Supporting Information

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1 Supporting Information Wiley-VCH Weinheim, Germany

2 Allosteric activation of HtrA protease DegP by stress signals in bacterial protein quality control Michael Meltzer, Sonja Hasenbein, Patrick Hauske, Nicolette Kucz, Melisa Merdanovic, Sandra Grau, Alexandra Beil, Dafydd Jones, Tobias Krojer, Tim Clausen, Michael Ehrmann,* and Markus Kaiser* Dipl.-Biol. M. Meltzer, Dr. S. Hasenbein, Dipl.-Chem. (FH) P. Hauske, Dipl.-Biol. N. Kucz, Dr. M. Merdanovic, Prof. Dr. M. Ehrmann Zentrum für Medizinische Biotechnologie, FB Biologie und Geographie, Universität Duisburg-Essen, Essen, Germany. Fax: +49 (201) , Dipl.-Chem. (FH) P. Hauske, Dr. M. Kaiser Chemical Genomics Centre der Max-Planck-Gesellschaft, Otto-Hahn-Str. 15, Dortmund, Germany. Fax: +49 (231) , Dr. S. Grau, Dr. A. Beil, Dr. D. Jones, Prof. Dr. M. Ehrmann Cardiff School of Biosciences, Cardiff University, Museum Ave, Cardiff CF10 3US, United Kingdom. Dr. T. Krojer, Dr. T. Clausen Research Institute for Molecular Pathology (IMP), Dr. Bohrgasse 7, 1030 Vienna, Austria.

3 A. Experimental procedures Synthesis of activating peptides and chromogenic substrate All solvents and reagents were from Fluka, Aldrich or Acros and were generally of peptide synthesis grade or p.a. quality and were used without further purification. Dry solvents were obtained from Aldrich while Fmoc amino acids were purchased from IRIS biotech. H-Val-pNA HCl was obtained from Bachem while 2-chlorotrityl and preloaded Wang resin was purchased from Novabiochem. LC-ESI-MS analysis was performed on a LCQ Advantage Max system from Thermo Finnigan, coupled with the Agilent 1100 series analytical HPLC. A standard gradient of 10% acetonitrile in water (0.1% formic acid) to 100% acetonitrile (0.1% formic acid) on a RP-C 18 column in 10 min was used for all analytical measurements. Preparative HPLC purifications were performed on a Varian ProStar HPLC, employing a 5 µ C 18 reverse phase HPLC column from Macherey-Nagel and a gradient of 5% acetontrile in water (0.1% TFA) to 60% acetonitrile in water (0.1% TFA) in 60 min. Peptides 4-13 were generated on an automated Syro II peptide synthesizer, using standard Fmoc chemistry. As a solid support, Wang resin, preloaded with Fmoc phenylalanine (loading 0.48 mmol g -1 ) was used. Release from solid support and deprotection was achieved by a TFA/H 2 O/EDT/TIS (94:2.5:2.5:1) cleavage cocktail. Crude peptides were precipitated by addition of cold diethylether and then purified by preparative HPLC to yield pure peptides 4-13 (purity >95%). Peptides were synthesized by manual Fmoc-solid phase synthesis as automated peptide synthesis failed to deliver the desired peptides. 2-Chlorotrityl resin was used as a solid support. Loading of the first amino acid was performed for 2 h with 0.7 eq. of the corresponding amino acid and 4 eq. DIEA in dry DCM, followed by capping with DIEA/MeOH/DCM (2:2:6). Initial loading efficiency was determined by UV absorption of the Fmoc cleavage product and obtained loadings were now used for later equivalent calculations. Fmoc cleavage during peptide synthesis was achieved by addition of 20% piperidine in DMF for 20 min and was repeated for driving the reaction to completeness. Further amino acid couplings were performed with 4 eq. of side chain protected Fmoc amino acids, 4 eq. of HOBt, 3.5 eq. HBTU and 4 eq. DIEA in DMF and a coupling time of 2 h. Coupling efficiency of each step was verified by a Kaiser test or in case of Fmoc-Pro-OH with a chloranil test. Double couplings were performed after obtaining positive results. Resin cleavage was obtained with AcOH/TFE/DCM (2:2:6), followed by azeotropic distillation with cyclohexane. Deprotection of the peptide was achieved with TFA/H 2 O/EDT/TIS (94:2.5:2.5:1) to yield crude peptides which were precipitated by addition of cold ether, followed by purification with preparative HPLC and analysis by LC-ESI-MS (all purities >95%).

4 Chromogenic substrate 1 was synthesized by a combination of Fmoc-solid phase synthesis and solution chemistry. Manual solid phase synthesis was used to synthesize side chain protected Boc-S(tBu)- PMFK(Boc)-G-OH on 2-chlorotrityl resin, using essentially the protocol for activating peptides Resin cleavage under preservation of the protecting groups was obtained with AcOH/TFE/DCM (2:2:6), followed by azeotropic distillation with cyclohexane. This fully protected precursor peptide was then coupled in solution over night with 2 eq. of H-Val-pNA HCl, using 1.5 eq. EDC HCl, 2 eq. HOBt and 3.5 eq. DIEA in DCM to which some drops of DMF for facilitating solubility of the reagents were added. For workup, a large excess of DCM was added and the resulting organic phase was washed with brine, dried over Na 2 SO 4, filtered and evaporated to dryness. Cleavage of remaining protecting groups was achieved by addition of a mixture of TFA/H 2 O/EDT/TIS (94:2.5:2.5:1) for 2 h, followed by precipitation of crude product by addition of cold diethylether and HPLC purification which led to chromogenic substrate 1 (purity >95%) in 17.5 mg yield (13%). HPLC-ESI-MS: t R 6.45 min, m/z [M+H] +, [M+2H] 2+, [2M+H] +, calcd for C 41 H 61 N 10 O 10 S +. The chromogenic substrates 2 and 3 were prepared accordingly. 2: yield = 16.8 mg (17%). HPLC-ESI-MS: t R 5.89 min, m/z [M+H] +, [2M+H] +, calcd for C 26 H 39 N 8 O : yield = 18.6 mg (7.3%). HPLC-ESI-MS: t R 5.61 min, m/z [M+H] +, [M+2H] 2+, calcd for C 66 H 105 N 18 O Protein purification His tagged DegP, DegPS 210 A and point mutations in PDZ domain 1 were purified under native conditions as described in [1] except for using E. coli degp ompc null mutant strain MA001. DegSΔTM (residues ) and the periplasmic fragment of RseA (residues ) were expressed in BL21(DE3) as described in [3]. pcs13 expresses signal sequenceless mals with a His tag under control of the P tac promoter. Purification of signal sequenceless MalS was done under denaturing conditions as described in [1], except for the following modification: elution buffer was 8 M urea, 100 mm NaH 2 PO 4, 50 mm citrate, 100 mm imidazole, ph 4.5. Eluted MalS was dialysed against 8 M urea, 100 mm NaH 2 PO 4, 50 mm citrate, ph 4.5 to remove imidazole.

5 DegP assays Protease assays using resorufin labelled casein (Roche) as a substrate were done as described in [1]. Protease assays using MalS as a substrate were performed in 50 mm NaH 2 PO 4 (ph 8.0) with 0.13 µm DegP and 1.25 µm MalS. When indicated, activating peptides (50 µm) were preincubated with DegP for 10 min before adding MalS. The assays were stopped by adding SDS sample buffer followed by boiling for 5 min. Samples were analyzed by SDS-PAGE. Protease assays using the synthetic pna-substrate SPMFKGV-pNA were performed in 50 mm NaH 2 PO 4 (ph 8.0) with 0.13 µm DegP and 0.5 mm substrate by measuring the changes in OD 405 (Tecan GENios Pro reader) continuously for 1 h. Activating peptides (50 µm), full length MalS (0.13 µm) or casein (15 µg) [2] were preincubated with DegP 10 min before adding pna substrate. Complete digests of 1.25 mg MalS were performed with 250 µg DegP for 2 h at 37 C. The reaction was terminated by adding 3 vol. of ice-cold acetone and the remaining protein was precipitated at -20 C for 2 h. The sample was centrifuged for 30 min at 16,000 g at 4 C. The supernatant containing the proteolytic products was evaporated in a speed vac (Eppendorf Concentrator 5301). The resulting pellet was dissolved in 100% DMSO. 1 µg of MalS fragments was used in proteolysis assays using pna substrate 1. Chaperone function of DegP was determined by following refolding of chemically denatured MalS as described previously in [1] except that refolding was done overnight at 22 C. The effect of activating peptides was tested by addition of 50 µm DNRDGNVYFF peptide (4). DegS Proteolytic cleavage of the RseA fragment by DegS was performed at 37 C in 100 mm NaH 2 PO 4, ph 8.0, 200 mm NaCl, 10% glycerol, 5 mm MgCl 2 and 1 mm DTT as previously described in [3,4]. 30 µm of the RseA fragment were cleaved by 10 µm DegS TM using various decapeptides DNRDGNVYXF (100 µm) for activation of DegS. The reaction was stopped after 4 h by adding Tris-tricine sample buffer and boiling. Samples were analyzed on 12.5% Tris-tricine SDS-PAGE leading to the separation of RseA, an NH 2 -terminal fragment (RseA N-term; 5.2 kda) and a C-terminal fragment (RseA C-term; 7.7 kda;). [4] The same assays were performed to test the activation of DegS by C-terminal fragments of periplasmic proteins.

6 ITC Isothermal-titration calorimetry (ITC) experiments were performed at 37 C using a MicroCal VP-ITC microcalorimeter. Purified DegPS 210 A mutant protein and peptides were diluted in 50 mm NaH 2 PO 4, (ph 8.0) and degassed for 15 min in a thermovac sample degasser before titration. ITC experiments were initiated with a 1.4 ml solution of protein in the temperature-controlled cell, a 0.3 ml solution of peptide in the titrator syringe and stirring at 394 rpm. Injections of 5 µl peptide were dispensed into the cell with a duration of 7.1 s and a 240 s equilibration time between injections. Data were analysed using Origin software and heats of binding were corrected for the heat of peptide dilution.

7 B. Experimental data Biochemical characterization of DegP Supporting Table 1. ph dependence of DegP activity. ph Buffer [a] Specific activity [b] 5.0 Propionic Acid Propionic Acid MES NaH 2 PO NaH 2 PO NaH 2 PO NaH 2 PO Bicine Bicine Ethanolamine Ethanolamine Ethanolamine 1.8 [a] Buffers were 50 mm, the ionic strength was adjusted to 150 mm with NaCl. MES is [2-(N-morpholine)-ethane sulphonic acid], N,N-Bis(2-hydroxyethyl) and Bicine is N,N-Bis(2-hydroxyethyl)glycine. Substrate and DegP concentrations were 0.5 mm and 0.13 µm, respectively. Standard deviation was typically <15%. [b] Specific activity is given as nmol pna-substrate hydrolysed per min and mg of DegP. In addition, we evaluated the influence of the ionic strength of the buffer on DegP proteolysis. We found a two-fold higher activity in buffers of low ionic strength when compared to high ionic strength. No influence of divalent ions on proteolysis could be detected. Consequently, we established 50 mm NaH 2 PO 4 at ph 8.0 as the most appropriate assay conditions.

8 Allosteric activation of DegP Supporting Figure 1. Activation of DegP by MalS fragments. fold induction Full length Complete digest C-term no addition MalS added DegP activation was followed by proteolysis of 1 by DegP. The assay was performed using 0.5 mm 1, 0.13 µm of either full length MalS, 1 µg completely digested MalS or 50 µm C-terminal MalS fragment for activation of DegP (0.13 µm). The activity of DegP without adding MalS was defined as 1 corresponding to 10.4 nmol pna substrate hydrolysed per min and mg DegP. Supporting Figure 2. Tris-tricine SDS-PAGE of RseA proteolysis by DegS. DegS RseA RseA C-term RseA N-term Cleavage of RseA was performed using 100 µm of peptides 4-16 for activation of DegS. Proteins were visualised by Coomassie blue staining.

9 Supporting Figure 3. Degradation of MalS under activating conditions. A min MalS DegP B min MalS DegP C min MalS DegP MalS was present in 1.25 µm and DegP in 0.13 µm concentrations. Samples were taken at the timepoints indicated. Following SDS PAGE, proteins were visualised by Coomassie blue staining. A. SDS-PAGE of MalS cleavage by DegP. B./C. SDS-PAGE of MalS cleavage by DegP with weakly activating peptide 8 (C-terminal YYF) at 50 µm (B) or with OmpC-peptide 15 at 50 µm (C).

10 Isothermal calorimetry with activating peptide 4 and non-activating peptide 6 Supporting Figure 4. Binding of 6 to DegPSA mutant. Time (min) µcal/sec kcal/mol of injectant Molar Ratio Binding of 5 µl aliquots of activating peptide 6 (c = 1 mm) to a solution of the proteolytically inactive DegP S210A mutant (c = 20 µm) monitored by isothermal titration calorimetry. Protein and peptide were diluted in 50 mm NaH 2 PO 4 (ph 8.0). The black line is a fit of the data with the model of three sequential binding sites with K d s of 40.65, 7.58 and µm. Isothermal titration calorimetry (ITC) experiments were performed to measure the affinity of activating peptides to DegP (DegPSA mutant). Data could be fitted with the 3 sequential binding sites indicating a cooperativity between three binding sites in DegP with a positive cooperativity between the first and the second binding site. K d s and the total enthalpy ΔH of binding are shown in Supporting Table 2. Affinity of the YFF-peptide 4 to the first binding site is approximately two times stronger than the affinity of

11 YLF-peptide 6 to this binding site. The K d s of the second and the third binding site do not differ significantly. Supporting Table 2. K d s and ΔH of activating peptides 4 and 6. activating peptide K d s [µm] ΔH [kcal/mol] YLF YFF

12 Activating Peptides bind to PDZ domain 1 Supporting Figure 5. Point mutations in PDZ domains interfere with activation of DegP. A min min MalS DegP R262A MalS DegP R262A MalS DegP V328S MalS DegP V328S No peptide added With OmpC Peptide 15 No peptide added With OmpC peptide 15 B 12 Specific activity [nmol mg -1 min -1 ] WT V328S R262 A A. Degradation of MalS (1.25 µm) by DegP point mutants R 262 A and V 328 S (0.13 µm) with and without OmpC-peptide 15 (50 µm). The reactions were done at 37 o C and were stopped at the timepoints indicated. Following SDS PAGE, proteins were visualised by Coomassie blue staining. B. DegP (0.13 µm) was incubated with resorufin-labelled casein at 37 C in 50 mm NaH 2 PO 4 (ph 8.0). Specific activity is expressed in nmol mg -1 min -1.

13 Activating peptides induce switching from chaperone to protease activity Supporting Figure 6. Refolding assays of wt DegP with and without activating peptides. Timepoints [h] MalS ox MalS red 0.13 µm chemically unfolded MalS was incubated with 0.36 µm wt DegP in 250 mm NaH 2 PO 4 ph 8.0 in absence (left-hand side) and presence (right-hand side) of 50 µm of activating peptide 4. Samples were incubated at 22 C. At indicated time points aliquots were taken and proteins were TCA precipitated, resuspended in sample buffer without DTT. Following SDS PAGE, the S-S bonded folded (ox) and unfolded reduced (red) forms of MalS were visualised by Western blotting with polyclonal antibodies against MalS.

14 C. References [1] C. Spiess, A. Beil, M. Ehrmann, Cell 1999, 97, [2] B. Lipinska, M. Zylicz, C. Georgopoulos J. Bacteriol. 1990, 172, [3] C. Wilken, K. Kitzing, R. Kurzbauer, M. Ehrmann, T. Clausen, Cell 2004, 117, [4] N. P. Walsh, B. M. Alba, B. Rose, C. A. Gross, R. T. Sauer, Cell 2003, 113,