^E is inhibited by its antisigma factor, RseA, a membrane-spanning protein whose cytoplasmic domain binds to σ^E with picomolar affinity (10-13). Accumulation of unassembled porin monomers serves as a signal to activate the DegS protease to cleave RseA in its periplasmic domain (14, 15). This initiates a proteolytic cascade in which RseP cleaves periplasmically truncated RseA near or within the cytoplasmic membrane to release the RseA_cytoplasmic-σ^E complex, and cytoplasmic ATP-dependent proteases complete the degradation of RseA thereby releasing active σ^E (16-19).RseB, a second negative regulator of the envelope stress response (11, 20, 21), binds to the periplasmic domain of RseA with nanomolar affinity. RseB is an important regulator of the response (2, 22, 23). It prevents RseP from degrading intact RseA, thereby ensuring that proteolysis is initiated only when the DegS protease is activated by a stress signal (21). Additionally, RseB prevents activated DegS from cleaving RseA, suggesting that interaction of RseB with RseA must be altered before the signal transduction cascade is activated (23).The goal of the present studies was to explore how RseB binds to RseA. The interaction partner of RseB is the unstructured periplasmic domain of RseA (RseA-peri). Within RseA-peri, amino acids ∼169-186 constitute a major binding determinant to RseB (23, 24). This peptide alone binds RseB with 6 μm affinity, and deleting this region abrogates binding to RseB (23). Additional regions of RseA-peri also contribute to RseB binding, as intact RseA-peri binds with 20 nm affinity to RseB (23). Much less is known about the regions of RseB required for interaction with RseA. RseB is homodimeric two-domain protein, whose large N-terminal domain shares structural homology with LolA, a protein that transports lipoproteins to outer membrane (24, 25). The smaller C-terminal domain is connected to the N-terminal domain by a linker, and the two domains share a large interface, which may facilitate interdomain signaling. Glutaraldehyde cross-linking studies indicate that the C-terminal domain interacts with RseA, but the regions of interaction were not identified (25).In the present report, we study the interaction of RseB and RseA. We establish that both domains of RseB interact with RseA-peri. Using a global sequence alignment, we discovered several regions in RseA and RseB that had high sequence similarity, despite the low overall sequence similarity between these two proteins, a finding that was independently confirmed by a local sequence similarity algorithm. This suggested that these regions were functionally dependent, and we performed a set of mutagenesis experiments designed to test this idea. Our studies of the binding properties of these mutants revealed that regions in both the N terminus and C terminus of RseB modulate interaction with RseA. Moreover, genetic suppression analysis and cysteine-mediated disulfide bond formation suggest that the region of RseA/B with highest similarity (RseA residues 165-191 (major binding determinant in RseA) and RseB residues 233-258) are interacting partners.     

RseB binding to the periplasmic domain of RseA modulates the RseA:sigmaE interaction in the cytoplasm and the availability of sigmaE.RNA polymerase.

The Escherichia coli sigmaE regulon has evolved to sense the presence of misfolded proteins in the bacterial envelope. Expression of periplasmic chaperones and folding catalysts is under the control of sigmaE RNA polymerase. The N-terminal domain of RseA sequesters sigmaE in the cytoplasmic membrane, preventing its association with core RNA polymerase. The C-terminal domain of RseA interacts with RseB, a periplasmic protein. The relative concentration of sigmaE:RseA:RseB is 2:5:1 and this ratio remains unaltered upon heat shock induction of the sigmaE regulon. Purification from crude cellular extracts yields cytoplasmic, soluble sigmaE RNA polymerase as well as membrane sequestered sigmaE.RseA and sigmaE.RseA.RseB. RseB binding to the C-terminal domain of RseA increases the affinity of RseA for sigmaE by 2- to 3-fold (Kd 50-100 nM). RseB binds also to the misfolded aggregates of MalE31, a variant of maltose binding protein that forms inclusion bodies in the periplasm. We discuss a model whereby the RseB-RiseA interaction represents a measure for misfolded polypeptides in the bacterial envelope, modulating the assembly of sigmaE RNA polymerase and the cellular heat shock response.     

degS (hhoB) is an essential Escherichia coli gene whose indispensable function is to provide σE activity

DegS (HhoB), a putative serine protease related to DegP/HtrA, regulates the basal and induced activity of the essential Escherichia coli sigma factor sigma (E), which is involved in the cellular response to extracytoplasmic stress. DegS promotes the destabilization of the sigma (E)-specific anti-sigma factor RseA, thereby releasing sigma (E) to direct gene expression. We demonstrate that degS is an essential E. coli gene and show that the essential function of DegS is to provide the cell with sigma (E) activity. We also show that the putative active site of DegS is periplasmic and that DegS requires its N-terminal transmembrane domain for its sigma (E)-related function.     

Down-regulation of porins by a small RNA bypasses the essentiality of the regulated intramembrane proteolysis protease RseP in Escherichia coli

Adaptation to extracytoplasmic stress in Escherichia coli depends on the activation of sigmaE, normally sequestered by the membrane protein RseA. SigmaE is released in response to stress through the successive RseA cleavage by DegS and the RIP protease RseP. SigmaE and proteases that free it from RseA are essential. We isolated a multicopy suppressor that alleviated RseP and DegS requirement. The suppressor encodes a novel small RNA, RseX. Its activity required the RNA-binding protein Hfq. We used the property that small RNAs are often involved in RNA-RNA interactions to capture RseX putative partners; ompA and ompC mRNA, which encode two major outer membrane proteins, were identified. RseX activity was shown to confer an Hfq-dependent coordinate OmpA and OmpC down-regulation. Because RseP is shown to be no longer essential in a strain lacking OmpA and OmpC, we conclude that RseP, which is required for normal sigmaE activation, prevents toxicity due to the presence of two specific outer membrane proteins that are down-regulated by RseX.     

A pair of circularly permutated PDZ domains control RseP, the S2P family intramembrane protease of Escherichia coli

The sigma(E) pathway of extracytoplasmic stress responses in Escherichia coli is activated through sequential cleavages of the anti-sigma(E) protein, RseA, by membrane proteases DegS and RseP. Without the first cleavage by DegS, RseP is unable to cleave full-length RseA. We previously showed that a PDZ-like domain in the RseP periplasmic region is essential for this negative regulation of RseP. We now isolated additional deregulated RseP mutants. Many of the mutations affected a periplasmic region that is N-terminal to the previously defined PDZ domain. We expressed these regions and determined their crystal structures. Consistent with a recent prediction, our results indicate that RseP has tandem, circularly permutated PDZ domains (PDZ-N and PDZ-C). Strikingly, almost all the strong mutations have been mapped around the ligand binding cleft region in PDZ-N. These results together with those of an in vitro reaction reproducing the two-step RseA cleavage suggest that the proteolytic function of RseP is controlled by ligand binding to PDZ-N.     

Conversion of a Regulatory into a Degradative Protease

The PDZ protease DegS senses mislocalized outer membrane proteins and initiates the sigmaE pathway in the bacterial periplasm. This unfolded protein response pathway is activated by processing of the anti-sigma factor RseA by DegS and other proteases acting downstream of DegS. DegS mediates the rate-limiting step of sigma E induction and its activity must be highly specific and tightly regulated. While DegS is structurally and biochemically well studied, the determinants of its pronounced substrate specificity are unknown. We therefore performed swapping experiments by introducing elements of the homologous but unspecific PDZ protease DegP. Introduction of loop L2 of DegP into DegS converted the enzyme into a non-specific protease, while swapping of PDZ domains did not. Therefore, loop L2 of the protease domain is a key determinant of substrate specificity. Interestingly, swapping of loop L2 did not affect the tight regulation of DegS. In addition, the combined introduction of loop L2 and PDZ domain 1 of DegP into DegS converted DegS even further into a DegP-like protease. These and other data suggest that homologous enzymes with distinct activities and regulatory features can be converted by simple genetic modifications.     

Acid stress activation of the σE stress response in Salmonella enterica serovar Typhimurium

The alternative sigma factor σ^E is activated by unfolded outer membrane proteins (OMPs) and plays an essential role in Salmonella pathogenesis. The canonical pathway of σ^E activation in response to envelope stress involves sequential proteolysis of the anti-sigma factor RseA by the PDZ proteases DegS and RseP. Here we show that σ^E in Salmonella enterica sv. Typhimurium can also be activated by acid stress. A σ^E-deficient mutant exhibits increased susceptibility to acid pH and reduced survival in an acidified phagosomal vacuole. Acid activation of σ^E-dependent gene expression is independent of the unfolded OMP signal or the DegS protease but requires processing of RseA by RseP. The RseP PDZ domain is indispensable for acid induction, suggesting that acid stress may disrupt an inhibitory interaction between RseA and the RseP PDZ domain to allow RseA proteolysis in the absence of antecedent action of DegS. These observations demonstrate a novel environmental stimulus and activation pathway for the σ^E regulon that appear to be critically important during Salmonella –host cell interactions.     

The structure of RseB: a sensor in periplasmic stress response of E. coli

An elegant network of signal transduction has evolved in the bacterial cell envelope to respond to environmental stress. It is initiated by sensing unfavourable and harmful changes in the periplasm. The stress signal is then transmitted by a controlled degradation of the transmembrane anti-sigma-factor RseA that leads to the activation of the alternative sigma factor sigma(E). The periplasmic protein RseB exerts a crucial role in modulating the stability of RseA. RseB from Escherichia coli has been crystallized and crystal structures were determined at 2.4 A and at 2.8 A resolution. The protein forms a homodimer, with the monomer composed of two domains. The large domain resembles an unclosed beta-barrel that is structurally remarkably similar to a protein family capable of binding the lipid anchor of lipoproteins. The small C-terminal domain, connected to the large domain by a partially unstructured loop, is responsible for interaction with RseA. On the basis of the structure of RseB, we suggest that it acts as a sensor of periplasmic stress with a dual functionality: it detects mislocalized lipoproteins and propagates the signal to induce the sigma(E)-response.     

Dispensable PDZ domain of Escherichia coli YaeL essential protease

In Escherichia coli, adaptation to extra-cytoplasmic stress relies on sigma(E) activation to induce a rescue pathway. Under non-stressed conditions, sigma(E) is sequestered by the inner membrane protein RseA. Extra-cytoplasmic stress activates DegS-dependent cleavage of RseA, rendering RseA sensitive to further degradation by the YaeL protease. YaeL contains two motifs characteristic of a family of metallo-proteases, as well as a periplasmic PDZ domain. We report results of mutational analyses of the YaeL domains. Surprisingly, expression in a strain depleted for wild-type YaeL or YaeL variants having a 40 amino acid deletion of the PDZ domain or amino acid substitutions of conserved amino acids of the YaeL PDZ domain did not affect cell viability. The proteolytic activity against RseA of these YaeL variants became independent of DegS. These observations suggest that the YaeL PDZ domain exerts a negative control on YaeL activity. Rather than being involved in substrate recognition, the PDZ domain of YaeL is likely to act as an inhibitor of proteolytic activity.     

Crystal structure of the DegS stress sensor: How a PDZ domain recognizes misfolded protein and activates a protease

Gram-negative bacteria respond to misfolded proteins in the cell envelope with the sigmaE-driven expression of periplasmic proteases/chaperones. Activation of sigmaE is controlled by a proteolytic cascade that is initiated by the DegS protease. DegS senses misfolded protein in the periplasm, undergoes autoactivation, and cleaves the antisigma factor RseA. Here, we present the crystal structures of three distinct states of DegS from E. coli. DegS alone exists in a catalytically inactive form. Binding of stress-signaling peptides to its PDZ domain induces a series of conformational changes that activates protease function. Backsoaking of crystals containing the DegS-activator complex revealed the presence of an active/inactive hybrid structure and demonstrated the reversibility of activation. Taken together, the structural data illustrate in molecular detail how DegS acts as a periplasmic stress sensor. Our results suggest a novel regulatory role for PDZ domains and unveil a novel mechanism of reversible protease activation.     

YaeL proteolysis of RseA is controlled by the PDZ domain of YaeL and a Gln-rich region of RseA

sigmaE is an alternative sigma factor involved in a pathway of extracytoplasmic stress responses in Escherichia coli. Under normal growth conditions, sigmaE activity is down-regulated by the membrane-bound anti-sigmaE protein, RseA. Extracytoplasmic stress signals induce degradation of RseA by two successive proteolytic events: DegS-catalyzed first cleavage at a periplasmic site followed by YaeL-mediated second proteolysis at an intramembrane region. Normally, the second reaction (site-2 proteolysis) only occurs after the first cleavage (site-1 cleavage). Here, we show that YaeL variants with the periplasmic PDZ domain deleted or mutated allows unregulated cleavage of RseA and consequent sigmaE activation. It was also found that a glutamine-rich region in the periplasmic domain of RseA was required for the avoidance of the YaeL-mediated proteolysis in the absence of site-1 cleavage. These results indicate that multiple negative elements both in the enzyme (PDZ domain) and in the substrate (glutamine-rich region) determine the strict dependence of the site-2 proteolysis on the site-1 cleavage.