Supporting InformationLi et al. 10.1073/pnas.0903289106SI Materials and MethodsProtein Preparation. The ORFs of full-length RseA, DegS, andRseP were cloned into pET21b (Novagen). To facilitatepurification, the carboxyl terminus of RseP was fused withGST. All proteins were overexpressed in E. coli BL21(DE3).Expression of DegS and RseP was induced in LB medium with0.2 mM isopropyl-�-D-thiogalactopyranoside at an OD600 of1.0–1.5. Expression of RseA was performed in M9 minimalmedium, and the cells were harvested 3 h after induction at22 °C. All cells were resuspended in a buffer containing 25 mMTris (pH 8.0) and 150 mM NaCl, and lysed by using sonication.The membrane fraction was collected and incubated with1.5%(wt/vol) N-dodecyl-�-D-maltopyranoside (DDM; Anatrace)for 1 h at 4 °C. After another ultracentrifugation step, thesupernatant was loaded to Ni2�-NTA resin (Qiagen) or GS4Baffinity column (GE Healthcare). The protein was eluted in abuffer containing 25 mM Tris (pH 8.0), 250 mM imidazole, and0.035% DDM, or 50 mM Tris (pH 8.7), 10 mM GSH, 150 mMNaCl, and 0.035% DDM. The protein was concentrated andapplied to Superdex-200 (GE Healthcare) in a buffer contain-ing 10 mM Tris (pH 8.0), 150 mM KCl, 0.02% DDM (wt/vol),and 5 mM DTT. The peak fraction was collected for biochem-ical assay. All point mutations were generated by using astandard PCR-based cloning strategy. All mutant proteinswere prepared as described above.

PDZ1 (residues 127–220), PDZ2 (residues 222–309), PDZ2-I304A (residues 222–309), and PDZ2 with a carboxyl-terminallyfused GKASPV (RseA residues 143–148) peptide were sub-cloned into pET15b (Novagen). The cells were homogenized ina buffer containing 25 mM Tris (pH 8.0) and 150 mM NaCl andwere lysed by using sonication. Cell debris was removed bycentrifugation. The supernatant was loaded to an Ni2�-NTAaffinity column (Qiagen). The protein was released from thecolumn by thrombin cleavage for 3 h at room temperature in abuffer containing 25 mM Tris (pH 8.0) and 150 mM NaCl. Theprotein was concentrated to �30 mg/mL and applied to Super-dex-200 (GE Healthcare) in a buffer containing 10 mM Hepes(pH 7.5) and 150 mM KCl. The peak fraction was collected forcrystallization.

Crystallization and Data Collection. All of the crystals were grownat 18 °C by using the hanging-drop vapor-diffusion method. Wellbuffer of PDZ1 contained 100 mM bis-Tris propane (pH 7.0) and2.5 M (NH4)2SO4. Crystals appeared in 1 to 3 days and grew tofull size in 1 week. Well buffer of the WT PDZ2 contained 4 Msodium formate and 4% methanol. Well buffer of PDZ2-I304Amutation contained 4 M sodium formate. Crystals appeared in1 day and grew to full size in 3 to 5 days. Well buffer ofPDZ2-GKASPV contained 0.2 M (NH4)2SO4 and 30% (wt/vol)Polyethylene Glycol 4000 (Fluka). Crystals appeared in 1 dayand grew to full size in 2 days. X-ray diffraction data werecollected in-house on a Bruker X8 Proteum system. A flash-cooled native single crystal was mounted in cryoprotectant in anarbitrary orientation at 100 K in the cold stream. For phasing ofPDZ1, the crystal was soaked into 0.2 M NaI for 15–20 secondsand flash-cooled in cold nitrogen stream. The 360° high-

redundancy, SAD data were measured with a single-axis rota-tion. All data except those for PDZ2-I304 and PDZ2-GKASPVwere collected in-house on a Bruker X8 Proteum system andwere processed by using SAINT� and SADABS (1). ThePDZ2-I304A and PDZ2-GKASPV data were collected at theSpring-8 beamline BL38B1 and SSRF beamline BL17U, respec-tively, and were processed by using HKL2000 (2). Full datacollection and processing statistics are shown in Table S1.

Structure Determination. Iodine-SAD phasing and solvent f lat-tening of PDZ1 were performed with HKL2MAP (3) programusing the underlying SHELX suite (4), which resulted in aninterpretable map. BUCCANEER (5) autotraced 87 residuesout of 94 aa. Manual model building and adjustment wereperformed subsequently with COOT (6). The final modelwas refined against the high-resolution native data by usingPHENIX.REFINE (7). PDZ2 WT and mutate structures weresolved with molecular replacement method by using 2ZPMcoordinates as the starting model. The structure refinementwas performed with PHENIX (7). The Ramachandran plotwas generated with PROCHECK (8), and rmsd bond lengthsand angles were obtained from WHATCHECK (9).

In Vitro DegS and RseP Cleavage Assays. The WT and mutant RseAproteins were used as the substrate. The assay was performed at37 °C for 90 min in a buffer containing 50 mM NaHPO4 (pH 8.4),400 mM NaCl, 0.02% DDM (wt/vol), and 3.3% glycerol. Thefinal concentrations of the substrate proteins were �0.1 �M. Thefinal concentrations of DegS and OMP peptide (DNRDGN-VYYF) were 0.05 and 10 �M, respectively. The final RsePconcentration was 0.05 �M. The reaction was stopped by SDSsample buffer, and the cleavage products were analyzed bySDS/PAGE and Coomassie staining. The digestion mixture aftercleavage was then purified by chromatography on a C8 HPLCcolumn (Vydac) using a mobile phase of 5–80% acetonitrile inwater (vol/vol, linear gradient) supplemented with 0.1% formicacid. Fractions containing cleaved peptide were analyzed byionic-trap MS and sequential Edman degradation.

In Vitro Cleavage of RseA 1–148 by RseP. The WT RseA 1–148protein was used as the substrate. The assay was performed at37 °C for 90 min in a buffer containing 10 mM Tris�HCl (pH 8.0),150 mM KCl, 0.02% DDM (wt/vol), and 4 mM DTT. The finalconcentrations of the substrate protein were �6 �M. The finalRseP concentration was 1.5 �M. The reaction was stopped bySDS sample buffer, and the cleavage products were analyzed bySDS/PAGE and Coomassie staining.

�-Galactosidase Assays. The oE activity was assayed by monitoring�-galactosidase activity expressed from a oE-dependent lacZreporter gene. After growing for 4 h at 37 °C in M9 medium, thetransformed cells were stressed by transferring to 42 °C for 3 h.The final OD600 of the cells was 1.0–1.5. All assays wereperformed at least twice reproducibly. Data from all sampleswith error were shown. Assays were performed as describedpreviously (10).

1. Bruker (2003) SAINT, SADABS, XPREP and SHELXTL/NT Software Reference Manual(Bruker AXS Inc., Madison, WI).

2. Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscil-lation mode. Methods Enzymol 276:307–326.

3. Pape T, Schneider TR (2004) HKL2MAP: A graphical user interface for macromolecularphasing with SHELX programs. J Appl Crystallogr 37:843–844.

4. Sheldrick GM (2008) A short history of SHELX. Acta Crystallogr A 64:112–122.

5. Cowtan K (2006) The Buccaneer software for automated model building. 1. Tracingprotein chains. Acta Crystallogr D Biol Crystallogr 62:1002–1011.

6. Emsley P, Cowtan K (2004) Coot: Model-building tools for molecular graphics. ActaCrystallogr D Biol Crystallogr 60:2126–2132.

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7. Adams PD, et al. (2002) PHENIX: Building new software for automated crystallo-graphic structure determination. Acta Crystallogr D Biol Crystallogr 58:1948 –1954.

8. Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK - a program tocheck the stereochemical quality of protein structures. J Appl Crystallogr 26:283–291.

9. Hooft RW, Vriend G, Sander C, Abola EE (1996) Errors in protein structures. Nature381:272.

10. Miller JH (1992) A Short Course in Bacterial Genetics: A Laboratory Manual andHandbook for Escherichia coli and Related Bacteria (Cold Spring Harbor LaboratoryPress, Plainview, NY).

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Fig. S1. Identification of cleavage fragments of RseA. After DegS and RseP cleavages, the indicated peptide fragments were sent to amino-terminal sequencingand MS analysis. Amino-terminal sequencing identified the DegS cleavage site to be between Val-148 and Ser-149 (Left). Analysis by MS confirmed the DegScleavage site and identified the RseP cleavage site to be between Ala-108 and Cys-109 (Right). The same analysis was applied to some of the mutant proteinswith identical cleavage positions by DegS and RseP.

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Fig. S2. RseP is inactivated by the zinc-chelating agent 1,10-phenanthroline in a concentration-dependent manner.

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Fig. S3. Comparison of protease cleavage of WT and V148N RseA. (A) Comparison of DegS cleavage of WT RseA (black line) and RseA-V148N (magenta line).Shown here is a time course of DegS cleavage of the full-length RseA. Percentage of cleaved RseA is shown as the y axis. The cleavage of RseA-V148N wasconsiderably slower than that for the WT RseA. (B) Comparison of RseP cleavage of WT RseA (black line) and RseA-V148N (magenta line). Shown here is a timecourse of RseP cleavage of the RseA fragment 1–148. Percentage of cleaved RseA 1–148 is shown as the y axis. The cleavage of RseA-V148N 1–148 was considerablyslower than that for the WT RseA 1–148.

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Fig. S4. Mutation of residue 147 of RseA has little impact on the Site-2 cleavege by RseP.

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Fig. S5. Mutation of residue 146 of RseA has little impact on the Site-2 cleavege by RseP.

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Fig. S6. Structural features of the PDZ domains of RseP. (A) Structural comparison of the WT PDZ1 (cyan) and WT PDZ2 (purple). Note that the peptide-bindingpocket of PDZ1 is blocked by an �-helical element. The peptide-binding pocket of PDZ2 is occupied by the carboxyl-terminal residue Ile-309 from the adjacentPDZ2 domain within the same homodimer. Overlay of these two structures shows that Ile-309 overlaps with Val-210 from the �-helical element. (B) Comparisonof the WT PDZ2 (purple) and PDZ2-I304A (magenta) reveals that the peptide-binding pocket is considerably narrowed by the mutation. (C) A close-up view ofthe interactions between Ile-309 and the surrounding residues in WT PDZ2. Note that there are two hydrogen bonds, indicated by red dashed lines.

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Table S1. Statistics of data collection and refinement

Data collection PDZ1 PDZ1�I-SAD PDZ2�WT PDZ2�I304A PDZ2�pep

Space group P3121 P3121 P43212 P21 P 31

Unit cell dimension, Å,°a 59.74 60.44 108.96 47.41 35.09b 38.15c 61.69 61.71 58.34 51.27 62.37� 102.84

Resolution range (outer shell), Å 30–1.67 (1.77–1.67) 30–1.9 (2–1.9) 30–3.09 (3.20–3.09) 30–2.01 (2.11–2.01) 30–1.6 (1.63–1.6)Total observations (unique) 89,799 (14,152) 165,642 (10,790) 24,453 (6,696) 18,873 (10,330) 10,945 (469)Redundancy (outer shell) 5.91 (1.75) 8.26 (1.62) 3.7 (3.2) 1.55 (0.61) 3.0 (1.8)Rsym (outer shell) 0.035 (0.39) 0.058 (0.439) 0.1 (0.393) 0.094 (0.335) 0.06 (0.246)Completeness (outer shell), % 93.1 (76.4) 95.4 (71.2) 97.6 (90.4) 85.2 (47.7) 97.5 (85)I/� (outer shell) 26.9 (3.2) 21.6 (2.3) 19.8 (3.24) 9.85 (3.21) 33.1 (2.8)Refinement

Rfactor/Rfree 0.189/0.235 0.205/0.27 0.197/0.222 0.192/0.219Total no. of atoms (water) 864 (131) 1378 (0) 1463 (141) 773 (70)B factors (overall), Å2 31.06 69.08 20.72 30.27

Main chain 24.73 67.13 17.27 25.37Side chain 34.23 70.49 23.61 33.68

rmsd bonds, Å 0.006 0.011 0.007 0.004rmsd angles, ° 1.053 1.409 0.936 0.883Ramachandran plot, %

Most favored 94.9 80.3 93.6 95.8Additional 5.1 14.8 6.4 4.2Generous 0 4.9 0 0Disallowed 0 0 0 0

Values in parentheses are for the highest-resolution shell. Rsym � �h�i�Ih,i � Ih�/�h�iIh,i, where Ih is the mean intensity of the i observations of symmetry-relatedreflections of h. R � ��Fobs � Fcalc�/�Fobs, where Fobs � Fp, and Fcalc is the calculated protein structure factor from the atomic model (Rfree was calculated with 5%of the reflections). The rmsd values in bond lengths and angles are the deviations from ideal values.

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