Regulation of the E stress response by DegS: how the PDZ domain...

Regulation of the �E stress responseby DegS: how the PDZ domain keepsthe protease inactive in the resting stateand allows integration of differentOMP-derived stress signals uponfolding stressHanna Hasselblatt,1,3 Robert Kurzbauer,1,3 Corinna Wilken,1 Tobias Krojer,1 Justyna Sawa,1

Juliane Kurt,1 Rebecca Kirk,1 Sonja Hasenbein,2 Michael Ehrmann,2 and Tim Clausen1,4

1Research Institute of Molecular Pathology—IMP, A-1030 Vienna, Austria; 2Centre for Medical Biotechnology, FB Biologyand Geography, University Duisburg-Essen, Universitaetsstrasse, D-45117 Essen, Germany

The unfolded protein response of Escherichia coli is triggered by the accumulation of unassembled outermembrane proteins (OMPs) in the cellular envelope. The PDZ-protease DegS recognizes these mislocalizedOMPs and initiates a proteolytic cascade that ultimately leads to the �E-driven expression of a variety offactors dealing with folding stress in the periplasm and OMP assembly. The general features of how OMPsactivate the protease function of DegS have not yet been systematically addressed. Furthermore, it isunknown how the PDZ domain keeps the protease inactive in the resting state, which is of crucialimportance for the functioning of the entire �E stress response. Here we show in atomic detail how DegS isable to integrate the information of distinct stress signals that originate from different OMPs containing a�-x-Phe C-terminal motif. A dedicated loop of the protease domain, loop L3, serves as a versatile sensor forallosteric ligands. L3 is capable of interacting differently with ligands but reorients in a conserved manner toactivate DegS. Our data also indicate that the PDZ domain directly inhibits protease function in the absenceof stress signals by wedging loop L3 in a conformation that ultimately disrupts the proteolytic site. Thus, thePDZ domain and loop L3 of DegS define a novel molecular switch allowing strict regulation of the �E stressresponse system.

[Keywords: Unfolded protein response; regulatory proteolysis; protein quality control; HtrA]

Received June 20, 2007; revised version accepted August 24, 2007.

All living organisms face a wide variety of environmen-tal stresses, which severely affect the stability of proteinmolecules and promote their unfolding. Damaged pro-teins might accumulate as large aggregates that ofteninterfere with cellular function as is evident in severalneurodegenerative diseases (for review, see Macario andConway de Macario 2005). To efficiently cope with mis-folded proteins, cells evolved a sophisticated proteinquality-control system that precisely monitors the cor-rect folding of individual proteins and coordinates repairand degradative functions (Gottesman et al. 1997; Wick-ner et al. 1999; Duguay and Silhavy 2004). Due to thevital importance of this process, all cells have multiple

stress response pathways that adjust the levels of mo-lecular chaperones and proteases (Raivio 2005). Withinthese pathways, compartment-specific signaling cas-cades sense the presence of misfolded protein and trans-mit this information to dedicated transcriptional regula-tors.

The envelope stress response of Gram-negative bacte-ria uses the mechanism of “regulated intramembraneproteolysis” (Brown et al. 2000) to transmit the stresssignal across the inner membrane to the cytoplasmictranscriptional system (Fig. 1A; Alba et al. 2002). Ulti-mately, the response is carried out by the alternative �factor E (�E), which orchestrates the expression of genesencoding periplasmic chaperones, folding catalysts, pro-teases, and enzymes involved in cell-wall homeostasis(Rouviere et al. 1995; Rhodius et al. 2006). Under non-stress conditions, �E is captured in an inactive state bythe cytoplasmic domain of the transmembrane anti-�

3These authors contributed equally to this work.4Corresponding author.E-MAIL [email protected]; FAX 43-1-798-7513.Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.445307.

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factor RseA. Folding stress causes mislocalization ofouter membrane proteins (OMPs) to the periplasm,thereby triggering a proteolytic cascade that leads to thecomplete digestion of RseA. Initially, the site-1 proteaseDegS, a membrane-tethered protease facing the periplas-mic space, becomes activated by the C termini of unas-sembled OMPs and introduces the first cut in RseA(Walsh et al. 2003). This cut removes the bulk of theperiplasmic domain of RseA, thereby abolishing inhibi-tion of the site-2 protease RseP by the RseB regulatorprotein (De Las Penas et al. 1997; Cezairliyan and Sauer2007; Kim et al. 2007). Subsequently, RseP cleaves RseAwithin its transmembrane segment, thereby releasingthe N-terminal domain of RseA with the tightly bound�E into the cytoplasm (Ades et al. 1999; Kanehara et al.2002; Akiyama et al. 2004). Further degradation events ofRseA catalyzed by Clp proteolytic complexes liberate �Eto interact with RNA polymerase and to up-regulatestress response promoters (Flynn et al. 2004; Levchenkoet al. 2005; Chaba et al. 2007).

DegS plays a key role within the �E pathway as itdirectly couples the sensing of the stress signal with thetriggering of the response pathway (Walsh et al. 2003;Young and Hartl 2003) and furthermore catalyzes therate-limiting step in the proteolytic cascade (Chaba et al.2007). DegS belongs to the widely conserved HtrA familyof serine proteases involved in various aspects of proteinquality control and cellular signaling (Clausen et al.2002). Like other family members, DegS is composed ofa trypsin-like protease domain and a C-terminal PDZdomain. PDZ domains are well-characterized protein–protein interaction motifs that bind to the C terminus oftarget proteins (Fanning and Anderson 1996). They con-tain specificity pockets for accommodating the C-termi-nal residues at the 0, −2, and −3 positions, where position0 refers to the C-terminal residue (Fig. 2A). A distinctivefeature of HtrA proteins is that their proteolytic activitycan be reversibly switched on and off (Ehrmann andClausen 2004; Hasenbein et al. 2007). For DegS, it wasshown that the OmpC C terminus binds to the PDZ

domain and turns on protease activity (Walsh et al.2003). Structural elements involved in the activationprocess comprise several active site loops of the proteasedomain. Loop L3 (residues 177–189; for nomenclature,see Fig. 1B and Perona and Craik 1995) senses the pres-ence of the PDZ-bound activator and transmits this in-formation to the activation domain that is formed byloops L1 (residues 195–200), L2 (residues 218–230), andLD (residues 161–166). This domain is homologous tothe classical activation domain of serine proteases,which generates the functional proteolytic site after pro-peptide cleavage (Huber and Bode 1978). The crystalstructure of DegS complexed with an activating peptidemimicking the C terminus of OmpC (Wilken et al. 2004)showed that the PDZ-bound activator interacts specifi-cally with the protease domain via the side chain of theglutamine in the −1 position, which forms a hydrogenbond with a main-chain oxygen of loop L3. This polarinteraction triggers reorientation of loop L3, thereby in-ducing remodeling of the activation domain into its ac-tive conformation. However, both polar and hydrophobicresidues are found in the −1 position of various OMPs(Struyve et al. 1991). Since several of the correspondingC termini are capable of activating DegS in vitro (Wilkenet al. 2004), it remained puzzling how DegS interactswith these OMPs and integrates the information fromdifferent peptides serving as stress signals. Furthermore,it has been shown that a PDZ deletion mutant ofDegS exhibits protease activity in vivo and in vitro,pointing to an inhibitory function of the PDZ domain(Walsh et al. 2003; Cezairliyan and Sauer 2007). Themechanism of how the PDZ domain keeps the proteaseinactive in the latent state and thus links DegS activitydirectly to the presence of the stress signal has not yetbeen determined. To study the molecular details of DegSregulation, which are of fundamental importance for theentire �E stress response, we performed a structure–function analysis of a PDZ-deletion mutant and of DegScomplexes with activating peptides derived from variousOMPs.

Figure 1. Function of DegS in the �E stress response. (A)Schematic presentation of the �E stress response. (B) Rib-bon presentation of one subunit of the DegS trimer high-lighting loop L3 (red) that mediates communication be-tween the PDZ and protease domains, and the activationdomain (loops L1, L2, and LD, green) that allows forma-tion of a functional catalytic triad comprising residuesHis96, Asp126, and Ser201, which are shown in ball-and-stick mode.

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Results

OMP-derived peptides with a �-x-Phe C-terminalmotif function as stress stimuli activating DegS

It has been proposed that proteins with a Tyr-x-Phe C-terminal sequence should be capable of interacting withDegS (Walsh et al. 2003). Furthermore, it has been shownthat most of the peptides carrying a Tyr-x-Phe motif arealso able to activate protease function (Wilken et al.2004). However, several OMPs of Escherichia coli do notcontain a corresponding consensus motif and exhibitrather diverse C-terminal sequences. Therefore, wewished to explore the binding and activation potential ofvarious E. coli OMPs. We performed isothermal titrationcalorimetry (ITC) binding studies and RseA cleavage as-says using derivatives of the NH2-DNRLGLVXXX-COOHpeptide, where the ultimate three residues matched thesequences of the individual OMPs indicated in Figure2A. In the following, we refer to the OMP-derived pep-tides on the basis of their C-terminal residues; e.g., the“YQF peptide” refers to the activating peptide with aTyr–Gln–Phe C terminus.

A typical ITC profile illustrating the binding of theFRF peptide to DegS is shown in Figure 2B. The ITCresults demonstrated that all activating peptides, with

the exception of the DLF peptide, can bind to DegS.Among the analyzed YxF peptides, DegS showed thehighest affinity for YYF and YWF (KD of 3–6 µM) (Table1); moderate affinity for YQF, YRF, and YTF (KD of 55–80µM); and the weakest affinity for YTF and YAF (KD of126 µM and 267 µM, respectively). These data suggestthat YxF peptides are mainly bound to the PDZ domainby the phenylalanine and tyrosine in the 0 and −2 posi-tions, whereas the residue in the −1 position appears tointeract differently with the protease domain, causingthe observed differences in affinity. Interestingly, pep-tides that deviate from the YxF consensus also interactedwith DegS. While the LKF and THF peptides were boundweakly to DegS (KD of 300 and 450 µM, respectively), theFRF peptide (KD of 18 µM) was bound 4.5 times morestrongly than the related YRF peptide. These data implythat the preferred residue in the −2 position might be aphenylalanine rather than a tyrosine and that small hy-drophobic residues are also tolerated in the −2 position.

To explore the relevance of the ITC data for DegS ac-tivation, we studied the capacity of the OMP-derivedpeptides to induce RseA cleavage (Fig. 2C). First, we in-cubated DegS with distinct activating peptides presentin 50 µM concentration. The cleavage assays clearlydemonstrated that the FRF peptide most efficiently ac-

Figure 2. Binding and activation potentialof different OMP-derived C termini. (A) Ac-tivating peptides used in this study. In thetop panel, the general binding mode to thePDZ domain is indicated together with thenomenclature used here. For the ligand, theC-terminal phenylalanine present in allOMPs is highlighted. The table shown inthe bottom panel summarizes the C ter-mini of the analyzed peptides (green), thecorresponding OMPs in E. coli, and the in-dividual dissociation constants and activa-tion capabilities. (B) ITC measurement ofthe binding of the FRF-peptide to the DegSwild-type protein. (Top panel) Ten-microli-ter aliquots of the FRF peptide (1 mM) wereinjected into the sample cell containing 85µM DegS. (Bottom panel) The area undereach peak was integrated and plottedagainst the molar ratio peptide/DegS insidethe sample cell. The black line representsthe fit to a binding isotherm, assuming onebinding site per protomer. (C) SDS-PAGE ofRseA cleavage by DegS. The periplasmicdomain of RseA was present in 30 µM,DegS was present in 10 µM, and the acti-vating peptides were present in the indi-cated concentrations. The reactions werestopped after 18 h. In the control reactions(first three lanes), the assay was conductedwithout activating peptide.

Regulation of �E stress response by DegS

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tivates protease function, whereas YAF, YTF, YQF, andYRF activate DegS to a significantly lesser extent. Forpeptides LKF, THF, and DLF, no activation could be de-tected under these conditions. In order to account for thedifferent KD values and to directly monitor the activa-tion potential of the individual peptides, we repeated theRseA degradation assays with activating peptides pre-sent in concentrations corresponding to their threefoldKD values. As shown in Figure 2C, all OMP-derived pep-tides, with the exception of the DLF peptide, were able toactivate the DegS protease. Taken together, all OMPswith a �-x-Phe C-terminal motif seem to be capable ofactivating DegS in vitro and thus could act as potentialstress stimuli in vivo, however to various degrees.

Detachment of the PDZ domain from the proteasedomain allows activator binding but impairsprotease activation

To analyze the requirement of a proper positioning of theprotease and PDZ domain for peptide binding, we stud-ied the binding characteristics of the D122A mutant. Inthe wild-type protein, Asp122 forms a short-distancedsalt bridge with Arg256 that is part of the segment link-ing the protease and PDZ domain. Together with a fewother polar interactions, it is this salt bridge that mainlydetermines the relative orientation of the two domainsenabling their communication. Notably, the D122A mu-tant showed the highest affinity for all analyzed peptidesas was evident from the KD values that are three- tofivefold lower than wild type. Interestingly, the bindingconstants determined for the D122A mutant resembleresults of a previous ITC analysis, where the interactionof the YQF and YYF peptides to the isolated PDZ domainof DegS was studied (Walsh et al. 2003). The reported KD

values of 15 and 0.63 µM, respectively, fit nicely to thecorresponding KD values of 19 and 1.3 µM of the D122Amutant determined in this study. This analogy impliesthat the PDZ domain of the D122A mutant behaves likean “isolated” PDZ domain that is not fixed to the protein

body, whereas in wild-type DegS, specific interactionsbetween the PDZ and protease domain seem to imposeconstraints on ligand binding. Further lines of evidencesupport this hypothesis. First, the D122A mutant exhib-ited an entirely different thermodynamic signature (�Hand −T�S values) in the ITC experiments from the otherDegS variants. Particularly the �H values were reducedby ∼2–4 kcal/mol. Although it is difficult to pinpoint themolecular basis for the different thermodynamic param-eters, the ITC experiments indicated that the D122Amutation strongly affects the binding of activating pep-tides. Second, it should be noted that the D122A mutantresisted, in contrast to other DegS mutants, any attemptto crystallize and also showed a strikingly different be-havior during NiNTA purification, most likely becausethe affinity tag was directly connected to the C terminusof the PDZ domain. Thus, our data suggest that the im-proved binding characteristics of the D122A mutantmight be due to a differently oriented or more flexiblePDZ domain. Since this mutant is able to bind the acti-vator but does not cleave the RseA substrate, we con-clude that a proper arrangement of protease and PDZdomain is essential to transfer the stress signal betweenthese domains.

Destabilization of the activation domain does notinfluence activator binding

The ITC studies also showed that wild-type and Y162ADegS undergo similar contacts with OMP-derived pep-tides as the values for KD, �H, and −T�S were almostidentical (Table 1). Tyr162 is a central component of theactivation domain of DegS and plays a key role in stabi-lizing its proteolytically active form upon binding theallosteric activator. Consistently, the Y162A mutantwas inactive in the RseA cleavage assay (Wilken et al.2004). The present results indicate that the conforma-tion of the activation domain does not affect binding ofthe activator to the PDZ domain. However, the congru-ent KD values imply that loop L3 of the Y162A mutantretains its interactions with the activator and shouldthus be present in its “peptide-bound” conformation.Therefore, activator binding and protease activation ap-pear to be two independent events coupled by the prote-ase loop L3, which senses activating peptides bound tothe PDZ domain and transmits this information to theprotease domain.

Crystal structure of the complex between the YWFactivator and DegS

To explore the molecular mechanism of how DegS rec-ognizes different stress signaling peptides, we set out todetermine the crystal structures of complexes with theYWF, YYF, and FRF peptides, which represent the mostpotent DegS activators (Fig. 2C; Wilken et al. 2004).While crystallization trials with YYF and FRF were notsuccessful, we managed to cocrystallize DegS with theYWF peptide. The structure of the complex was deter-

Table 1. Binding constants of different activating peptidesdetermined by ITC measurements

DegS ActKD

(µM)�H

(kcal/mol)−T�S

(kcal/mol) na

Wild type YAF 267 −7.03 2.05 0.82YTF 126 −7.15 1.72 0.93YQF 53 −7.31 1.38 0.90YRF 79 −7.90 2.19 0.98YYF 6.5 −7.89 0.42 1.11YWF 3.2 −7.60 −0.06 1.13

Y162A YQF 63 −7.45 1.62 0.99YYF 5.5 −7.86 0.57 1.07YWF 2.2 −7.69 −0.17 1.06

D122A YQF 19 −11.51 4.97 0.91YYF 1.3 −10.55 2.38 0.92YWF 0.9 −9.35 0.99 1.07

an refers to the stoichiometry between the ligand and targetprotein of the ITC fit.

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mined at 2.5 Å resolution (R-factor 20.4%, Rfree 23.4%)(Table 2) and revealed the binding mode of the activator(Fig. 3A).

The stress peptide was bound as an additional �-strandto the small �-sheet of the PDZ domain with its carbox-ylate group being anchored by the carboxylate-bindingloop Tyr258–Ile259–Gly260–Ile261 of DegS. The struc-ture indicated that the 0 pocket, which is formed byresidues Ile259, Thr318, Met319, and Val322, is the mainanchoring site for the activator, since this pocket isclearly defined by electron density as is the bound phe-nylalanine side chain. In contrast, the −2 and −3 pocketsand their bound residues are present in a rather flexiblestate. In analogy to the previously solved structure of theDegS/YQF complex, the residue in the −1 position, thetryptophan, interacts with the protease domain (Fig. 3A).Its indol side chain is wedged between the terminal�-strand of the PDZ domain and loop L3 of the proteasedomain. Particularly, residues Tyr351 and Gly185 arelocated in close proximity to the indol group (distancesbetween 3.0 and 4.5 Å) and should support ligand bindingby van der Waals interactions. Furthermore, the phenolicgroup of Tyr351 is oriented nearly parallel to the indolring system of the −1 tryptophan of the activator and isthus in a good position for ring-stacking interactions.

Structural comparison with the DegS/YQF complexshowed that binding of both activators triggers similarconformational changes in the protease domain leadingultimately to the formation of a functional proteolyticsite. Consistently, the protease domains of both acti-vated forms fit nicely to each other as indicated by theroot-mean-square deviation (RMSD) of 0.61 Å for 208 C�atoms, whereas superposition with the resting DegSyielded a RMSD of 0.90 Å for 185 C� atoms. Structuraldifferences between the two activated forms are mainly

restricted to loop L3, which interacts differently with the−1 residue of the activator. While the glutamine of theYQF peptide forms a hydrogen bond to the main-chaincarbonyl of Thr184, the tryptophan of YWF was boundby nondirected van der Waals interactions.

Protease loop L3 is able to integrate the informationof different stress signaling peptides

Binding of the YWF peptide to DegS generated an asym-metric particle, in which the activator was bound differ-ently in the three subunits (Fig. 3B). Although the acti-vator generally underwent van der Waals interactionswith Gly185, the backbone conformation of loop L3 dif-fered significantly. In contrast, the active site loops L1,L2, and LD obtained almost identical conformations inthe three protomers, yielding an entirely activated tri-meric DegS particle. Superposition of the three YWF- andthe YQF-complex structures highlights the structuralflexibility of loop L3 in interacting with different acti-vating peptides (Fig. 3B). Particularly, the central part ofthis loop comprising residues 180–186 was present infour different conformations. This inherent flexibilityshould form the basis to bind to different −1 residues ofvarious activating peptides. Most interestingly, however,the stem regions of loop L3 comprising residues 177–179and 187–189 adopt a similar fold in all activated DegSprotomers. This conformation appears to be crucial totransmit the stress signal from the PDZ to the proteasedomain. In particular, the reoriented residues of the N-terminal stem segment are important for protease acti-vation, as Ile179 induces formation of a hydrophobiccluster that forms the core of the functional activationdomain, and Arg178 induces reorientation of the activa-tion loop LD. Taken together, OMP-derived activating

Table 2. Data collection and refinement statistics

DegS/YWF DegS�PDZ

Data collectionSpace group and cell constants C2 with a = 205.7 Å,

b = 142.6 Å, c = 41.1 Å, � = 90.7°C2 with a = 113.8 Å, b = 66.2 Å,

c = 84.5 Å, � = 95.7°Resolution (Å)a 30–2.5 (2.59–2.50) 20–2.6 (2.7–2.6)Unique reflections 39,228 (3644) 18,520 (1651)Data redundancy 2.8 (2.6) 2.7 (2.4)I/�(I) 13.2 (2.0) 9.8 (2.5)Completeness (%) 95.0 (88.2) 96.0 (85.5)Rsym (%)b 4.7 (33.0) 11.3 (32.8)

RefinementRcryst/Rfree (%)c 20.4/23.4 19.8/26.7Atoms in refinement protein/solvent 6291/157 4282/241RMSD bonds/angles/B-values 0.007 Å/1.45°/2.74 Å2 0.006 Å/1.36°/1.67 Å2

Residues (%) inMost favored 83.9 87.4Additionally allowed 15.1 11.6Generously allowed 1.0 1.1Disallowed region of Ramachandran plot 0.0 0.0

aThe numbers for the last resolution shell are given in parentheses.bRsym is the unweighted R-value on I between symmetry mates.cRcryst = ∑hkl||Fobs(hkl)| − k|Fcalc(hkl)||/∑hkl|Fobs(hkl)| for the working set of reflections; Rfree is the R-value for 5% of the reflectionsexcluded from refinement.






peptides can interact differently with DegS, but retain acommon activation mechanism that depends on the re-positioning of loop L3 in a conserved manner.

Activation potential of different activating peptides

It has been proposed that aromatic residues in the −1position of allosteric ligands are most effective in acti-vating the protease function of DegS (Wilken et al. 2004).Our ITC data indicate that the corresponding YYF andYWF activators have KD values that are one order ofmagnitude lower than other activators such as YQF andYRF. Thus the varying activation capability could sim-ply reflect the affinity of peptide ligands to DegS. In orderto test the activation potential of different ligands havinga �-x-Phe C terminus, we performed RseA degradationassays using peptide concentrations that were directlyrelated to individual KD values. Surprisingly, the in-duced DegS activity varied gradually (Fig. 2C), suggestingthat different allosteric ligands generate different alloste-rically activated complexes. To confirm these results, werepeated the degradation assays using activator peptidesin saturating concentrations (i.e., 10-fold KD). As shownin Figure 4A, different activators, indeed, exhibited dif-ferent capabilities to switch on DegS protease function.While the YWF activator was slightly more effectivethan the YQF activator, the FRF peptide clearly yieldedthe most active DegS protease. Unfortunately, it was notpossible to obtain structural data of a DegS/FRF com-plex. However comparison of the YQF- and YWF-com-plex structures revealed structural features that might be

mechanistically relevant and determine the activationpotential of allosteric ligands. In contrast to the YQFpeptide, binding of YWF to resting DegS would lead to asterical clash between its tryptophan side chain andPro183 of loop L3 (Fig. 4B). Hence the higher efficiency ofYWF to stimulate DegS might be due to its ability toimmediately displace loop L3 from its “inhibitory” po-sition. Furthermore, the thermal motion factors of theactivation domain of the YWF-activated DegS were con-siderably lower than of the YQF complex (Fig. 4C). Par-ticularly, loop L2, which assembles the S1 specificitypocket and the main-chain-binding patch, was more flex-ible in the YQF complex. Thus it appears that binding ofthe YWF peptide generates a more rigid proteolytic site,which should have a direct impact on proteolytic activ-ity. Similar to classical trypsin proteases, where the tran-sition from zymogen to active protease is accompaniedby a disorder–order transition (Huber and Bode 1978),such a well-defined proteolytic site should be crucial forcleaving the RseA substrate efficiently. In sum, thesedata indicate that additional factors other than the indi-vidual binding constants of stress peptides seem to affectthe structural integrity of the activation domain and de-termine the degree of protease activation.

Deletion of the PDZ domain yields a DegS proteasewith reduced activity that cannot be stimulatedby activating peptides

Previous biochemical and structural data highlighted theregulatory function of the PDZ domain of DegS. The

Figure 3. Structure of the complex between DegS andYWF. (A) Stereo presentation of the omit density of thebound YWF activator and of the main-chain-bindingsegment of the PDZ domain. The 3Fo − 2Fc electron-density map was calculated at 2.5 Å resolution (con-toured at 1.0 �.) after omitting the PDZ domain and theYWF activator from the refined model. Only the fourC-terminal residues of the activator were defined byelectron density. The YWF peptide (green)and the DegSresidues that interact with the tryptophan in the −1position are shown as a stick model. Furthermore, theArg256:Asp122 salt bridge, which mainly defines theorientation of protease (brown) and the PDZ domain(gray), is indicated. (B) Ribbon plot of the superimposedstructures of resting and activated DegS. The boundYWF peptide is shown as a stick model to indicate theposition of the activator-binding site. Loop L3 is high-lighted by color (resting DegS in red, YQF-activated inblue, and YWF-activated in green). Although the centralsegment of loop L3 differs in all activated forms, thestem regions show a mostly conserved conformation.

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crystal structure of DegS in complex with the YQF pep-tide indicated that the PDZ domain offers a binding sitefor an allosteric activator that stimulates protease activ-ity (Ehrmann and Clausen 2004; Wilken et al. 2004). Incontrast to this “activation model,” the alternative“PDZ-inhibitory model” postulates that binding ofOMP-like peptides to DegS relieves the inhibitory effectof the PDZ domain on protease function (Walsh et al.2003). One important prediction of the latter model isthat a PDZ deletion mutant should be fully active,whereas the activation model suggests that such a mu-tant should be proteolytically less active. Recent workcould not clarify this point, as DegS PDZ deletion mu-tants showed different behaviors in vivo and in vitro(Walsh et al. 2003; Cezairliyan and Sauer 2007).

To distinguish between the two opposing models andto study the precise role of the PDZ domain in this pro-cess, we performed a structure–function analysis of aPDZ-deletion mutant DegS�PDZ comprising residues 42–255. Like wild-type DegS, the purified DegS�PDZ oc-curred as a trimer in solution but showed a lower solu-bility. It was, for example, not possible to concentratethe protein to >0.05 mM. However, the low solubilitywas not an indication of decreased stability, as the mu-tant did not aggregate and did not lose enzymatic activ-ity over time. Protease assays revealed that DegS�PDZ

was able to cleave the RseA substrate at the same posi-tion as the parent construct. However, the proteolyticactivity was significantly weaker and did not depend onthe presence of an activating peptide (Fig. 5A). Next, wedetermined the crystal structure of DegS�PDZ, whichwas refined at 2.6 Å resolution to an R-factor of 19.8%(Rfree = 26.7%) and exhibited good stereochemistry. Theprotease was present in a trimeric state, and each of the

intersubunit interfaces was conserved with the wild-type protein. Alignment of DegS�PDZ with the inactiveand active forms of DegS yielded RMSD values of 0.99 Å(for 182 aligned C�s) and 0.59 Å (for 191 aligned C�s),respectively, indicating that the DegS�PDZ structure wasmore similar to the activated form of DegS. Consis-tently, the activation domain of the deletion mutant waspresent in its functional state (Fig. 5B), promoting theformation of a properly formed oxyanion hole and S1-specifity pocket. However, the catalytic triad showed ahigher conformational flexibility than activated DegS(Fig. 5B). Particularly the side chain of His96 was poorlydefined by electron density and obtained different orien-tations in the distinct active sites of the DegS trimer.Furthermore Asp126 was located more distantly toHis96 and thus should not be fully capable of activatingthe His96/Ser201 couple for nucleophilic attack on theRseA substrate, as was evident from RseA cleavage as-says.

The PDZ domain of resting DegS captures loop L3in a conformation that disrupts the activation domainof the protease

Next, we aimed to identify structural features thatcaused the observed differences in regulation and activ-ity. Structural alignment of inactive and activated DegSwith DegS�PDZ indicated characteristic differences inthe fold of the sensor loop L3. Although the three struc-tures show significant variations in the central part ofloop L3, the L3 stem segments of the PDZ deletion mu-tant and activated DegS aligned well to each other (Fig.5C). As mentioned previously, these regions are impor-tant for propagating the stress signal from PDZ to prote-

Figure 4. Differences in the YQF- and YWF-activatedDegS. (A) SDS-PAGE of the cleavage of the periplasmicdomain of RseA (30 µM) by DegS (10 µM), applying satu-rating activator concentrations. For each activator, the 10-fold concentration of the individual KD value was applied.The reactions were carried out at 30°C and stopped after3 h. The control reaction was carried out without activat-ing peptide. (B) Comparison of YQF- and YWF-bindingmode. Alignment of the PDZ domains with bound acti-vator (YQF, blue; YWF, green) onto the resting DegS struc-ture (red) illustrates the different binding positions of the−1 glutamine and tryptophan side chains, respectively. Incontrast to the YQF peptide, binding of the indol ring ofthe YWF activator leads to a sterical clash with loop L3 ofthe resting protease. (C) Ribbon plot showing the proteasedomain of DegS with mapped thermal motion factors:(blue) rigid parts; (red) flexible parts. The relevant activesite loops are labeled. For both activated complexes, theactive site loops L1, L2, and LD are better defined as inresting DegS. However, loop L2 was more flexible in theDegS/YQF structure than in the DegS/YWF structure.The differences between the average thermal motion fac-tors of loops LD/L1/L2 and the protease domain are asfollows: 46.1, 29.7, and n.d. for resting DegS; −10.3, −9.9,and 49.7 for YQF-activated DegS; and −12.9, −9.7, and 36.3Å2 for YWF-activated DegS.






ase domain. It is important to note that the sensor loopL3 of DegS�PDZ is not oriented by the PDZ domain oractivating peptide and should thus represent its “unre-strained” conformation. In full-length DegS, the PDZ do-main captures loop L3 by wedging it in the interdomainspace. Here, L3 undergoes numerous interactions withthe PDZ domain including van der Waals interactions(L3 residues 179–183 with PDZ residues 319–323), hy-drophobic interactions (Leu181 bound in hydrophobicpocket lined with residues 244, 254, and 256), and ashort-distanced salt bridge between Arg178 and Asp320.Combined, these interactions bend loop L3 into a con-formation that is strikingly different from its unre-strained conformation and that ultimately disrupts theactivation domain (Fig. 5C). For example, Gln187 of loopL3 forms a hydrogen bond with Asp221, a residue defin-ing the orientation of loop L2. By the Gln187:Asp221interaction, loop L2 is pulled toward the PDZ domainand the activation domain is disrupted. The switch inactivity is further advanced by Phe220 (L2), the residueadjacent to Asp221. In activated DegS and in DegS�PDZ,Phe220 forms, together with Tyr162 (loop LD), the hy-drophobic core of the functional activation domain,whereas in latent DegS, the side chain of Phe220 is ori-ented to the other side of the protein backbone, where itundergoes hydrophobic interactions with residuesGly185 and Phe189 of the PDZ-bound loop L3. Bindingof activating peptides triggers a rearrangement of loopL3, which switches around by almost 180°, thereby re-

leasing loop L2 to interact with loops L1 and LD and toset up the functional activation domain. Taken together,our results show that the PDZ domain directly preventsformation of a functional protease. It captures loop L3 ina conformation that destabilizes the activation domain.Binding of activating peptides triggers a conformationalswitch in loop L3 that relieves the destabilizing effect onthe activation domain and ultimately enables DegS tocleave RseA.

Discussion

The envelope stress response of E. coli is mediated by apeptidic stress signal inducing a proteolytic cascade, inwhich DegS, RseP, and cytoplasmic ATP-dependent pro-teases sequentially degrade the anti-� factor RseA,thereby activating �E, the transcriptional regulator ofthe �E regulon. Upon detection of mislocalized OMPs,DegS carries out the first proteolytic cut of RseA, whichis also the rate-determining step in the entire signalingcascade. Furthermore, the amount of OMP-activatedDegS is directly linked to the degree of �E activation(Chaba et al. 2007). Therefore, the tight regulation of theprotease activity of DegS appears to be crucial for therobustness and performance of the �E stress response.The aim of this work was to elucidate the moleculardetails of the corresponding regulatory mechanisms.

First, we were interested to better understand how theprotease function of DegS is activated in general by

Figure 5. Structure–function analysis of the PDZ de-letion mutant DegS�PDZ. (A) SDS-PAGE of RseA cleav-age by DegS. The periplasmic domain of RseA was pres-ent in 30 µM, DegS and DegS�PDZ were present in 10µM, and the YYF peptide was present in 100 µM. Thereaction was stopped after 5 h. (B) Overall fold of theDegS�PDZ trimer. Mechanistic important loops L1, L2,L3, and LD of one monomer are colored orange. To in-dicate the position of the proteolytic site, the individualcatalytic triads are shown as a stick model. (Left inset)Superposition of the catalytic triad of activated DegS(green) and DegS�PDZ (orange). For the PDZ deletionmutant, the triad was observed in a different configura-tion in each subunit of the trimer, illustrating its flex-ibility. (Right inset) Superposition of active site loops ofDegS�PDZ (orange), resting DegS (red), and activatedDegS (green). Notably, the loops of the activation do-main (L1, L2, LD) of DegS�PDZ and activated DegS fitnicely to each other. (C) Stereoview of the superpositionof DegS�PDZ (orange) with YWF-activated (green) andresting DegS (red), highlighting the different conforma-tions of loop L3. The presence of the PDZ domain aswell as the binding of activating peptides reorient loopL3 in a distinct but specific manner. Key residues forthe interaction between loop L3 and loops L2 and LD ofthe activation domain are labeled and shown as a stickmodel. The superposition also illustrates the inherentflexibility of the central part of loop L3, which obtainsa different conformation in each structure.

Hasselblatt et al.





OMPs. The biogenesis of OMPs requires ∼60% of pro-teins encoded in the �E regulon, many of which are chap-erones and folding catalysts (Rhodius et al. 2006). Uponfolding stress in the periplasm, misfolded proteins com-pete with unassembled OMPs for protein quality-controlfactors, which ultimately leads to increased amounts offree OMPs in the cellular envelope. Since the C terminiof different OMPs are directly involved in the formationof the porin �-barrel and thus not accessible in the nativestate, the presence of free OMP C termini in theperiplasm is an excellent indicator of folding stress. Thelast 10 residues of most OMPs have a similar amino acidcomposition and form an amphipathic �-strand with hy-drophobic residues in the 0, −2, −4, −6, and −8 positions.Interestingly, such sequences are not present in periplas-mic proteins, allowing the specific identification of mis-localized OMPs. Comparison of OMPs with differentfunctions (e.g., receptors, enzymes, porins) from differentbacteria indicates that the C-terminal 0-residue, a phe-nylalanine, is widely conserved, most likely due to itsessential role in OMP assembly in the outer membrane(Struyve et al. 1991). Other residues are less conserved,and only position −2 has a slight preference for tyrosine.The present study indicates that the binding mode ofpotential activators to the PDZ domain of DegS matchesthis conserved pattern, since binding of the C-terminalphenylalanine appears to be the key interaction with thePDZ domain, and phenylalanine and tyrosine residuesare preferred in the −2 position. However, some OMP Ctermini, which have a small hydrophobic residue in thisposition, are also capable of activating DegS, and thusthe consensus sequence of an allosteric activator of DegSshould be �-x-Phe. As this motif is present in the major-ity of all OMPs, DegS seems to be capable of recognizingand getting activated by different mislocalized OMPs ina highly specific manner.

We also wished to study the binding mode of an acti-vating peptide that does not contain the OmpC-likeYQF C terminus and determined the crystal structure ofDegS in complex with the YWF activating peptide. Thestructural data showed that the PDZ-bound YQF andYWF activators undergo different interactions with theprotease domain but ultimately trigger activation ofproteolytic function in the same manner. The integra-tion of the stress signal is achieved by loop L3, which iscomposed of two functional parts. The structurallyflexible central segment is able to variably interact withresidues in the −1 position of different activatingpeptides, whereas the stem regions of loop L3 reorient ina largely conserved manner to trigger protease activa-tion. Thus the specific composition of loop L3 enablesDegS to become activated by different misfoldedOMPs and to channel the corresponding informationinto the �E stress response (Fig. 6A). Our biochemicaland structural data pinpoint several key residues that arecrucial for the regulation of DegS and for propagating thestress signal through the molecule (Fig. 6B). In the rest-ing state, the PDZ domain keeps DegS inactive by cap-turing loop L3 in a position that enables Gln187 to in-teract with Asp221 and that disrupts the hydrophobic

core of the activation domain of the protease. Upon fold-ing stress, OMP C termini are recognized by the PDZdomain and interact via their −1 residue with loop L3 ofthe protease domain. Rearrangement of L3 abolishes theinteraction between Gln187 and Asp221 and allows for-mation of a functional activation domain, in whichTyr162 (LD), Leu164 (LD), Ile179 (L3), Phe220 (L2), andIle232 (L2) form the hydrophobic core. The relocatedTyr162 is now capable of undergoing main-chain inter-actions with His198 and to flip the peptide bond betweenresidues 198 and 199. Remodeling of the backbone ofresidues 197–201 establishes a properly formed oxyanionhole and catalytic triad.

Since DegS catalyzes the rate-limiting step in the�E cascade, its activity has to be tightly controlledto remain inactive under nonstress conditions and be-come activated only in the presence of exposed OMPC termini. The structure of the DegS�PDZ mutantillustrates that the PDZ domain is absolutely required toinhibit proteolytic activity of DegS in the resting stateby fixing loop L3 in a specific conformation that abol-ishes protease activity. In addition to its inhibitoryfunction, the PDZ domain specifically recognizes thecompartmental �-x-Phe stress stimulus and thus enablesDegS to regulate its protease function in strict de-pendence of the stress signal and to react immediately tovarying amounts of mislocalized OMPs. The crystalstructures of DegS in complex with YQF and YWF im-ply that OMP-like activating peptides use a commonmechanism and abolish the inhibitory effect of thePDZ domain by releasing loop L3. However, clear differ-ences could be observed in the degree of activation.For example, the FRF peptide was generating an acti-vated DegS form with much higher proteolytic activitythan the YxF peptides. Although the molecular detailsof this mechanism remain to be elucidated, our struc-tural data reveal that different activating peptides inducedifferent rearrangements of loop L3, which have adifferent effect on the active site geometry and rigid-ity. Similarly, loop L3 of the PDZ deletion mutant ob-tained a unique conformation that ultimately generateda less-ordered active site with reduced proteolytic activ-ity.

Taken together, our data indicate that the PDZ do-main of DegS exerts both inhibitory and activating func-tions. In the absence of allosteric ligands, it inhibits pro-tease function by capturing loop L3, whereas upon fold-ing stress, it offers a binding site for allosteric activatorsthat release loop L3 and turn on protease function in aspecific manner. Thus the PDZ domain and loop L3 ofDegS set up a novel molecular switch that regulates theactivity of DegS in strict dependence on the presence ofmislocalized OMPs. We have shown previously that theswitch in activity is reversible (Wilken et al. 2004). Thereversible allosteric activation of a rate-limiting proteasein a signal transduction pathway, such as DegS in the �Estress response, seems to represent a vital regulatorymechanism that might be compared, on the conceptuallevel, to phosphorylation/dephosphorylation switches ofclassical signaling cascades.






Materials and methods

Protein expression and purification

The ORF encoding the protease and PDZ domain (residues 42–354) of DegS and the D122A and Tyr162A mutants were ex-pressed as C-terminal His tag proteins and purified as describedpreviously (Wilken et al. 2004). Briefly, cells transformed withcorresponding pET-15b plasmids were grown at 37°C in LB me-dium and overexpression was induced at OD600 = 0.6 with 1mM IPTG. After 3 h of incubation, cells were harvested bycentrifugation and lysed by sonication in 50 mM NaPO4 buffer(pH 7.5) and 200 mM NaCl. Proteins were purified using Ni-NTA resin (Qiagen) and a Superdex 200 column (GE Health-care). Prior to crystallization, the protein buffer was exchangedby a NAP10 desalting column (GE Healthcare) to 10 mM NaPO4

(pH 7.5). Prior to ITC measurements and protease assays, theprotein buffer was exchanged to 100 mM NaPO4 (pH 7.5), 150mM NaCl, and 5 mM MgCl2.

The protease domain of DegS (residues 42–255) lacking thePDZ domain was amplified using the previously described degSconstruct as a template and cloned into pET21a (Novagen). TheC-terminal His6-tagged degS�PDZ was expressed in E. coliBL21(DE3) for 3 h at 37°C. The cultures were harvested by cen-trifugation, resuspended in 50 mM NaPO4 (pH 8.0) and 300 mMNaCl, and lysed by freeze–thawing and subsequent sonicationon ice. To prevent proteolytic digestion, 0.1 mM PMSF, whichdoes not inhibit DegS, was added during the initial purificationsteps. The PDZ deletion mutant was purified by Ni-NTA affin-ity chromatography. A Superdex-75 column was instrumentalfor further purification and to adjust the protein buffer to 20mM Tris (pH 7.5) and 500 mM NaCl. Prior to crystallization,DegS�PDZ was concentrated to 1.2 mg/mL.

Crystallization

For crystallization of the DegS–activator complex, the peptideNH2-DNRLGLVYWF-COOH (100 µM) was added to DegS andincubated for 30 min before setting up the cocrystallization tri-als. Crystals of the complex were grown in sitting drops at 19°Cby mixing 4 µL of DegS/YWF with 2 µL of a crystallizationsolution containing 0.1 M HEPES (pH 7.5), 6% PEG 6000, 9%MPD, and 10 mM MgCl2. Crystal trials were set up in cryschemplates with a reservoir volume of 400 µL. For cryo measure-ments, crystals were transferred from the crystallization drop tothe mother liquor supplemented with 18% MPD as cryo pro-tectant and 100 µM YWF peptide.

Sitting-drop crystallization trials of DegS�PDZ (1.2 mg/mL)were carried out at 19°C in 96-well plates with a reservoir vol-ume of 100 µL. Crystals of DegS�PDZ appeared after 2 d in 28%PEG400, 0.1 M HEPES (pH 7.5), and 0.2 M Li-sulfate after mix-ing 200 nL of protein with 100 nL of precipitant. For cryo mea-surement, crystals were directly transferred from the drop intothe nitrogen gas stream.

Structure solution and quality of the structures

High-resolution data of the activator complex and the DegS�PDZ

mutant were collected in-house on a MarResearch imageplate. Data were integrated using DENZO and scaled withSCALEPACK (Otwinowski and Minor 1997). Both crystal formswere of the monoclinic space group C2 with one DegS trimer inthe asymmetric unit. The structure of the DegS�PDZ mutantwas determined by molecular replacement using the programMOLREP of the CCP4 package (Collaborative ComputationalProject in Macromolecular Crystallography 2002) and the pro-tease domain (residues 43–251) of DegS as a search model (PDB

Figure 6. Model for the integration of different OMP-derived stress signals by DegS. (A) The left panel illus-trates activation of DegS by different OMP C termini.Loop L3 is highlighted with its inhibitory (red) and ac-tivating (green) structural elements. Molecular detailsof both inhibitory and activating processes are given inB. In latent DegS, loop L3 directly inhibits proteasefunction by disrupting the activation domain. Bindingof the allosteric activator to the PDZ domain triggers aswitch of this loop into its active position, where it nowsupports the setup of a functional proteolytic site. Theright panel illustrates the cellular function of DegS act-ing as a mechanistic funnel to integrate the informationfrom different mislocalized OMPs into the �E stressresponse. (B) Working model for how DegS switchesfrom the resting to the activated state. Key residues thatare important for regulation and for signal propagationare labeled. Loop L3 of the resting DegS is drawn in red,loop L3 of the active DegS is in green. Details of thesignal transduction through the DegS molecule leadingto the functional protease are described in the text.

Hasselblatt et al.





ID 1soz). The structure of the YWF activator complex wassolved by using the refined structure of the YQF-activated DegS,from which loops L1, L2, L3, and LD were omitted. For bothstructures, electron-density maps based on the coefficients2Fo − Fc and 3Fo − 2Fc were calculated from the phases of theinitial model. The resulting maps were used to build atomicmodels in O (Jones et al. 1991). Refinement, model rebuilding,and water incorporation proceeded smoothly via rigid body, po-sitional, and later B-factor optimization in CNS (Brunger et al.1998). Finally, the structures were checked using simulated an-nealing composite omit maps. Some protein segments includingresidues 221–229, 264–280, and 336–341 were hardly visible inthese maps and were therefore omitted from the model. Duringrefinement, clear electron density developed in the 3Fo − 2Fc

omit density map for two of three activator molecules. The datacollection and refinement parameters are summarized in Table2. All graphical presentations were prepared using the programPYMOL (DeLano 2002).

In vitro cleavage assay

Proteolytic activity of wild-type DegS and DegS�PDZ was mea-sured by following cleavage of the periplasmatic domain of E.coli RseA (residues 121–216), which was expressed with an N-terminal His6 tag. After purification by NiNTA affinity chro-matography, the RseA substrate was concentrated to 7.5 mg/mL. Cleavage assays were performed at 30°C in 100 mM NaPO4

(pH 7.5), 200 mM NaCl, 10% glycerol, 5 mM MgCl2, and 1 mMDTT. For the cleavage reaction, DegS and DegS�PDZ were ad-justed to 10 µM and RseA to 30 µM. The reactions were stoppedby adding SDS-sample buffer and were analyzed by SDS-PAGE.

Isothermal titration calorimetry

The thermodynamic values of the interaction between DegSand different activating peptides were determined by isothermaltitration calorimetry (MCS-ITC; Microcal). All experimentswere conducted in overflow mode at 30°C. Solution (1.4-mL) ofDegS (80–90 µM) was placed in the temperature-controlledsample cell and titrated with different OMP-derived peptides (1mM), which were loaded in the 300-µL mixing syringe. For allexperiments, 100 mM NaPO4 buffer (pH 7.5) supplementedwith 150 mM NaCl and 5 mM MgCl2 was used as the buffer.Injections of 10 µL of peptide were dispensed into the samplecell using a 120-sec equilibration time between experimentsand stirring at 300 rpm. Control experiments using the identicalexperimental setup were carried out in order to measure andcorrect the heat of dilution. Ultimately the data were analyzedusing ORIGIN software following the instructions of the manu-facturer.

Accession numbers

The Protein Data Bank accession numbers for the YWF-acti-vated and the PDZ deletion forms of DegS are 2R3Y and 2R3U,respectively.

Acknowledgments

We thank Peggy Stolt-Bergner and the Clausen laboratory forcritical reading of the manuscript and helpful discussions. TheResearch Institute of Molecular Pathology (IMP) is fundedby Boehringer Ingelheim. M.E. was supported by the BritishBiology and Biotechnology Research Council, Deutsche For-schungsgemeinschaft, and the Fonds der Chemischen Industrie;

T.C. was supported by the EMBO Young Investigator Program;and H.H, T.K., and J.S. were supported by the Austrian ScienceFund (FWF P17881-B10).

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10.1101/gad.445307Access the most recent version at doi: 21:2007, Genes Dev.

Hanna Hasselblatt, Robert Kurzbauer, Corinna Wilken, et al. of different OMP-derived stress signals upon folding stresskeeps the protease inactive in the resting state and allows integration

E stress response by DegS: how the PDZ domainσRegulation of the

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