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Structural Insights on the Bacteriolytic and Self-protection Mechanism of Muramidase Effector Tse3 in Pseudomonas aeruginosa

Lianbo Li, Weili Zhang, Qisong Liu, Yu Gao, Ying Gao, Yun Wang, David Zhigang Wang*, Zigang Li*,

Tao Wang*

Lab of Computational Chemistry and Drug Design, Key Laboratory of Chemical Genomics, School of Chemical Biology & Biotechnology, Peking University, Shenzhen Graduate School, Shenzhen 518055,

China

Running title: Crystal Structure of Muramidase Effector Tse3 with Tsi3 Key words: Pseudomonas aeruginosa; Crystal structure; Toxins; Peptidoglycan; Protein complexes; Type VI secretion system; Muramidase Effector Tse3; Immune protein Tsi3; Antitoxins Background: Muramidase effector Tse3 from Pseudomonas aeruginosa kills rival bacteria but is neutralized by Tsi3 for self-protection. Results: Tse3 contains two domains and Tsi3 binds with it via hydrogen bond network. Conclusion: The peptidoglycan hydrolysis activity of Tse3 depends on Ca2+ ions and Tsi3 obstructs its catalytic pocket for neutralization. Significance: Elucidate how P. aeruginosa benefits from Tse3:Tsi3 for fitness. ABSTRACT The warfare among microbial species as well as between pathogens and hosts is fierce, complicated and continuous. In Pseudomonas aeruginosa, the muramidase effector Tse3 (Type VI Secretion Exported 3) can be injected into the periplasm of neighboring bacterial competitors by a Type VI secretion apparatus, eventually leading to cell lysis and death. However P. aeruginosa protect itself from lysis by expressing immune protein Tsi3 (Type Six secretion Immunity 3). Here, we report the crystal structure of the Tse3:Tsi3 complex at 1.8 Å resolution, revealing that Tse3 possesses one open accessible, goose-type lysozyme like domain with peptidoglycan hydrolysis activity. Calcium ions bind specifically in the Tse3 active site and are identified to be crucial for its bacteriolytic activity. In combination with biochemical studies, the structural basis of self-protection mechanism of Tsi3 is also elucidated, thus providing an understanding and new insights into the effectors of Type VI secretion system.

Secretion systems are used by bacteria to deliver toxins/effectors evoking cell lysis，thus providing benefits for bacterial survival (1,2). The recently reported Type VI secretion systems (T6SSs) in Pseudomonas aeruginosa, which facilitate the colonization of P. aeruginosa for its growth advantage, are also widely distributed in many bacterial species (3-8). P. aeruginosa kills neighboring bacterial cells by delivering toxic effectors through its tail-like T6SS apparatus directly into target cells, disrupting the processes of life cycles of target bacteria and expressing immune proteins for self-protection (8-15). For these T6SS effectors and their immune partners, several crystal structures were recently reported: Tse1 (13,16-19), Tae3 (20) and Tae4 (21,22), possessing the cell lysis activity by cleaving the D-Glu-mDAP1 of peptidoglycan in the Gram-negative bacteria, were structurally characterized as endopeptidases with classical NlpC/P60 fold. Tse2 has been characterized as a toxin but with unknown mechanism, and its cognate partner Tsi2 is rationalized to be a cytoplasm neutralizer by physical interaction with Tse2 (23-25). Tse3 is delivered to the periplasm of recipient bacteria and promotes cell lysis, and is characterized with muramidase activity that cleaves the β-1,4 bond between MurNAc1 and GlcNAc1 in peptidoglycan. However, P. aeruginosa itself could express periplasmic immune protein Tsi3 that neutralizes the lethal action of this toxin (11). Here we report the first structural study of the Tse3:Tsi3 toxin/antitoxin complex at 1.8 Å resolution. Together with mutagenesis studies and

http://www.jbc.org/cgi/doi/10.1074/jbc.C113.506097The latest version is at JBC Papers in Press. Published on September 11, 2013 as Manuscript C113.506097

Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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a biochemical analysis, we provide structural insights into the bacteriolytic activity of Tse3 and further investigate how Tsi3 neutralizes Tse3 toxicity: Tse3 consists of two domains, the C-terminal catalytic domain adopts an extended goose-type lysozyme like structure where Ca2+

ions play essential roles for its cell lysis activity. The immune protein Tsi3 interacts with Tse3 via a hydrogen bond network, occupies the catalytic site of Tse3 to block the substrate binding. These studies are significant by not only contributing to a better understanding of T6SS secretion system in P. aeruginosa, but also by providing new insights into niche competition among pathogenic bacteria. EXPERIMENTAL PROCEDURES

Cloning, expression and purification of proteins—Full-length Tse3 (residues 1-408) and the N-terminal signal peptide deleted Tsi3 (residues 23-145, Tsi3-ΔN) were inserted into pET-15b respectively between the restriction enzyme sites of NdeI/BamHI with a 6XHis-tag and a thrombin cleavage site. Transformed BL21 (DE3) competent cells were cultured in LB medium at 37˚C and induced by 0.5mM IPTG at 30˚C for 6 hours. Bacterial cells were resuspended in buffer A (25mM Tris-HCl [pH8.2], 500mM NaCl, 10mM imidazole) and lysed by passing through a French press twice. Cell debris was removed by centrifugation at 16,000 g for 60min at 4°C. The fusion protein was purified by Ni-NTA cartridge (Qiagen) and the 6XHis-tag was removed by thrombin cleavage. Target proteins were further purified by size exclusion chromatography (Superdex-200, GE Healthcare) with buffer B (20mM Tris-HCl [pH8.2], 150mM NaCl, 1mM DTT). For Tse3:Tsi3-ΔN complex formation, Tse3 was mixed with excessive Tsi3-ΔN, followed by gel filtration to remove the unbounded Tsi3-ΔN.

Crystallization, Data collection and structure determination—Hanging-drop vapor diffusion crystallization trials were set up at 16 °C. 2µl of purified Tse3:Tsi3-ΔN (~10mg/ml) was mixed with well solution (100mM HEPES, 2M NH4COOH, 50µM CaCl2, pH7.2) in a 1:1 ratio. The micro-seeding method was used to initiate crystal formation followed by two times macro-seeding to generate plate crystals with good diffraction. Crystals were flash frozen with 25% glycerol in mother liquor for screening and data

collection, or derivatized in mother liquor with 1M sodium iodide for 30 seconds before flash freezing. Back soaking was also applied to improve clarity.

Two crystal forms (C2 and P21) of the Tse3:Tsi3-ΔN complex were initially obtained but only the P21 crystals diffracted very well and were used for final structure determination. Most crystals were anisotropic so hundreds of crystals were screened on an in-house Rigaku X-ray source. High-resolution native datasets were collected at 100K on the BL17U beamline of the Shanghai Synchrotron Radiation Facility (SSRF). The datasets were integrated and processed using crystalclear-1.44 (26) or HKL2000 (27) for Synchrotron. Phasing and auto model building were undertaken by SHELXC/D/E (28) and PHENIX suite (29). Model improvement and refinements were finalized in COOT (30) and REFMAC in CCP4 (31) (Table 1).

Isothermal titration calorimetry (ITC) measurement—Experiments were performed using the iTC200 system (GE Healthcare) at 20 °C with titration buffer C (20mM HEPES [pH7.4], 100mM NaCl). 80µM 6XHis-tag removed Tse3 in 400µl titration buffer was placed in the sample cell, and 680µM 6XHis-tag removed Tsi3-ΔN in 40ul titration buffer C was loaded into the injection syringe. A 120s delay at the start of the experiment was followed by 20 injections containing 40µl of the solution with 240s intervals. All measured samples were stirred at 500 rpm. Blank injections of the Tsi3 protein into buffer C were subtracted from the experimental titration and the data were analyzed in Origin7.0 (MicroCal®).

Whole-Cell lysis and Tse3 activity rescue experiment—Overnight culture of E. coli B strain was sub-inoculated into fresh LB media and grown to the late logarithmic phase (OD600=1.5). Harvested cells were washed and resuspended in permeabilization buffer E (0.5M sucrose, 0.2% v/v Tween-20, and 40mM HEPES [pH6.8]). To prepare a Ca2+ free Tse3 sample, 0.8mg/ml fresh purified Tse3 was incubated with 150µM EDTA overnight in buffer F (10mM HEPES [pH7.2], 100mM NaCl (99.999%)). Multi-steps of ultra-filtration using buffer F were applied to remove EDTA and metal ions. To prepare Ca2+-rescued Tse3, CaCl2 was added to the Ca2+-free Tse3 sample with a final concentration of 500µM and incubated on ice for 3 hours to let Ca2+ incorporate back into the protein.

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For activity rescue experiments, the concentration of wild type Tse3, Ca2+-free Tse3 and Ca2+-rescued Tse3 were adjusted to 1.5mg/ml. 150µl of each sample were added into 3ml E.coli B strain cell in permeabilization buffer E (OD600=1.1), resulting of 1.7µM Tse3, 7.5µM EDTA or 25µM CaCl2 for rescue. The cell lysis rate was reflected by decrease of OD600 at 27 degree.

RESULTS

Crystal Structure of the Tse3:Tsi3 Complex—The crystal structure of Tse3:Tsi3-ΔN was determined by Single Isomorphous Replacement with Anomalous Scattering (SIRAS) of the iodide derivative and refined to 1.8Å without residues located in outlier region by Ramachandran plot (Table 1). It contains full length Tse3 (1-408) and Tsi3-ΔN (23-145) with clear and continuous electron density map. The final model contains two Tse3:Tsi3-ΔN heterodimer per asymmetric unit. Therefore, only one of the Tse3:Tsi3-ΔN complexes is displayed and discussed in this paper. Tse3 is mainly composed of α-helices and short loops, which consist of a small N-terminal domain with a C-terminal catalytic domain (Fig. 1A). The N-terminal domain (residues 1-125) contains seven α-helices (α1-α7), which are packed in a rod-like helical repeat motif and tightly interacting with the C-terminal domain (Fig.1B). The C-terminal catalytic domain (residues 147 to 408) contains two major lobes (Lobe1, Lobe2) forming an open accessible catalytic groove with three Ca2+

binding cations (Fig. 1A). Tsi3 is mainly folded as a seven-strand (β3-β9), highly curved anti-parallel β-sheet, with a small β-sheet (residues 23 to 39, β1-β2) and α-helix (residues 133 to 142, α1-Tsi3) back-flanked on it (Fig. 1A). DALI search results show that Tsi3 shares structural similarities with Mog1p (Z score=8.8, PDB: 1EQ6) (32) and CyanoP (Z score=7.4, PDB: 2LNJ) (33) but bears no functional relevance. In Tse3:Tsi3-ΔN complex structure, three loops of Tsi3 protrude to Tse3 and fully occupy the enzymatic groove, and Arg-60 is located in the center of loop and buried into the Tse3 enzymatic pocket (Fig. 1A). The C-terminal domain of Tse3 interacts with N-terminal domain mainly via extensive hydrophobic interactions and hydrogen bonds. A

long loop (128-146) is mainly composed of Ala and Gly (128-AAAGATGVASQA-139), link the N-terminal domain to the C-terminal domain. Phe-127 and Phe-144 anchor to the pocket of the core C-terminal domain and act as a hinge, providing the flexibilities to the N- and C-terminal domains (Fig. 1A;1B). The α7 helix (108-125) is buried into the hydrophobic groove of C-terminal domain and interacts with the N-terminal domain by forming hydrophobic interactions (Fig. 1C; 1D). Two pairs of salt bonds (Asp-120 with Arg-80, Asp-113 with Arg-84) reinforce the interaction between α7 and the N-terminal domain (Fig. 1E). All of these interactions suggest that the α7 helix plays a central role for the structural integrity of the protein by linking the two domains and in a position necessary for enzymatic activity. DALI search results show that the overall fold of the N-terminal domain of Tse3 is similar to the eukaryotic ARM1 repeat and the HEAT repeat proteins (34). Sequence alignments of the N-domain with HEAT repeat proteins show that the N-terminal domain contains a set of conserved hydrophobic residues for inter-helix interactions. Specifically, α1-α6 constitute the conserved ARM and HEAT like repeat domain, but α7 in Tse3 is less conserved.

Tse3 features a goose-type lysozyme like catalytic C domain—The C-terminal domain of Tse3 is mainly composed of two lobes in a V-shape (Lobe1: residues 225-253 and 315-408; Lobe2: residues 147-221 and 255-314) (Fig. 2A). The enzymatic groove is in the center part of C-terminal domain, which is mainly formed by hydrophobic residues and hydrophilic residues with Ca2+ I, II binding, thus forming a hydrophobic environment with negatively charged catalytic key residue Glu-250 (11) (Fig. 2B).

In Tse3, only Lobe1 of the C-terminal domain share high similarities to homologues, while Lobe2 is less conserved and specific. DALI results show that lytic transglycosylase MltE (Z score=10.7, PDB: 2Y8P) (35), goose-type lysozyme (Z score=10.0, PDB: 3GXK) (36) and lytic transglycosylase Slt70 (Z score=9.8, PDB: 1QTE) (37) share structural similarities with the C-terminal domain. Sequence alignments and structure superimpositions (Data not shown) indicate that only Lobe 1 of the C-terminal domain adopts a goose-type lysozyme like structure, confirming the known bacteriolytic muramidase

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activity of Tse3 (11). There are three types of lysozymes:

chicken-type, goose-type and lysozyme from T4 bacteriophage (36,38,39). The goose-type lysozymes adopt a α+β fold consisting of seven α-helices and three β-strands (36). In Tse3, the central helix α14 contains the highly conserved glutamic acid Glu-250 (Glu-73 in goose-type lysozyme, Glu-478 in Slt70 and Glu-64 in MltE), which is proposed to act as a general-acid catalyst (36); Another β-hairpin also exhibits high conservation, the conserved glutamine Gln-280 of Tse3 (Gln-99 in goose-type lysozyme, Gln-496 in Slt70, Gln-82 in MltE) is located on this β-hairpin and coordinats with Ca2+ cations (Fig. 2B), even though no metal ion binding was reported in these homologues (35-37).

Tsi3 interacts with Tse3 and obstructs the catalytic pocket—An inspection of the electrostatic potential mapped onto the molecular surfaces reveals that the contact surfaces of Tse3:Tsi3-ΔN are complementary not only in shape, but also in electrostatic surface charge (Fig. 2B). Three loops of Tsi3 (Glu-55 to Gln-64 between β4 and β5, Asp-95 to Ser-102 between β7 and β8, Ser-125 to Glu-129 between α1-Tsi3 and β9) bind in the enzymatic groove and interact with Tse3 by an extensive network of hydrogen bonds, with additional intra-molecular hydrogen bonds between Gln-124, Asn-61 and Gly-101, fully occupy the enzymatic groove of Tse3 (Fig. 1A; 2B). More interestingly, the side chain of Arg-60 in Tsi3 forms a strong hydrogen bond network with negatively charged residues (Glu-250, Asp-253 and Thr-377 from Tse3, Ser-99 from Tsi3) and water molecules (H2O-I, H2O-II), thus blocks the catalytic site of Tse3 (Fig. 2C). Single site mutation (R60A or R60E) of Tsi3 can completely abolish the interaction of Tse3:Tsi3-ΔN, which is testified by gel filtration (Data not shown), confirming that Arg-60 plays dominant role for Tsi3 immune function. The ITC result also show that wild type Tsi3-ΔN binds to Tse3 moderately with an equilibrium dissociation constant of 33µM, hints that the interaction between Tse3 and Tsi3 is weak (Fig. 2D).

Calcium is essential for enzymatic activity of Tse3—The most striking discovery in Tse3:Tsi3-ΔN complex structure is that three calcium ions bind with Tse3: two calcium ions (Ca2+-I, Ca2+-II) bind adjacent to Glu-250 in the

middle part of catalytic groove, and the third calcium ion (Ca2+-III) binds in the loop between helix α21 and α22 (Fig. 1A; 2B). Ca2+-I coordinates with two water molecules (H2O-I, H2O-II) and four negatively charged residues from Tse3 (side chain of Asn-181, Gln-254 and Glu-258; main chain of Asp-253) (Figure. 2B; 3A). Ca2+-II also coordinates with four residues from Tse3 (side chain of Glu-258, Asp-262 and Gln-280; side chain and main chain of Ser-275) and the side chain of Glu-126 from Tsi3, with a distance of 4.3 Å to Ca2+-I (Fig. 2B; 3A). The third calcium ion (Ca2+-III) is coordinated with one water molecule (H2O-III) and 5 residues located in the 12-residues loop between α21 and α22 (side chain of Glu-375, Ser-378 and Asp-382; main chain of Arg-379 and Asn-384) (Fig. 2B; 3A).

The presences of calcium ions in the catalytic site lead us to question whether they are enzymatic factors. Gel filtration results show that the Tse3:Tsi3-ΔN heterodimer could be completely disrupted in the presence of 7.5µM EDTA, but adding of 25µM CaCl2 could rescue the formation of complex (Fig. 3B). The presence of calcium ions in the Tse3:Tsi3-ΔN complex was also experimentally confirmed by ICP-OES1, the results show that calcium ions bind to the Tse3:Tsi3-ΔN complex with a molar ratio of 2:1 when it with 10µM CaCl2 in solvent, but only 1:1 without 10µM CaCl2 present (Data not shown). In vitro whole-cell lysis assays also indicate that calcium ions are necessary for Tse3 enzymatic activity: wild type Tse3 could easily hydrolyze the peptidoglycan of bacteria and cause cell lysis, which is indicated by the fast dropping of the OD600 absorption. In contrast, EDTA treated Tse3 (Ca2+ free) lost this ability but addition of excess CaCl2

could rescue its cell lysis activity (Fig. 3C).

DISCUSSION Two T6SS toxins (Tse1, Tae4) structures were

recently reported and both belong to the cysteine peptidase NlpC/P60 superfamily (16,18,19,21,22). Whereas Tse3 shows its specific features that it contains an ARM/HEAT-like N-terminal domain and a goose-type lysozyme like C-terminal catalytic domain (Fig. 1A). In the Tse3:Tsi3-ΔN complex structure described here, the interactions between the N- and C-terminal domains of Tse3 are mainly mediated by the α7 helix and Lobe2 (Fig. 1B; 1C; 1D; 1E). The significant differences

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in the N-terminal domain and Lobe2 may introduce unique features of Tse3 and contribute to the regulation of enzymatic activity of the C-terminal domain. Considering that ARM- and HEAT -like motifs are commonly found to mediate protein-protein interactions by their modular recognition of extended peptide regions (34), it is reasonable to speculate that the N-terminal domain of Tse3 is crucial for the proper folding and conformational stabilities of the Tse3. Although some enzymes that catalyze a hydrolytic reaction, such as phospholipase A2 and staphylococcal nuclease, bind with Ca2+ in their active site as a catalytic factor (40,41), Ca2+ binding and involvement of enzymatic activity are not observed in the reported T6SS toxins (15,16,18-22) or the goose-type lysozyme homologues (35-37). Based on the high-resolution structure and biochemical studies presented here, we identified three Ca2+ ions that bind with Tse3:

two Ca2+ ions directly bind in the catalytic site and adjacent to the catalytic residue Glu-250 (Fig. 2C; 3A). Structural superimpositions with homologues indicate that the structural features of Lobe2 in Tse3 mainly contribute to this unique Ca2+ binding ability. Enzymatic studies also show that Tse3 requires Ca2+ for cell lysis activity (Fig. 3C), demonstrating that muramidase effector Tse3 is different from other T6SS toxins. Furthermore, the ICP-OES results show that the binding molar ratio of calcium to Tse3:Tsi3-ΔN could vary from 2:1 to 1:1 (data not shown), strongly suggesting that the binding of Ca2+ ions to Tse3 is weak and dynamic. Even though the Ca2+ ions are found to be essential for the enzymatic activity of Tse3, the details of peptidoglycan substrate binding and hydrolysis reaction mechanism still remain unclear and await to be addressed by further investigations.

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FOOTNOTES This work was funded by MOST of China 2013CB911501, NSFC31300600, SZSTI program ZDSY20120614144410389, JCYJ20120614150904060, and Startup fund from PKUSZ to T.W.; NSFC21102007, SZSTI program SW201110060, SW201110018 and Peacock Program KQTD201103 to Z.G.L.; MOST of China 2012CB722602, NSFC20872004, NSFC20972008, NSFC21290180, SZSTI “Shuang Bai Project” to D.Z.W.. We thank the staff of beamline BL17U at SSRF for the technical assistance during data collection; we thank Prof. Olaf G. Wiest for proofreading; we thank Yankui Lin, Feng Xiao and Zhi Yan in the Shenzhen Entry-Exit inspection and Quarantine Bureau for the instrumental assistance of ICP-OES elemental analysis. *Correspondence and requests for materials should be addressed to: Tao Wang (tau@pku.edu.cn); Zigang Li (lizg@pkusz.edu.cn); or David Zhigang Wang (dzw@pkusz.edu.cn). School of Chemical Biology & Biotechnology, Peking University, Shenzhen Graduate School, Lishui Road, Nanshan District, Shenzhen, 518055, China. Tel: 86-755-26032703; Fax: 86-755-26032511. 1The abbreviations used are: D-Glu-mDAP, gamma-D-glutamyl-L-meso-diaminopimelic acid; MurNac，N-acetylmuramic acid；GlcNac，N-acetylglucosamine；ARM, armadillo; ICP-OES, Inductively Coupled Plasma-Optical Emission Spectrometer. Atomic coordinates and structure factors (code 4LUQ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org).

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FIGURE LEGENDS FIGURE 1. Overall structure of Tse3:Tsi3-ΔN complex. A, Ribbon and surface representations of the Tse3:Tsi3-ΔN complex structure, three loops of Tsi3 protruded to Tse3 and fully occupied the enzymatic groove (yellow: Tsi3 β-sheet; green: Tsi3 α-helix; cyan: Tse3 C-terminal domain (residues 147-408); blue: Tse3 N-terminal domain; purple: linker loop (residues127-146); orange: α7-helix of Tse3), calcium ions are indicated by arrow. B, α7 helix mediated the interactions between the N-terminal domain and The Lobe2 of C-terminal domain, Trp-103, Phe-106 and Phe-144 anchored in the pocket of the C-terminal domain and shown in stick. C, Hydrophobic interactions between α7-helix and the C-terminal domain. D, Hydrophobic interactions between α7-helix and the N-terminal domain. E, Salt bonds reinforce the interactions between α7-helix and the N-terminal domain (dash lines marked with distance (Å)). FIGURE 2. Tse3 features a two-lobe catalytic C domain and Tsi3 obstructs the catalytic pocket. A, Surface representations of the enzymatic groove from top and side views (yellow: Lobe1; green: Lobe2; red: conserved residues Glu-250 and Gln-280). B, Top view of the enzymatic groove of Tse3, hydrophobic residues marked in black, catalytic key residues shown in red, Ca2+ ions indicated in purple, the electrostatic potential is depicted with surface coloration from red (negative) to blue (positive). C, Close-up view of the network of hydrogen bonds between Arg-60 of Tsi3 and the neighboring residues in catalytic site of Tse3 (water molecule in red; Ca2+ in purple), hydrogen bonds are shown in yellow dash lines with distance (Å). D, Affinity of Tse3:Tsi3-ΔN is measured by Isothermal Titration Calorimetry (ITC), deviation of dissociation constants Kd from the ITC data was 6.4%. FIGURE 3. Ca2+ ions bind with Tse3 and play crucial role for the cell lysis activity of Tse3. A, Hydrogen bonds network of Ca2+ binding in Tse3, H2O in red; Ca2+ in purple; residues of Tse3 shown in cyan; Arg-60 and Glu-126 from Tsi3 shown in yellow, hydrogen bonds are shown as yellow or blue dash line with distance (Å). B, Size-exclusion chromatography experiments for testing the Calcium ion-mediated Tse3:Tsi3-ΔN interaction. The peak position of Tse3, Tsi3-ΔN and Tse3:Tsi3-ΔN complexes are corresponding to elution volume of 11.6ml, 12.7ml and 11.1ml, Tse3:Tsi3-ΔN complex dissociated completely with EDTA treatment and calcium add-in is capable of recovering the complex formation. C, In vitro cell lysis experiment for examination of the activity of Tse3, OD600 growth curves of E. coli reflected the cell lysis activity, EDTA treated Tse3 lost its function but calcium ions rescued this activity. (E*: 7.5µM EDTA; Ca2+: 25µM CaCl2).

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TABLES Table 1 Data collection and refinement statistics of Tse3-Tsi3-ΔN complex Native KI-Derivative Data collection Space group P21 P21 Cell dimensions a, b, c (Å) 63.33，81.29，111.41 62.57, 81.34, 110.99 a, b, g (°) 90，93.64，90 90, 93.02, 90 Wavelength(Å) 0.9791 1.5418 Resolution (Å) 50-1.77(1.8-1.77) 24.87-2.2(2.28-2.2) Rmerge 0.057(0.253) 0.108(0.679) I /σI 25.44(7.08) 5.76(2.16) Completeness (%) 99.23(96.10) 99.10(92.28) Redundancy 3.7(3.7) 7.0(4.4) Refinement Resolution (Å) 40.49-1.77 No. reflections 96128 Rwork / Rfree 0.153/0.198 No. atoms 8997 Protein 8226 Ca2+ ion 6 Water 765 B factors 25.93 Protein 24.90 Ca2+ ion 14.53 Water 32.40 r.m.s. deviations Bond lengths (Å) 0.019 Bond angles (°) 1.963 *Values in parentheses are for highest-resolution shell. Rmerge = Σ |I - <I>|/Σ I, where I = observed intensity and <I> = average intensity of multiple observations of symmetry-related reflections. Rwork = Shkl ||Fobs| - |Fcalc|| / Shkl |Fobs|. Rfree is defined as above but calculated for 5% of reflections randomly selected from the dataset.

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FIGURES Figure 1

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Figure 2

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Figure 3

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Zigang Li and Tao WangLianbo Li, Weili Zhang, Qisong Liu, Yu Gao, Ying Gao, Yun Wang, David Zhigang Wang,

Muramidase Effector Tse3 in Pseudomonas aeruginosaStructural Insights on the Bacteriolytic and Self-protection Mechanism of

published online September 11, 2013J. Biol. Chem. 

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