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

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: We elucidate how P. aeruginosa benefits from Tse3-Tsi3 for fitness. 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 protects 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.

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 protects 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, goosetype 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) 4 in Pseudomonas aeruginosa, which facilitate the colonization of P. aeruginosa for its growth advantage, are also widely distributed in many bacterial species (3)(4)(5)(6)(7)(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 cell lysis activity by cleaving the ␥-D-glutamyl-L-meso-diaminopimelic acid 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)(24)(25). Tse3 is delivered to the periplasm of recipient bacteria and promotes cell lysis, and is characterized with muramidase activity that cleaves the ␤-1,4 bond between N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) 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 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 Ca 2ϩ ions play essential roles for its cell lysis activity. The immune protein Tsi3 interacts with Tse3 via a hydrogen-bond network and 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-Fulllength Tse3 (residues 1-408) and the N-terminal signal peptide-deleted Tsi3 (residues 23-145, Tsi3-⌬N), respectively, were inserted into pET-15b between the restriction enzyme sites of NdeI/BamHI with a His 6  site. Transformed BL21 (DE3) competent cells were cultured in LB medium at 37°C and induced by 0.5 mM isopropyl-1-thio-␤-D-galactopyranoside at 30°C for 6 h. Bacterial cells were resuspended in buffer A (25 mM Tris-HCl (pH 8.2), 500 mM NaCl, 10 mM imidazole) and lysed by passing through a French press twice. Cell debris was removed by centrifugation at 16,000 ϫ g for 60 min at 4°C. The fusion protein was purified by nickel-nitrilotriacetic acid cartridge (Qiagen), and the His 6 tag was removed by thrombin cleavage. Target proteins were further purified by size exclusion chromatography (Superdex-200, GE Healthcare) with buffer B (20 mM Tris-HCl (pH 8.2), 150 mM NaCl, 1 mM DTT). For Tse3-Tsi3-⌬N complex formation, Tse3 was mixed with excessive Tsi3-⌬N followed by gel filtration to remove the unbounded Tsi3-⌬N. Crystallization, Data Collection, and Structure Determination-Hanging-drop vapor diffusion crystallization trials were set up at 16°C. 2 l of purified Tse3-Tsi3-⌬N (ϳ10 mg/ml) was mixed with well solution (100 mM HEPES, 2 M NH 4 COOH, 50 M CaCl 2 , pH 7.2) in a 1:1 ratio. The micro-seeding method was used to initiate crystal formation followed by macro-seeding two times 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 1 M potassium iodide (KI) for 30 s before flash freezing. Back soaking was also applied to improve clarity.
Two crystal forms (C2 and P2 1 ) of the Tse3-Tsi3-⌬N complex were initially obtained, but only the P2 1 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 100 K 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) (see Table 1).
Isothermal Titration Calorimetry (ITC) Measurement-Experiments were performed using the iTC200 system (GE Healthcare) at 20°C with titration buffer C (20 mM HEPES (pH 7.4), 100 mM NaCl). 80 M His 6 tag-removed Tse3 in 400 l of titration buffer was placed in the sample cell, and 680 M His 6 tag-removed Tsi3-⌬N in 40 l of titration buffer C was loaded into the injection syringe. A 120-s delay at the start of the experiment was followed by 20 injections containing 40 l of the solution with 240-s 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 Escherichia coli B strain was sub-inoculated into fresh LB medium and grown to the late logarithmic phase (A 600 ϭ 1.5). Harvested cells were washed and resuspended in permeabilization buffer E (0.5 M sucrose, 0.2% v/v Tween 20, and 40 mM HEPES (pH 6.8)). To prepare a Ca 2ϩ -free Tse3 sample, 0.8 mg/ml fresh purified Tse3 was incubated with 150 M EDTA overnight in buffer F (10 mM HEPES (pH 7.2), 100 mM NaCl (99.999%)). Multiple steps of ultrafiltration using buffer F were applied to remove EDTA and metal ions. To prepare Ca 2ϩ -rescued Tse3, CaCl 2 was added to the Ca 2ϩ -free Tse3 sample with a final concentration of 500 M and incubated on ice for 3 h to let Ca 2ϩ incorporate back into the protein.
For activity rescue experiments, the concentrations of wild type Tse3, Ca 2ϩ -free Tse3, and Ca 2ϩ -rescued Tse3 were adjusted to 1.5 mg/ml. 150 l of each sample was added into 3 ml of E. coli B strain cell in permeabilization buffer E (A 600 ϭ 1.1), resulting in 1.7 M Tse3, 7.5 M EDTA, or 25 M CaCl 2 for rescue. The cell lysis rate was reflected by the decrease of A 600 at 27°C.

RESULTS
Crystal Structure of the Tse3-Tsi3 Complex-The crystal structure of Tse3-Tsi3-⌬N was determined by single isomorphous replacement with anomalous scattering (SIRAS) of the iodide derivative and refined to 1.8 Å without residues located in the outlier region by Ramachandran plot (Table 1). It contains full-length Tse3-(1-408) and Tsi3-⌬N-(23-145) with clear and continuous electron density map. The final model contains two Tse3-Tsi3-⌬N heterodimers per asymmetric unit. Therefore, only one of the Tse3-Tsi3-⌬N complexes is displayed and discussed in this study.
The C-terminal domain of Tse3 interacts with N-terminal domain mainly via extensive hydrophobic interactions and hydrogen bonds. A long loop (residues 128 -146), mainly composed of Ala and Gly ( 128 AAAGATGVASQA 139 ), links 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. 1, A and B). The ␣7 helix (residues 108 -125) is buried into the hydrophobic groove of the C-terminal domain and interacts with the N-terminal domain by forming hydrophobic interactions (Fig. 1, C and D). Two pairs of salt bonds (Asp-120 with Arg-80, Asp-113 with Arg-84) reinforce the interaction between ␣7 and the N-terminal domain (Fig. 1E). All of these interactions suggest that the ␣7 helix plays a central role for the structural integrity of the protein by linking the two domains 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 ARM 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 interhelix interactions. Specifically, ␣1-␣6 constitute the conserved ARM and HEAT-like repeat domain, but ␣7 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 Ca 2ϩ -I and -II binding, thus forming a hydrophobic environment with negatively charged catalytic key residue Glu-250 (11) (Fig. 2B).
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-⌬N are complementary not only in shape, but also in electrostatic surface charge (Fig. 2B). Three loops of Tsi3 (Glu-55 to Gln-64 between ␤4 and ␤5, Asp-95 to Ser-102 between ␤7 and ␤8, and Ser-125 to Glu-129 between ␣1-Tsi3 and ␤9) bind in the enzymatic groove and interact with Tse3 by an extensive network of hydrogen bonds, with additional intramolecular hydrogen bonds between Gln-124, Asn-61, and Gly-101, fully 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 are marked in black, catalytic key residues shown in red, Ca 2ϩ ions are indicated in purple, and 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; Ca 2ϩ in purple). Hydrogen bonds are shown in yellow dashed lines with distance (in Å). D, affinity of Tse3-Tsi3-⌬N is measured by ITC; deviation of dissociation constants K d from the ITC data was 6.4%.
occupying the enzymatic groove of Tse3 (Figs. 1A and 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 and Ser-99 from Tsi3) and water molecules (H 2 O-I and H 2 O-II) and 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-⌬N, which is verified by gel filtration (data not shown), confirming that Arg-60 plays a dominant role for Tsi3 immune function. The ITC results also show that wild type Tsi3-⌬N binds to Tse3 moderately with an equilibrium dissociation constant of 33 M, hinting that the interaction between Tse3 and Tsi3 is weak (Fig. 2D).
Calcium Is Essential for Enzymatic Activity of Tse3-The most striking discovery in the Tse3-Tsi3-⌬N complex structure is that three calcium ions bind with Tse3; two calcium ions (Ca 2ϩ -I and Ca 2ϩ -II) bind adjacent to Glu-250 in the middle part of catalytic groove, and the third calcium ion (Ca 2ϩ -III) binds in the loop between helixes ␣21 and ␣22 The presence of calcium ions in the catalytic site led us to question whether they are enzymatic factors. Gel filtration results show that the Tse3-Tsi3-⌬N heterodimer could be completely disrupted in the presence of 7.5 M EDTA, but adding 25 M CaCl 2 could rescue the formation of complex (Fig. 3B). The presence of calcium ions in the Tse3-Tsi3-⌬N complex was also experimentally confirmed by ICP-OES; the results show that calcium ions bind to the Tse3-Tsi3-⌬N complex with a molar ratio of 2:1 with 10 M CaCl 2 in solvent, but only 1:1 without 10 M CaCl 2 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 A 600 absorption. In contrast, EDTAtreated Tse3 (Ca 2ϩ free) lost this ability, but the addition of excess CaCl 2 could rescue its cell lysis activity (Fig. 3C).

DISCUSSION
The structures of two T6SS toxins (Tse1 and Tae4) were recently reported. Both belong to the cysteine peptidase NlpC/ P60 superfamily (16,18,19,21,22), whereas Tse3 contains an ARM/HEAT-like N-terminal domain and a goose-type lysozyme-like C-terminal catalytic domain (Fig. 1A). In the Tse3-Tsi3-⌬N complex structure described here, the interactions between the N-and C-terminal domains of Tse3 are mainly mediated by the ␣7 helix and Lobe2 (Fig. 1, B-E). The significant differences 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 Ca 2ϩ in their active site as a catalytic factor (40,41), Ca 2ϩ 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)(36)(37). Based on the high-resolution structure and biochemical studies presented here, we identified three Ca 2ϩ ions that bind with Tse3; two Ca 2ϩ ions bind directly in the catalytic site and adjacent to the catalytic residue Glu-250 (Figs. 2C and 3A), and the third Ca 2ϩ ion binds in the loop between helixes ␣21 and ␣22. Structural superimpositions with homologues indicate that the structural features of Lobe2 in Tse3 mainly contribute to this unique Ca 2ϩ binding ability. Enzymatic studies also show that Tse3 requires Ca 2ϩ 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-⌬N could vary from 2:1 to 1:1 (data not shown), strongly suggesting that the binding of Ca 2ϩ ions to Tse3 is weak and dynamic. Although the Ca 2ϩ 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.