The Structure of the Toxin and Type Six Secretion System Substrate Tse2 in Complex with Its Immunity Protein

Short Article

Highlights Authors d Tse2 from P. aeruginosa has structural features similar to Craig S. Robb, Melissa Robb, ADP-ribosylating toxins Francis E. Nano, Alisdair B. Boraston d The active-site of the Tse2 toxin is consistent with an NAD- Correspondence dependent activity [email protected] d Tse2 forms an inactivated heterotetrameric complex with the Tsi2 anti-toxin In Brief Tse2 is a Pseudomonas aeruginosa type 6 secretion system substrate and cytoactive toxin whose structural features suggest a relationship with ADP- ribosylating toxins and an active-site arrangement consistent with NAD- dependent activity, providing the ﬁrst insight into the molecular basis of Tse2 toxicity.

Accession Numbers 5AKO

The Structure of the Toxin and Type Six Secretion System Substrate Tse2 in Complex with Its Immunity Protein

Craig S. Robb,1 Melissa Robb,1 Francis E. Nano,1 and Alisdair B. Boraston1,* 1Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC V8W 3P6, Canada *Correspondence: [email protected] http://dx.doi.org/10.1016/j.str.2015.11.012

SUMMARY putative needle tip complex made of VgrG and PAAR-repeat proteins or the ring-shaped needle body protein Hcp (Shneider Tse2 is a cytoactive toxin secreted by a type six et al., 2013; Silverman et al., 2013). secretion apparatus of Pseudomonas aeruginosa. Pseudomonas aeruginosa, an opportunistic pathogen, is a The Tse2 toxin naturally attacks a target in the cyto- model organism for the study of the T6SS and its effectors. plasm of bacterial cells, and can cause toxicity if ar- The first three discovered secreted substrates from the H1- tificially introduced into eukaryotic cells. The X-ray T6SS from P. aeruginosa are translocated into competing bacte- crystal structure of the complex of Tse2 and its rial cells, resulting in death of the recipients (Hood et al., 2010; Russell et al., 2011). Each of these toxins has an associated im- cognate immunity protein Tsi2 revealed a heterote- munity protein, termed type six immunity (referred to as Tsi 1 trameric structure with an extensive binding inter- through 3), which protects P. aeruginosa from self-intoxication face. Structural identity was found between Tse2 or sister cell intoxication (Russell et al., 2011, 2014). Type six and NAD-dependent enzymes, especially ADP-ribo- effectors Tse 1 and 3 are hydrolases that degrade the peptido- sylating toxins, which facilitated the identification of glycan layer of neighboring bacteria (Russell et al., 2011). Ectop- the Tse2 active site and revealed it to be occluded ically expressed Tse2 has been shown to be toxic to yeast and upon binding the inhibitor Tsi2. The structural mammalian cells, but the toxic effect of Tse2 directly from identity shared with NAD-dependent enzymes, P. aeruginosa has only been observed against other bacteria including conserved catalytic residues, suggests (Hood et al., 2010). The toxic activity of Tse2 is directed toward that the mechanism of Tse2 toxicity may be NAD the cytoplasmic compartment of targeted bacterial cells (Li et al., dependent. 2012), indicating that the toxin has to cross the two membranes of both the attacking and targeted cell. Tse2 shares no sequence identity with any protein of known function and, although the structure of its immunity protein is INTRODUCTION known, the structure and molecular mechanism of Tsi2 inhibition of Tse2 are unknown. Here, the X-ray crystal structure of Gram-negative bacteria employ type six secretion systems the complex of Tse2 and Tsi2 reveals the molecular me- (T6SS) to transfer effector molecules, often toxins, to adjacent chanism of inhibition. The structure of Tse2 also led to the dis- bacteria and host cells (Ho et al., 2014). Many of the character- covery of unique structural features that differentiate this ized T6SS toxins target competing bacteria, although there are cytoactive toxin from the other structurally characterized toxins also examples of eukaryotic targeting (Hood et al., 2010; Jiang that are active in the periplasm. Structural identity with bacte- et al., 2014). Gene clusters encoding T6SSs are found widely rial ADP-ribosyltransferase toxins, which informed structure- in the genomes of both pathogenic and environmental Gram- guided mutagenesis, provided critical insight into the active negative bacteria (Boyer et al., 2009). Secretion systems that site of Tse2. are involved in targeting bacteria are primarily involved in maintaining competitive fitness in polymicrobial communities, but may also function as signaling systems (Russell et al., RESULTS 2014). The T6SS is analogous to a contractile phage-tail complex that Structure of the Tse2/Tsi2 Complex is attached to the bacterial inner membrane and injects effectors Co-expression of the P. aeruginosa tse2 and tsi2 genes in into adjacent cells (Basler and Mekalanos, 2012; Leiman et al., Escherichia coli allowed the production and purification of 2009). Secretion is thought to be driven by a two-component the protein complex, the structure of which was determined contractile tube of TssB and TssC that provides energy to force to a resolution of 2.4 A˚ by molecular replacement using the the needle tip, needle body, and associated effectors through structure of Tsi2 as a search model (Table 1). The asymmetric the membrane complex of the donor cell and into the target unit contained a single heterotetramer comprising two mole- cell (Basler et al., 2012; Clemens et al., 2015). Secretion of an cules each of Tse2 and Tsi2 (Figure 1A). At the core of the effector molecule is dependent on an interaction with either the tetramer is a dimer of Tsi2 (Figure 1A), with the two identical

Structure 24, 277–284, February 2, 2016 ª2016 Elsevier Ltd All rights reserved 277 Table 1. Data Collection and Structural Reﬁnement Statistics identical sets of two interfaces mediating the interaction of the two Tse2 molecules with the Tsi2 dimer. The Tsi2 dimer interface PaTse2Tsi2 is 818 A˚ 2, which is roughly 15% of the total Tsi2 monomer Data Collection surface area, and is predicted to have a binding energy of Beamline SSRL 12-2 19.3 kcal/mol. The interface of the Tsi2 dimer with a Tse2 Wavelength (A˚ ) 1.000 monomer displays charge complementarity (Figure 1B) and Temperature (K) 100 this binding surface, which involves both Tsi2 molecules, can Resolution (A˚ )a 38.34–2.40 (2.53–2.40) be divided into two regions whereby each monomer of the Tsi2

Space group P212121 dimer has an interface with the one Tse2 molecule (Figure 1C). Unit cell dimensions The larger interface, which we refer to as the primary interface, occurs between the b sheet of Tse2 and a helix 2 of Tsi2 (i.e. be- a, b, c (A˚ ) 37.72, 103.14, 114.64 tween chain C of Tse2 and chain B of Tsi2, and between chain D R 0.089 (0.732) merge of Tse2 and chain A of Tsi2). This interface comprises 658 A˚ 2 of s I/ I 13.4 (2.3) surface area (12% of the total Tse2 surface area) and is pre- Completeness (%) 97.6 (99.1) dicted to have a binding energy of 11.2 kcal/mol. Consistent Redundancy 4.0 (4.2) with this large predicted binding energy is the formation of five Refinement hydrogen bonds as well as salt bridges between three residues Resolution (A˚ ) 35.83–2.40 on the basic surface of Tse2 (Arg-89, Arg-90, and Arg-119) and No. of reflections 16,778 three on the acidic surface of Tsi2 (Glu-38, Asp-45, and Asp- 49) (Figure 1D). Indeed, supporting the importance of the ionic Model contents A: Tsi2 (2–75) B: Tsi2 (1–75) component of this interface, Li et al. mutated Asp-45 and Glu- C: Tse2 (3–158, D38–40) 38 in Tsi2, which pair with Arg-119 and Arg-90, respectively, in D: Tse2 (3–158, D38–40) Tse2, and this abrogated binding of Tsi2 and Tse2 in vitro and eliminated the ability of Tsi2 to detoxify Tse2 in cell-based Rwork/Rfree 0.20/0.25 assays (Li et al., 2012). No. of atoms The secondary interface is formed by the extended loop of Protein 3,437 Tse2 enveloping the N and C termini of the second Tsi2 mole- Water 74 cule in the Tsi2 dimer (i.e. between chain C of Tse2 and chain B factors A of Tsi2, and between chain D of Tse2 and chain B of Tsi2). Overall 41.9 This interface comprises 616 A˚ 2 ofsurfacearea(11% of Protein 43.5 the total Tse2 surface area) with 14 hydrogen bonds, and is Water 37.6 predicted to have a binding energy of 8.7 kcal/mol (Fig- Rmsd ure 1E). Thus, the total interface between a single Tse2 molecule and the Tsi2 dimer is substantial at 1,274 A˚ 2 (24% of the Bond lengths (A˚ ) 0.015 total Tse2 surface area is buried at the interface) with a large Bond angles () 1.568 number of specific intermolecular interactions (i.e. hydrogen Ramachandran plot (%) bonds and salt bridges), all of which is consistent with the ho- Most favored 97 motetramer observed in the asymmetric unit being an explicit Outliers 0.93 biological assembly. Furthermore, the complete heterote- PDB accession code 5AKO tramer as it occurs in the asymmetric unit is predicted to be aValues in parentheses correspond to the highest-resolution shell (2.53– very stable with a Gibbs free energy of dissociation of 2.40 A˚ ). 23 kcal/mol, which again supports a multimeric structure that is specific and not an assembly that is driven by the formation of a crystal lattice. surfaces on either side of the Tsi2 dimer forming binding sites for Tse2 monomers, resulting in a heterotetrameric complex The Structure of Tse2 having C2 symmetry. Comparison of the Tsi2 dimer in the com- The structure of Tse2 from the complex comprises an anti-paral- plex with existing structures of Tsi2 (PDB: 3STQ, 3RQ9, and lel five-stranded b sheet with strands in the order of 3-1-2-5-4 3VPV) gives a root-mean-square deviation (rmsd) of 0.98– that is packed against a single a helix at the C terminus (Figure 2). 1.27 A˚ (Li et al., 2012; Zou et al., 2012), implying that Tsi2 Significant extended loops project out from the structure and are does not undergo any significant conformational changes stabilized by interactions with the immunity protein in the com- upon binding to Tse2. plex structure. In particular, the loop between strands 1 and 2

The symmetry and compact nature of the heterotetrameric also includes a pair of 310 helices and accounts for a full third complex making up the contents of the asymmetric unit sug- of the polypeptide. This somewhat sprawling conformation suggests that this is a natural and stable multimer. To provide addi- gests that Tse2 has regions with high ﬂexibility, which is in tional support for this, we performed a more detailed analysis of contrast to the structures other T6SS effectors adopt in compact the interfaces making up the complex using PISA (Krissinel and globular folds (Benz et al., 2012; Dong et al., 2013; Srikannatha- Henrick, 2007). Overall, ﬁve interfaces are involved in forming the san et al., 2013; Wang et al., 2013; Whitney et al., 2013; Zhang heterotetramer: one forming the dimer interface for Tsi2 and two et al., 2013).

278 Structure 24, 277–284, February 2, 2016 ª2016 Elsevier Ltd All rights reserved Figure 1. The Structure of Tse2 and Tsi2 Complex (A) Cartoon representation of the Tse2/Tsi2 heterotetramer looking down the two-fold axis of symmetry. The dimer of immunity protein molecules (Tsi2, green/cyan) is able to bind two toxin molecules (Tse2, purple). The dimensions of the heterotetramer are indicated. (B) A heterotrimer portion of the Tse2/Tsi2 complex (top; Tse2, purple; Tsi2, green and cyan) with the interface of this dimer opened along the vertical plane (shown by the dashed line) to expose the interacting surfaces of Tsi2 and Tse2 (bottom left and right, respectively). The surfaces are colored by electrostatic potential. (C) The hydrogen bonding network of the two interfaces highlighting the contributing residues. (D) Details of the primary interface with coloring as in (A). (E) Details of the secondary interface with coloring as in (A).

Arg, Ser-Thr-Ser, and Glu-X-Glu while the diphtheria toxin group has consensus

His, Tyr-X10-Tyr, and Glu. The diphtheria toxin itself was the most similar protein structure to Tse2 while the most structurally similar ADPRT from the cholera toxin group was HopU (PDB: 3UOJ), a T3SS effector from Pseudomonas syringae (Z score 2.7, rmsd of 3.5 A˚ over 69 matched Ca atoms, and 14% amino acid sequence identity) (Jeong et al., 2011). The structural motif shared between Tse2 and ADP-ribosyltransferases is the characteristic b sheet of this family of toxins (Figure 3A). This structural element houses the catalytic machinery of these toxins while the regions sur- We compared the structure of Tse2 with existing structures in rounding this structural core differ between the toxins, contrib- the databases by a Dali structure similarity search (Holm and uting to wide sequence variation among ADPRTs (Fieldhouse Sander, 1998). The structures returned as most similar were and Merrill, 2008). In addition to sharing the b sheet, three puta- nicotinamide adenine dinucleotide (NAD)-dependent enzymes tive catalytic residues were also structurally conserved between from bacteria, notably the mono-ADP-ribosyltransferase toxins the cholera toxin group of ADPRTs and Tse2 (Figures 3A and 3B). (ADPRTs). The most similar protein structure found was the diph- The residues Arg-14 and Ser-80 from Tse2 are structurally equiv- theria toxin (PDB: 1SGK) with a Z score of 4.3, an rmsd of 3.8 A˚ alent to Arg-128 and Ser-174 from C3 from Clostridium over 91 matched Ca atoms, and 10% amino acid sequence iden- botulinum. However, one catalytic residue shared between the tity (Bell and Eisenberg, 1997). The structural identity between two main groups of ADPRTs is a glutamate (Glu-214 in C3) that the diphtheria toxin (535 amino acids) and Tse2 (155 amino in Tse2 is replaced in the structure by His-122 (Figure 3C). The acids) was primarily limited to the ﬁve-stranded b sheet at the protein ranked second most similar to Tse2 was an NAD-depen- core of both folds (see below). The lack of greater structural iden- dent tRNA phosphotransferase (Z score 3.6, rmsd of 3.4 A˚ for tity with other available structures indicates that the remaining 73 matched Ca atoms, and 12% amino acid sequence arrangement of loops and the a helix in Tse2 creates an overall identity) (Kato-Murayama et al., 2005). The reaction catalyzed fold that is presently unique to the structural family of toxins to by this enzyme results in the overall transfer of phosphate which Tse2 belongs. from the nascent tRNA to ADP-ribose with loss of the nicotin- ADPRTs, which transfer ADP-ribose to target molecules amide group from NAD. This family of proteins is involved in thereby inactivating them, are most commonly divided into two the last step of tRNA maturation and is thought to bind NAD in groups based on different subsets of catalytic residues: the a manner analogous to the diphtheria toxin. An alignment of cholera toxin and the diphtheria toxin groups (Holbourn et al., Tse2, the two groups of ADPRTs, and the tRNA phospho- 2006). The cholera toxin group has the consensus sequence transferase reveals partial active-site conservation between the

Structure 24, 277–284, February 2, 2016 ª2016 Elsevier Ltd All rights reserved 279 Figure 2. The Structure of Tse2 from the Complex The structure is shown as a cartoon colored blue to red from the N terminus to the C terminus. The secondary structure elements of Tse2 are labeled sequentially by type. The dimensions of the Tse2 monomer are indicated.

feature also acts as the support for the catalytic centers of these NAD-dependent enzymes, which is largely also conserved in Tse2. The abrogation of toxicity for the R14A, S80A, and H122A mutant Tse2 proteins indicates the importance of these amino acids in the mechanism of Tse2 toxicity and, given their conservation with NAD-reactive enzymes, also supports their assignment phosphotransferase, ADPRTs, and Tse2 (Figure 3C). In the tRNA as being involved in a catalytic reaction the we propose involves phosphotransferase the conserved ADPRT glutamate is re- NAD. Asp-112 lies beside the putative catalytic His-122 and placed with a valine, whereas in Tse2 this position is occupied could contribute to catalysis, but appears to be dispensable by a histidine. for full toxicity of Tse2. Notably, when the amino acid sequence conservation among Tse2 homologs is mapped onto the surface Identification of a Putative Active Site of the Tse2 structure, the region containing the these functionally Our structural comparisons of Tse2 with NAD-dependent pro- important putative catalytic residues and, indeed, many of the teins predict that the set of residues in Tse2 (Arg-14, Ser-80, residues around the putative catalytic center are highly His-122) that are structurally conserved with the catalytic resi- conserved (Figure 3F). That these residues are conserved dues in the NAD-dependent enzymes have a role in the toxicity throughout the Tse2 family points to the general importance of of Tse2. To test this hypothesis, we performed structure-guided these amino acids and likely common function for these family mutagenesis to generate alanine substitution mutations for Arg- members. 14, Ser-80, and His-122. Due to its proximity to His-122, we also The deployment of such a highly toxic molecule requires that mutated Asp-112 (Figure 3A). We compared the growth of E. coli the bacterium protect itself from Tse2, which P. aeruginosa expressing the wild-type and mutant Tse2 genes, which were does using the Tsi2 anti-toxin. The large buried surface area of under isopropyl b-D-1-thiogalactopyranoside (IPTG)-inducible the Tse2/Tsi2 complex suggests a tight interaction between T7 RNA polymerase control, in the presence or absence of the the two components when forming the inhibited heterotetramer. inducer. E. coli expressing wild-type Tse2 was unable to grow Notably, the cleft housing the putative catalytic residues of Tse2 in the presence of IPTG, but in the absence of IPTG grew the sits directly between the primary and secondary Tse2/Tsi2 same as the control culture expressing Tsi2, which had the binding interfaces and is completely occluded upon binding same growth properties with and without the inducer (Figure 3D). the formation of the complex, providing a simple molecular-level The growth of the strains expressing Tse2 with the mutations explanation for the efficient means of Tsi2 inhibiting the effects of R14A, S80A, and H122A were the same as the uninduced Tse2 on the donor cell. Unlike the residues at the putative cata- wild-type and Tsi2 cultures, regardless of whether IPTG was lytic center, the majority of surface-exposed amino acids on added to cultures or not. Under the conditions to induce expres- Tse2 that are involved in binding Tsi2 shows are not conserved sion of the D112A mutant protein we observed a reduction of among the Tse2 family (compare Figures 3F and 3G). This im- growth, but not as profound a reduction as when native Tse2 is plies that the recognition of the toxin by anti-toxin is species expressed (Figure 3D). To demonstrate the level of expression dependent, in keeping with the concept that this is a specific of the Tse2 mutated proteins, we performed western blotting. form of self-recognition to prevent self-intoxication. The three Tse2 variants that showed a reversal of the growth The secreted chaperone Hcp is needed for the intracellular defect were produced in a soluble form indicating that they are stability of Tse2 in P. aeruginosa, and evidence indicates that it non-toxic, whereas Tse2 and Tse2 D112A were not detectable, is likely bound to the interior of the Hcp ring (Silverman et al., consistent with the nearly complete lack of growth of these 2013). The Tse2/Tsi2 heterotetramer is a lozenge shape that is strains (Figure 3E). 93 A˚ long and 41 A˚ in diameter (Figure 1A) and thus may thread lengthwise into the 46-A˚ diameter central cavity of the DISCUSSION Hcp hexameric ring. However, this possible mode of chaperoning the inactive Tse2/Tsi2 heterotetramer necessitates that Determination of the Tse2/Tsi2 complex structure showed Tse2 there be a mechanism for the segregation of Tse2 and Tsi2 dur- to possess a b-sheet platform that is structurally conserved with ing the secretion process so that active Tse2 may be delivered to ADPRTs and tRNA phosphotransferases. Notably, this structural the recipient cell. This is formally possible, but we find it unlikely

280 Structure 24, 277–284, February 2, 2016 ª2016 Elsevier Ltd All rights reserved Figure 3. Identiﬁcation of the Active Site of Tse2 (A) An overlay of Tse2 in green and C3 toxin from Clostridium botulinum is shown with the conserved catalytic residues highlighted (Me´ ne´ trey et al., 2002). (B) An overlay of the kinked b-sheet motif of Tse2 and C3 toxin with conserved residues and co-factor shown for reference. (C) A focused structure-based sequence alignment of structurally related proteins from the Dali server. Representative structures from the cholera toxin-type ADP-ribosyltransferases and Tse2 are highlighted in blue. The alignment includes structures of HopU from Pseudomonas syringae, pertussis toxin from Bordetella pertussis, and exoenzyme C3 from Clostridium limosum (Hazes et al., 1996; Tsuge et al., 2008; Vogelsgesang et al., 2008; Jeong et al., 2011). (legend continued on next page)

Structure 24, 277–284, February 2, 2016 ª2016 Elsevier Ltd All rights reserved 281 given the large amounts of energy that would likely be required to out in Phenix and resulted in a nearly complete model (Adams et al., 2010). dissociate the Tse2/Tsi2 heterotetramer. The heterotetrameric Rounds of restrained refinement in Refmac and manual building in Coot arrangement of the Tse2/Tsi2 toxin-anti-toxin complex is remi- were used to complete the model (Emsley et al., 2010; Murshudov et al., 2011). Structure validation was carried out using MolProbity, and Ramachan- niscent of the quaternary structure formed by two other T6SS dran statistics were taken from this analysis (Chen et al., 2010). The coordi- toxin-anti-toxin pairs (Yang et al., 2014; Zhang et al., 2013). In nates and structure factors have been deposited in the PDB (PDB: 5AKO). these cases, the high affinity of complex formation represented a dead end for the toxins. Thus, we favor the concept that that Mutagenesis and Toxicity Experiments the inhibited form of Tse2 found in the crystal structure repre- Mutagenesis of the putative active site was performed as previously described sents a secretion-incompetent form and that Hcp is responsible (Liu and Naismith, 2008). In brief, partially overlapping oligonucleotides were used to amplify by PCR the entire pET28-Tse2 plasmid, which was then trans- for chaperoning the Tse2 monomer. The length and diameter of a ˚ ˚ formed into competent E. coli DH5 (Table S1). The point mutations were the folded Tse2 monomer are 52 A and 41 A (Figure 2), confirmed by sequencing. respectively, and therefore of appropriate size to essentially fill E. coli BL21 (DE3) pLysS cells were transformed with either pET28-Tse2, the center of the Hcp ring, as was observed in electron micro- mutants of pET28-Tse2, or pET28-Tsi2. Starter cultures were grown for 6 hr scopy studies by Silverman et al. (2013). The structural features in LB medium at 37C with shaking before being diluted 25 times in 250 mlof of Tse2 are consistent with a mechanism of toxicity that is NAD medium in triplicate in a 96-well tray. The trays were incubated 37 C with pe- dependent and may provide ADP-ribosyltransferase activity, riodic shaking in the plate reader (5 s every 15 min). Measurements of absor- bance at 595 nm were collected every 15 min for 8 hr. Growth was measured although this remains to be conclusively determined. This poten- for each strain with or without 1 mM IPTG. Cells were resuspended in Laemmli tial activity, which parallels the cytotoxin effectors of the type III buffer and heated to 90C for 30 min. Samples were centrifuged and separated secretion system (Deng and Barbieri, 2008), distinguishes Tse2 by SDS-PAGE on a 15% polyacrylamide gel. Following electrophoresis, the from other T6SS effectors and, with an appropriately conserved samples were transferred to nitrocellulose at 30 V for 1 hr. The nitrocellulose target molecule, would explain the ability of Tse2 to act as a cyto- membrane was blocked using 5% milk in PBS with 1% Tween 20 (PBS-T) toxin toward both prokaryotic and eukaryotic cells. overnight at 16 C. The blocked membrane was probed with rabbit polyclonal immunoglobulin G against polyhistidine (Santa Cruz Biotechnology) in PBS-T with 1% milk for 1 hr at room temperature. After washing, a goat anti-rabbit EXPERIMENTAL PROCEDURES monoclonal with a fluorescent dye was applied in PBS-T with 1% milk for 1 hr at room temperature. The membrane was scanned at 700 and 800 nm Cloning, Production, Purification, Crystallization, and Structure to measure the fluorescence. Solving The genes for Tse2 and Tsi2 (PA2702, PA2703) were cloned into pET28 by ACCESSION NUMBERS amplifying the complete open reading frame of each gene (encoding residues 1–158 and 1–77) using genomic P. aeruginosa PAO1 DNA (ATCC 47085D-5) and appropriate primers (Table S1). These amplicons were digested with The coordinates and structure factors of the complex of Tse2 and Tsi2 from appropriate restriction enzymes and ligated into pET-28 or pET-22b making Pseudomonas aeruginosa reported in this paper have been deposited in the pET28-Tse2, pET22-Tse2, and pET28-Tsi2. For co-expression, Tse2 was re- PDB (PDB: 5AKO). amplified from pET28-Tse2 including the N-terminal His-tag and thrombin cleavage site, digested, and ligated into the NcoI-NotI site of pETDUET. This SUPPLEMENTAL INFORMATION vector was re-digested and Tsi2 was ligated into the second multiple cloning site at the NdeI-XhoI site to generate the complete vector pETDUET-Tse2Tsi2. Supplemental Information includes one figure and one table and can be found BL21 (DE3) harboring respective vectors were grown in LB medium supple- with this article online at http://dx.doi.org/10.1016/j.str.2015.11.012. mented with kanamycin or ampicillin. The cultures were grown at 37C for 6 hr and induced at 16C for 16 hr with 0.5 mM IPTG, cells harvested by centri- AUTHOR CONTRIBUTIONS fugation, and chemically lysed (Robb et al., 2012). Protein was purified from clarified by immobilized metal affinity chromatography, exchanged by dialysis Conceptualization: C.S.R. and A.B.B.; methodology: C.S.R. and A.B.B.; inves- into 20 mM Tris (pH 8.0) and 150 mM NaCl, and concentrated to 10 mg/ml for tigation: C.S.R. and M.C.; writing—original draft: C.S.R.; writing—review and crystallization. Crystals were obtained by the hanging-drop vapor diffusion editing: C.S.R., F.E.N., and A.B.B.; funding acquisition: A.B.B. and F.E.N.; su- method in 20 mM Tris (pH 8.5), 0.2 M NaCl, and 22% polyethylene glycol pervision: A.B.B. and F.E.N. 4000. Crystals were cryo-protected in crystallization solution supplemented with 25% ethylene glycol and placed directly in a cryo stream at 113 K. ACKNOWLEDGMENTS A diffraction dataset was collected at a wavelength of 1 A˚ . Data processing was carried out in XDS and SCALA from the CCP4 package in the space group This work was supported by a Canadian Institutes for Health Research (CIHR)

P212121. The structure was solved by molecular replacement searching for Operating Grant (FRN 89812). We thank the beamline staff at the Stanford Syn- two Tsi2 monomers (PDB: 3VPV) using Phaser-MR. Autobuilding was carried chrotron Research Laboratory BL12-2. A.B.B. thanks a Canada Research

Highlighted in purple, the diphtheria toxin, exotoxin A from Pseudomonas aeruginosa, and an NAD-dependent tRNA phosphotransferase are included for comparison (Bennett and Eisenberg, 1994; Kato-Murayama et al., 2005; Wedekind et al., 2001). The secondary structure assignments from (B) are shown above the alignment. The residues that line the putative catalytic site of Tse2 that were mutated to alanine are denoted with a star. Residues outlined in red are those known to be catalytic residues in the ADP-ribosylating toxins. (D) Growth curves of E. coli harboring plasmids that carry Tse2, Tse2 mutants, or Tsi2. The induced cultures are shown in blue compared with uninduced cultures in red for each strain. The dashed lines show the SD of triplicate measurements. WT, wild-type. (E) A western blot probing for histidine-tagged Tse2 from E. coli samples from the growth curves in (D). (F) Sequence conservation mapped onto the structure of Tse2. The colors of the residues are coded depending on conservation of a particular residue. The mutated residues from the toxicity experiments are highlighted. Sequence alignment used for this analysis is presented in Figure S1. (G) The structure of Tse2 with the surfaces forming the binding interface with Tsi2 colored green and cyan. The orientation of the protein is identical to that in (A). See also Figure S1.

282 Structure 24, 277–284, February 2, 2016 ª2016 Elsevier Ltd All rights reserved Chair in Molecular Interactions, an MSFHR Scholar Award, and an EWR Stea- Jiang, F., Waterfield, N.R., Yang, J.,Yang, G., and Jin, Q. (2014). A Pseudomonas cie Memorial Fellowship for support. aeruginosa type VI secretion phospholipase D effector targets both prokaryotic and eukaryotic cells. Cell Host Microbe 15, 600–610. Received: August 31, 2015 Kato-Murayama, M., Bessho, Y., Shirouzu, M., and Yokoyama, S. (2005). Revised: November 4, 2015 Crystal structure of the RNA 20-phosphotransferase from Aeropyrum pernix Accepted: November 23, 2015 K1. J. Mol. Biol. 348, 295–305. Published: December 31, 2015 Krissinel, E., and Henrick, K. (2007). Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797. REFERENCES Leiman, P.G., Basler, M., Ramagopal, U.A., Bonanno, J.B., Sauder, J.M., Pukatzki, S., Burley, S.K., Almo, S.C., and Mekalanos, J.J. (2009). 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