Crystal structure of the Agrobacterium tumefaciens type VI effector–immunity complex

The type VI secretion system (T6SS) comprises needle-shaped multisubunit complexes that play a role in the microbial defense systems of Gram-negative bacteria. Some Gram-negative bacteria harboring a T6SS deliver toxic effector proteins into the cytoplasm or periplasm of competing bacteria in order to lyse and kill them. To avoid self-cell disruption, these bacteria have cognate immunity proteins that inhibit their toxic effector proteins. T6SS amidase effector protein 4 (Tae4) and T6SS amidase immunity protein 4 (Tai4) are a representative of the toxic effector–immunity pairs of the T6SS. Here, the three-dimensional structures of Tai4 and the Tae4–Tai4 complex from Agrobacterium tumefaciens are reported at 1.55 and 1.9 Å resolution, respectively. A structural comparison with other Tae4–Tai4 homologs revealed similarities and differences in the catalytic and inhibitory mechanisms among the Tae4 and Tai4 family proteins.

1. Introduction

The type VI secretion systems (T6SSs) of Gram-negative bacteria inject various toxic effectors into the periplasmic or cytoplasmic space of the target cells and induce cell lysis of enemy cells (Hood et al., 2010; MacIntyre et al., 2010; Schwarz et al., 2010; Murdoch et al., 2011; Russell et al., 2011, 2012). The various T6SS-related amidase effector proteins (Taes) are classified into four families (Tae1, Tae2, Tae3 and Tae4) based on their cleavage specificities (Russell et al., 2012). These effectors and the unique bacterial secretion system, T6SS, which responds to enemy bacteria, enable Gram-negative bacteria to attack targeted heterologous cells (Russell et al., 2012). In addition to these toxic effectors, Gram-negative bacteria have four amidase immunity proteins (Tai1, Tai2, Tai3 and Tai4). Tai1, Tai2, Tai3 and Tai4 neutralize the endogenous toxic effectors Tae1, Tae2, Tae3 and Tae4, respectively. These effector–immunity pairs (Tae1–Tai1, Tae2–Tai2, Tae3–Tai3 and Tae4–Tai4) generally originate from the same operons. The presence of cognate effector–immunity pairs suggests that self-protection systems with the co-expression of effector proteins and immunity proteins are a common feature in Gram-negative bacteria possessing a T6SS (Russell et al., 2012).

Tae4–Tai4 is the fourth T6SS-related effector–immunity pair to be structurally determined. Previous studies reported the structures of the Tae4–Tai4 complexes from Enterobacter cloacae, Salmonella typhimurium and Serratia marcescens (Zhang, Gao et al., 2013; Zhang, Zhang et al., 2013; Benz et al., 2013; Srikannathasan et al., 2013). Comparisons of these structures revealed that S. marcescens Tai4 is structurally different from E. cloacae Tai4 and S. typhimurium Tai4, whereas the Tae4 proteins from the different species are highly conserved (Srikannathasan et al., 2013). However, the crystal structures of Tae4–Tai4 complexes from other species have remained unknown.

To gain insight into the Tae4 family proteins, we determined the crystal structures of Tai4 and of the Tae4–Tai4 complex from Agrobacterium tumefaciens at 1.55 and 1.9 Å resolution, respectively.

2. Materials and methods

2.1. Macromolecule production

The genes encoding the Tai4 and Tae4 proteins from A. tumefaciens (ATU4346 and ATU4347, respectively) were codon-optimized for Escherichia coli and synthesized by Invitrogen. The SignalP 4.1 server (Petersen et al., 2011) was used to predict the signal peptide of A. tumefaciens Tai4 (AtTai4). The AtTai4 gene segment (residues 26–129) without the putative signal sequence was PCR-amplified and inserted into the pCold-GST vector. The plasmid was transformed into E. coli Rosetta 2 (DE3) cells for protein expression.

The cells were grown in Luria–Bertani (LB) medium at 310 K until the OD₆₀₀ reached 0.8; gene expression was then induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) following a reduction in the temperature to 277 K. Cell growth was continued for 24 h at 288 K. The N-terminally His₆-GST-tagged AtTai4 was affinity-purified using an Ni–NTA column (Qiagen). The N-terminal His₆-GST tag was removed by incubation with Turbo3C protease (Nacalai Tesque) for 16 h at 277 K. After rechromatography on the Ni–NTA column, further purification was conducted by ion-exchange chromatography on a Resource Q column (GE Healthcare) and gel-filtration chromatography on a HiLoad Superdex 75 column (GE Healthcare). The purified samples were concentrated to 8.6 mg ml⁻¹ for crystallization.

For co-expression of the AtTae4–AtTai4 complex, the AtTai4 gene segment (residues 26–129) was cloned into the first multiple cloning site of the pETDuet-1 vector (Novagen) and the AtTae4 gene segment (residues 1–163) was subsequently cloned into the second multiple cloning site. A Tobacco etch virus (TEV) protease-recognition sequence was introduced between the His₆ tag and the AtTai4 sequence by a PCR-based method. The plasmid was transformed into E. coli Rosetta 2 (DE3) cells for overexpression. The cells were cultured in LB medium at 310 K until the OD₆₀₀ reached 0.8; gene expression was then induced with 0.5 mM IPTG following a temperature reduction to 277 K. The cells were further cultured at 291 K for 24 h. The AtTae4–AtTai4 complex was affinity-purified using an Ni–NTA column (Qiagen). The N-terminal His₆ tag was removed by incubation with TEV protease for 24 h at 277 K. After rechromatography on the Ni–NTA column, the complex was further purified by ion-exchange chromatography on a Resource Q column and subsequent gel-filtration chromatography on a HiLoad Superdex 75 column. The purified complex was concentrated to 13 mg ml⁻¹ for crystallization trials.

2.2. Crystallization

Initial crystallization trials were performed at 293 K by the sitting-drop vapor-diffusion method in a 96-well crystallization plate using various commercially available screening kits. Crystallization drops were prepared by mixing 200 nl purified protein solution and 200 nl reservoir solution using a Mosquito crystallization robot (TTP Labtech). The initial crystals of AtTai4 were optimized at 293 K by varying the concentrations of PEG and salt in the reservoir solution using an Additive Screen kit (Hampton Research). Plate-shaped crystals of AtTai4 were obtained in 33% PEG 6000, 1.5 M lithium chloride, 100 mM sodium acetate. The AtTae4–AtTai4 complex formed thick plate-shaped crystals using MemGold reservoir condition E11 consisting of 35% PEG 400, 0.05 M Tris pH 8.5, 0.05 M sodium sulfate, 0.05 M lithium sulfate.

2.3. Data collection and processing

All crystals were cryoprotected in reservoir solution supplemented with 25% ethylene glycol and flash-cooled in a nitrogen-gas stream. X-ray diffraction data for AtTai4 and for the AtTae4–AtTai4 complex were collected on beamlines BL41XU and BL32XU at SPring-8, Hyogo, Japan using a PILATUS3 6M detector (Dectris) and an MX225HS detector (Rayonix), respectively. The continuous helical data-collection scheme was applied using 12 × 8 µm (AtTai4) and 18 × 1 µm (AtTae4-AtTai4 complex) beams. Diffraction data were integrated with DIALS (Waterman et al., 2016) and scaled with AIMLESS (Evans & Murshudov, 2013). The data-collection statistics are shown in Table 1.

Table 1
Data-collection and refinement statistics

Values in parentheses are for the outer shell.

	AtTai4	AtTae4–AtTai4 complex
Data collection
Beamline	BL41XU, SPring-8	BL32XU, SPring-8
Wavelength (Å)	1.0000	1.0000
Crystal-to-detector distance (mm)	300	200
Rotation range per image (°)	0.5	0.5
Exposure time per image (s)	0.5	1.0
Oscillation range per crystal (°)	180	180
Helical translation step (µm)	0.5	0.3
No. of crystals	1	1
Space group	P212121	P61
Unit-cell parameters (Å)	a = 53.92, b = 57.76, c = 71.47	a = b = 72.03, c = 194.35
Resolution (Å)	53.92–1.55 (1.58–1.55)	97.18–1.90 (1.94–1.90)
Rp.i.m.	0.029 (0.313)	0.022 (0.544)
〈I/σ(I)〉	13.0 (2.3)	15.0 (1.3)
Completeness (%)	99.5 (94.7)	99.6 (96.3)
Multiplicity	6.2 (4.2)	10.1 (7.3)
CC1/2	0.998 (0.807)	0.999 (0.528)
Mosaicity (°)	0.12	0.24
Refinement
Resolution (Å)	53.92–1.55	97.18–1.90
No. of reflections	33087	44759
Rwork/Rfree	0.1742/0.1975	0.1903/0.2125
No. of atoms
Protein	1546	4050
Ligand	28	21
Solvent	141	122
Average B factors (Å^2)
Protein	26.9	51.5
Ligand	48.4	81.2
Solvent	35.2	51.3
R.m.s. deviations
Bond lengths (Å)	0.013	0.0089
Bond angles (°)	1.79	1.57
Ramachandran plot
Favored (%)	97.45	96.91
Allowed (%)	2.55	2.90
Outliers (%)	0	0.19

2.4. Structure determination

The structures of AtTai4 and the AtTae4–AtTai4 complex were solved by molecular replacement with MOLREP (Vagin & Teplyakov, 2010) using the structures of Tai4 from S. marcescens (PDB entry 3zfi; Srikannathasan et al., 2013) and the Tae4–Tai4 complex from S. marcescens (PDB entry 4bi8; Srikannathasan et al., 2013), respectively, as search models. Model building and structure refinement were performed using Coot (Emsley et al., 2010) and REFMAC5 (Murshudov et al., 2011), respectively. Ramachandran plot analysis was performed using MolProbity (Chen et al., 2010). The refinement statistics are shown in Table 1. The atomic coordinates and structure factors of AtTai4 and the AtTae4–AtTai4 complex have been deposited in the Protein Data Bank (PDB) with accession codes 6ije and 6ijf, respectively. X-ray diffraction images have been also deposited in the Zenodo data repository (https://doi.org/10.5281/zenodo.1453302).

3. Results and discussion

3.1. Overall structure

The crystal structure of AtTai4 was determined at 1.55 Å resolution. AtTai4 forms a homodimer composed of five α-helices (α1–α5; Fig. 1a). The α2 helix (residues 52–74) contributes to dimer formation in the asymmetric unit, which is consistent with the size-exclusion chromatography results indicating that AtTai4 exists as a dimer in solution. A disulfide bond is formed between Cys80 and Cys124 in each protomer (Fig. 1a). In addition, we determined the crystal structure of the AtTae4–AtTai4 complex at 1.9 Å resolution (Fig. 1b). The structure revealed that the AtTai4 dimer binds two AtTae4 molecules to form a heterotetramer in the asymmetric unit, which is consistent with the size-exclusion chromatography results indicating that the AtTae4–AtTai4 complex exists in a heterotetrameric form in solution. The crystal structure revealed that AtTae4 forms an intramolecular disulfide bond between Cys144 and Cys148, which may confer structural stability (Fig. 1b). Superimposition of AtTai4 alone and AtTai4 bound to AtTae4 resulted in a root-mean-square deviation (r.m.s.d.) value of 0.8 Å, indicating that no structural changes occur upon complex formation.

Figure 1 Crystal structures of the AtTai4 homodimer and the AtTae4–AtTai4 complex. (a) Structural overview of the AtTai4 homodimer. The intramolecular disulfide bonds between Cys80 and Cys124 are shown as yellow spheres. (b) Overall structure of the AtTae4–AtTai4 complex. The intramolecular disulfide bonds between Cys144 and Cys148 are shown as yellow spheres.

3.2. Structure comparison

A search for structural homologs was conducted using the DALI server (Holm & Laakso, 2016). The top-scoring structural homolog of AtTai4 was the Rap1a protein from S. marcescens (SmTai4; PDB entry 3zfi; Srikannathasan et al., 2013), with a Z-score of 13.5 and an r.m.s.d. of 1.7 Å. The structural homologs of AtTae4 are the following proteins: the Ssp1 protein from S. marcescens (SmTae4; PDB entry 4bi3; Srikannathasan et al., 2013), the Tae4 protein from E. cloacae (EcTae4; PDB entry 4hfk; Zhang, Zhang et al., 2013) and the Tae4 protein from S. typhimurium (StTae4; PDB entry 4j30; Benz et al., 2013). The most similar structural homolog was the SmTae4 protein, with a Z-score of 25.4 and an r.m.s.d. of 1.6 Å. Amino-acid sequence alignments of AtTai4 and AtTae4 with their homologs are shown in Figs. 2(a) and 2(b). AtTai4 shares 32.3% amino-acid sequence identity with SmTai4. AtTae4 shares 41.5%, 21.9% and 20.5% sequence identity with SmTae4, StTae4 and EcTae4, respectively. Therefore, the AtTae4–AtTai4 complex structure deepens our understanding of the structurally distinct interactions of the Tae4 and Tai4 family proteins.

Figure 2 Structure-based sequence alignments of AtTai4 and AtTae4 with their homologs performed with Clustal Omega and ESPript3. (a) Sequence alignment of AtTai4 with SmTai4 (PDB entry 3zfi; Srikannathasan et al., 2013). (b) Sequence alignment of AtTae4 with SmTae4 (PDB entry 4bi3; Srikannathasan et al., 2013), EcTae4 (PDB entry 4hfk; Zhang, Zhang et al., 2013) and StTae4 (PDB entry 4j30; Benz et al., 2013). The potential catalytic triad residues, Cys47, His131 and Asp133, are indicated by blue triangles.

A comparison of AtTai4 with SmTai4 revealed that AtTai4 contains a longer α2 helix and a longer loop between the α1 and α2 helices (Fig. 3a). As the longer α2 helix and loop between the α1 and α2 helices interact with two AtTae4 molecules in the asymmetric unit (Fig. 1b), AtTae4 and AtTai4 have structurally distinct interactions compared with the SmTae4–SmTai4 complex. In addition, neither the StTae4–StTai4 complex nor the EcTae4–EcTai4 complex has these interactions. Glu53 and Arg56 in the α2 helix form hydrogen bonds to Gln143 and Ser18 in one of the AtTae4 molecules in the asymmetric unit, respectively (Fig. 3b). Pro47, Asp48 and Val49 in the loop of AtTai4 form hydrogen bonds to Arg108, Thr142 and Arg108 in the other AtTae4 protomer in the asymmetric unit, respectively. In addition, Ser50 in the loop of AtTai4 interacts with Ser139 and Glu140 in AtTae4 (Fig. 3b). Thus, these specific interactions between AtTae4 and AtTai4 may contribute towards stabilizing the formation of the AtTae4–AtTai4 complex.

Figure 3 Structural comparison of the AtTae4–AtTai4 complex with the SmTae4–SmTai4 complex. (a) A superimposition of AtTai4 and SmTai4 indicated that AtTai4 contains extensions in the α2 helix and in the loop between the α1 and α2 helices. (b) The residues involved in the interaction between AtTai4 and AtTae4. (c) The crystal structure of the SmTae4–SmTai4 complex revealed that Gln84 of SmTai4 interacts with His133 of SmTae4 and blocks the active site (PDB entry 4bi8; Srikannathasan et al., 2013). (d) The crystal structure of the AtTae4–AtTai4 complex lacks the interaction between the expected catalytic His131 of AtTae4 and the corresponding residue of AtTai4. The glutamine is not conserved in AtTai4 and is replaced by an alanine in AtTai4. (e) The AtTai4 homodimer is positioned close to the AtTae4 active-site surface and may block substrate binding.

The structure of the SmTae4–SmTai4 complex revealed that SmTai4 is located at the entrance to the active site of SmTae4, where it blocks substrate access to the active site (Srikannathasan et al., 2013). The catalytic Gln84 of SmTai4 forms a hydrogen bond to His133 of SmTae4 and blocks the active site (Fig. 3c; Srikannathasan et al., 2013). Amino-acid sequence alignment of SmTai4 and AtTai4 demonstrated that Gln84 of SmTai4 is not conserved and is replaced by Ala86 in AtTai4 (Fig. 2a). In the present structure of the AtTae4–AtTai4 complex, although the potentially catalytic His131 residue of AtTae4 does not interact with any residues of AtTai4 (Fig. 3d), AtTai4 blocks the entrance to the substrate-binding pocket of AtTae4 and prevents substrate access to the active site (Fig. 3e).

Previous studies have suggested that AtTai4 and SmTai4 neutralize the activities of AtTae4 and SmTae4, respectively. The morphological abnormality mediated by SmTae4 was neutralized by SmTai4 (Srikannathasan et al., 2013). The growth inhibition of E. coli DH10B owing to the expression of AtTae4 was rescued by the co-expression of AtTai4 (Ma et al., 2014). While there is no direct interaction between the potentially catalytic His131 of AtTae4 and Ala86 of AtTai4, which corresponds to Gln84 of SmTai4, the structural comparison suggests that AtTai4 effectively neutralizes the activity of AtTae4 by blocking the entrance to its substrate-binding pocket.

3.3. Catalytic site

The Tae4 family proteins have conserved catalytic residues (Cys–His–Asp) that are responsible for their peptidoglycan amidase activity. Cys46, His128 and Asp139 of EcTae4 and Cys44, His126 and Asp137 of StTae4 form the catalytic triads, which are similar to the canonical catalytic triad in the papain-like cysteine peptidase (PDB entry 1bp4; LaLonde et al., 1998) (Zhang, Gao et al., 2013; Zhang, Zhang et al., 2013; Benz et al., 2013; Fig. 4a). SmTae4 also has a catalytic triad formed by Cys50, His133 and Asp135. While Asp139 of EcTae4 and Asp137 of StTae4 are replaced by Ser148 in SmTae4, Asp135 of SmTae4, which corresponds to Thr130 of EcTae4 and Thr128 of StTae4, is located at a position similar to those of Asp139 of EcTae4 and Asp137 of StTae4 in the SmTae4 structure (Figs. 4a and 4b). These observations suggested that Asp135 serves as the third aspartic acid residue in the catalytic triad in SmTae4 (Srikannathasan et al., 2013). In the present structure of the AtTae4–AtTai4 complex, Cys47, His131 and Asp133 also form a catalytic triad, as in the SmTae4–SmTai4 complex structure (Fig. 4b). Thus, the present structure re­inforces the idea that the Tae4 family proteins have two types of structurally distinct catalytic triads.

Figure 4 Structural differences in the catalytic triad. (a) EcTae4 and StTae4 have a conserved catalytic active center containing the catalytic residues (Cys–His–Asp), which have a similar arrangement to the catalytic triad of papain (PDB entry 1bp4; LaLonde et al., 1998). (b) SmTae4 and AtTae4 have the conserved catalytic residues (Cys–His–Asp), but the third aspartic acid in the catalytic triad has a distinct spatial arrangement.

4. Conclusion

In this work, we determined the crystal structures of AtTai4 and the AtTae4–AtTai4 complex. Comparisons of these structures with those of homologous proteins revealed that the AtTae4–AtTai4 complex shares structural similarity with the SmTae4–SmTai4 complex. A structural comparison of AtTai4 with SmTai4 showed that AtTai4 contains more extended helices and loops, which may enforce the interaction between AtTai4 and the adjacent AtTae4. A structural superimposition highlighted the differences in the spatial arrangement of the aspartic acid residue in the catalytic triad (Cys–His–Asp) among the Tae4 family proteins. The present structures enhance our understanding of the catalytic and inhibitory mechanisms of the Tae4 and Tai4 family proteins.