Crystal structure of the putative cytoplasmic protein STM0279 (Hcp2) from Salmonella typhimurium

The crystal structure of Hcp2 from S. typhimurium and its oligomeric state in solution are reported.

Abstract

STM0279 is a putative cytoplasmic protein from Salmonella typhimurium and was recently renamed haemolysin co-regulated protein 2 (Hcp2), with the neighbouring STM0276 being Hcp1. Both of them are encoded by the type VI secretion system (T6SS) of the Salmonella pathogenicity island 6 (SPI-6) locus and have high sequence identity. The Hcp proteins may function as a vital component of the T6SS nanotube and as a transporter and chaperone of diverse effectors from the bacterial T6SS. In this study, the crystal structure and the oligomeric state in solution of Hcp2 from S. typhimurium (StHcp2) were investigated. The crystal structure refined to 3.0 Å resolution showed that the protein is composed of a β-barrel domain with extended loops and can form hexameric rings as observed in known Hcp homologues. Mutation of the extended loop was found to partly destabilize the hexameric conformation into monomers or cause the production of inclusion bodies, suggesting it has an important role in hexameric ring formation.

1. Introduction  

The type VI secretion system (T6SS) is a novel multi-protein needle-like apparatus which is distributed widely in Gram-negative bacteria (Cascales & Cambillau, 2012 ▸; Silverman et al., 2012 ▸). It plays an important role in many processes in bacterial life cycles, such as interspecies competition, biofilm formation and virulence-related processes (Hood et al., 2010 ▸; Russell et al., 2011 ▸; Ho et al., 2014 ▸; Jiang et al., 2014 ▸; Vettiger & Basler, 2016 ▸). The haemolysin co-regulated protein (Hcp) secreted by all characterized T6SSs binds specifically to cognate effector molecules as a chaperone and receptor of substrates, as well as being postulated to form part of the T6SS secretion tube. The structures of several Hcp homologues have been reported, such as Hcp1 and Hcp3 from Pseudomonas aeruginosa (Douzi et al., 2014 ▸; Federico et al., 2015 ▸; Lim et al., 2015 ▸; Jobichen et al., 2010 ▸; Osipiuk et al., 2011 ▸). Significantly, all of them are composed of a β-barrel domain forming hexameric ring oligomers with an inner diameter of ∼40 Å. The internal pore can only accommodate small folded proteins (<20 kDa) or unfolded proteins, such as Tse1–3 from P. aeruginosa and EvpC from Edwardsiella tarda (Zheng & Leung, 2007 ▸). Negative-staining electron microscopy and mutational studies revealed that P. aeruginosa Tse1–3 bind to the inner surface of the Hcp hexamer (Silverman et al., 2013 ▸).

STM0276 and STM0279 are encoded by the type VI secretion system (T6SS) of the Salmonella pathogenicity island 6 (SPI-6) locus and share high sequence identity. They belong to the Hcp family and function as vital components of the T6SS nanotube, and were renamed StHcp1 and StHcp2, respectively. Moreover, StHcp1, rather than StHcp2, is required to kill Klebsiella oxytoca in vitro by specific inter­action with the T6SS peptidoglycan (PG) amidase effector Tae4 (Sana et al., 2016 ▸). In this study, we present the crystal structure and oligomerization study in solution of StHcp2.

2. Materials and methods  

2.1. Macromolecule production  

The gene encoding full-length StHcp2 was amplified from the S. typhimurium genomic DNA, and was cloned into pET-28at-plus (introducing an N-terminal TEV cleavage site, constructed by our laboratory). The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) cells for expression. Protein expression was induced by adding 0.5 mM isopropyl β-d-1-thiogalactopyranoside at 289 K. The supernatant was loaded onto a 2 ml Ni–NTA resin column (GE Healthcare) and eluted with buffer B [25 mM Tris–HCl pH 8.0, 50 mM NaCl, 5%(v/v) glycerol] containing 250 mM imidazole. The proteins were further purified by ion-exchange chromatography and subsequent gel-filtration chromatography. Macromolecule-production information is summarized in Table 1 ▸.

Table 1. Macromolecule-production information.

Source organism	S. typhimurium
DNA source	Genomic DNA
Forward primer	CGCGGATCCATGTCTTATGACATTTTTCTG
Reverse primer	CCGCTCGAGTTAAATTTCTTTGTTGGCC
Cloning vector	pET-28a
Expression vector	pET-28a
Expression host	E. coli BL21 (DE3)
Complete amino-acid sequence of the construct produced	MGSSHHHHHHSSGENLYFEHMASMTGGQQMGRGMSYDIFLKIDGIDGESMDDKHKNEIEVLSWRWNIHQESTMHAGSGLGSGKVSVTNLSFEHYIDRASPNLFKYCSSGKHIPQAILVMRKAGGNPLEYLKYTFTDLIIAMVSPSGSQGGEIASRESIELSFSTVKQEYVVQNQQGGSGGTITAGYDFKANKEI

2.2. Crystallization  

StHcp2 was concentrated to ∼10 mg ml⁻¹ using Millipore Amicon Ultra 10 kDa centrifugal filters. Crystallization screening was performed with kits from Hampton Research and Qiagen using the sitting-drop vapour-diffusion method at 293 K. The initial crystal was grown by mixing 1 µl protein solution with 1 µl reservoir solution consisting of 2.0 M ammonium sulfate, 0.1 M ammonium acetate pH 4.6. The optimal crystallization conditions were refined to 2.2 M ammonium sulfate, 0.1 M ammonium acetate pH 5.6. 20% glycerol added to the crystallization condition was used as a cryoprotective solution to flash-cool the crystals in liquid nitrogen. Crystallization information is summarized in Table 2 ▸.

Table 2. Crystallization.

Method	Sitting-drop vapour diffusion
Plate type	48-well plate
Temperature (K)	293
Protein concentration (mg ml−1)	10
Buffer composition of protein solution	20 mM Tris–HCl pH 8.0, 150 mM NaCl
Composition of reservoir solution	2.0 M ammonium sulfate, 0.1 M ammonium acetate pH 5.6
Volume and ratio of drop	1 µl, 1:1
Volume of reservoir (µl)	1

2.3. Data collection and processing  

Before data collection, crystals were soaked for 5 s in a cryoprotectant consisting of 20%(v/v) glycerol in the crystal mother liquor and were then flash-cooled in liquid nitrogen. The temperature was held at 100 K in a cold nitrogen-gas stream during data collection. X-ray diffraction data were collected using a PILATUS 6M detector on beamline BL19U1 at the Shanghai Synchrotron Radiation Facility (SSRF), Shanghai, People’s Republic of China. All data sets were processed with HKL-2000 (Otwinowski & Minor, 1997 ▸). Data-collection and processing statistics are summarized in Table 3 ▸.

Table 3. Data collection and processing.

Values in parentheses are for the outer shell.

Diffraction source	BL19U1, SSRF
Wavelength (Å)	0.9792
Temperature (K)	100
Detector	PILATUS 6M
Crystal-to-detector distance (mm)	400
Rotation range per image (°)	1.0
Total rotation range (°)	360
Exposure time per image (s)	0.5
Space group	C2
a, b, c (Å)	102.645, 147.786, 85.687
α, β, γ (°)	90.00, 123.74, 90.00
Mosaicity (°)	1.2
Resolution range (Å)	50.0–3.00
Total No. of reflections	58666
No. of unique reflections	21754
Completeness (%)	99.1 (99.5)
Multiplicity	2.7
〈I/σ(I)〉	16.2 (1.5)
R p.i.m.	0.054 (0.395)
R meas	0.090 (0.664)
Overall B factor from Wilson plot (Å2)	81.1

2.4. Structure solution and refinement  

The initial phases were calculated using Phaser (McCoy et al., 2007 ▸) with Burkholderia pseudomallei Hcp1 (PDB entry 3wx6; Lim et al., 2015 ▸) as the search model. The phases from Phaser and the structure factors from HKL-2000 were combined in ARP/wARP (Perrakis et al., 1999 ▸). Some biases were reduced manually in Coot (Emsley & Cowtan, 2004 ▸). The program phenix.refine (Afonine et al., 2012 ▸) was used to refine the structure and to append water molecules. MolProbity (Chen et al., 2010 ▸) was used to validate the structure. Refinement statistics are summarized in Table 4 ▸. PyMOL (http://www.pymol.org) was used to prepare structural figures.

Table 4. Structure refinement.

Values in parentheses are for the outer shell.

Resolution range (Å)	32.8–3.00
Completeness (%)	98.6
σ Cutoff	1.34
No. of reflections, working set	19915
No. of reflections, test set	1055
Final R cryst (%)	23.9
Final R free (%)	28.0
No. of non-H atoms	5934
R.m.s. deviations
Bonds (Å)	0.009
Angles (°)	1.220
Average B factor (Å2)	104
Ramachandran plot
Favoured regions (%)	88.9
Additionally allowed (%)	10.7
Outliers (%)	0.4

3. Results  

3.1. Overall structure of S. typhimurium Hcp2  

The structure of StHcp2 was solved by molecular replacement using the structure of Hcp1 (PDB entry 3wx6) from B. pseudomallei in space group C2 as a search model, and was refined to final R and R _free factors of 0.24 and 0.28, respectively, at 3.0 Å resolution (Table 1 ▸). The asymmetric unit contains six StHcp2 molecules with overall dimensions of ∼103 × 148 × 86 Å comprising 158 residues (Tyr3–Glu160). The residues Gln36–Gly49 inclusive, as well as Gly90–Pro93, Glu118–Ile119 and Gln139–Gly144, were not observed in the electron-density map and were not included in the current model.

The StHcp2 structure revealed a tight β-barrel domain (12 Å diameter) formed by two β-sheets that are comprised of four and five strands each (Fig. 1 ▸ a). The barrel is flanked by an 11-residue α-helix (Ala65–Ser75) and an extended loop formed by Arg31–Leu56 (some residues are missing; discussed below). Similar loops that were defined in several Hcp homologues can protrude more than 20 Å away from the β-barrel core. The hexameric rings could be generated by sixfold symmetry (Fig. 1 ▸ b). This stacking of hexameric rings can form a tube-like assembly (Fig. 1 ▸ c), with an outer diameter of 80 Å and an inner diameter of 40 Å. A similar hexameric ring stacking which forms a nanotube has been reported in Hcp homologues (Douzi et al., 2014 ▸; Federico et al., 2015 ▸; Lim et al., 2015 ▸; Jobichen et al., 2010 ▸; Osipiuk et al., 2011 ▸).

Figure 1.

Overall structure of StHcp2. (a) Cartoon representation showing the StHcp2 structure in rainbow (from the N-terminus in blue to the C-terminus in red). The missing residues are roughly depicted using dotted lines. (b) The StHcp2 hexameric ring: a cartoon representation of one hexamer generated by the sixfold crystallographic symmetry (top view). The α-helix of each monomer is placed almost parallel to the crystallographic axis. The diameter of the internal ring is 40 Å, while the external diameter is 80 Å. (c) Stacking of two representive StHcp2 hexameric rings (shown as light and dark grey surfaces) which form a tube-like architecture. (d) Structure-based sequence alignment of representative Hcp family members, including those from Salmonella typhimurium (St_Hcp2), Pseudomonas aeruginosa (Pa_Hcp1), Edwardsiella tarda (Et_Hcp1), Acinetobacter baumannii (Ab_Hcp1), Escherichia coli (Ec_Hcp1) and Burkholderia pseudomallei (Bp_Hcp1), performed using ClustalX (v.1.81) and ESPript 3.0. Conserved residues are boxed in blue; identical and conserved residues are highlighted on a red background and as red letters, respectively.

A DALI search (http://ekhidna.biocenter.helsinki.fi/dali_server) for globally similar proteins was performed within the Protein Data Bank (PDB). Significant structural similarity was found between StHcp2 and Hcp homologues from other species, such as Yersinia pestis Hcp (PDB entry 3v4h; r.m.s.d. of 1.0 Å for 125 C^α atoms; Center for Structural Genomics of Infectious Diseases, unpublished work), P. aeruginosa Hcp1 (PDB entry 1y12; r.m.s.d. of 1.1 Å for 131 C^α atoms; Mougous et al., 2006 ▸), E. tarda EvpC (PDB entry 3eaa; r.m.s.d. of 1.2 Å for 131 C^α atoms; Jobichen et al., 2010 ▸) and enteroaggregative E. coli Hcp1 (PDB entry 4hkh; r.m.s.d. of 1.5 Å for 124 C^α atoms; Douzi et al., 2014 ▸).

3.2. Oligomerization  

The oligomerization state of wild-type StHcp2 in solution was studied by analytical ultracentrifugation, using the purified protein at ∼1.0 mg ml⁻¹. The results showed two peaks corresponding to the apparent molecular weights of tetrameric (86.2 kDa; ∼25%) and hexameric (143.0 kDa; ∼75%) StHcp2, while a dodecamer was not observed (Fig. 2 ▸ a). Therefore, the model of ring interface interactions for this structure is likely to be the result of the crystal packing in this particular condition (Fig. 1 ▸ c). In solution, EvpC (a homologue of StHcp2) from E. tarda exists as a dimer at low concentration and as a hexamer at higher concentration (Jobichen et al., 2010 ▸). In addition, the Hcp from A. baumannii exists exclusively as a hexamer (Federico et al., 2015 ▸), while that from B. pseudomallei exists predominantly as a hexamer (with a small amount of dodecamer; Lim et al., 2015 ▸). It should be noted that the variable oligomerization state in solution may not reflect the intact status of Hcp in vivo, and the hexamer is predominant in all species that are associated with nanotube formation.

Figure 2.

Mutation studies on the extended loop of StHcp2 reveal its important role in stabilizing the overall structure of the hexameric rings. (a) Analytical ultracentrifugation analysis of the oligomerization state of wild-type StHcp2 and the E37A mutant in solution, showing the partial disassembly of the hexameric ring into monomers (20.4 kDa) for the E37A mutant. (b) Structural superimposition of StHcp2 (magenta) with P. aeruginosa Hcp1 (PaHCp1; PDB entry 1y12; green) and B. pseudomallei Hcp1 (BpHCp1; PDB entry 3wx6; yellow). The neighbouring subunit of the StHcp2 ring is shown as a surface in wheat. The extended loop (Arg31–Leu56) in StHcp2 can be defined based on the high similarity between the Hcp structures, although this loop conformation is variable in different species. The missing residues (Gln36–Gly49) are roughly depicted using dotted lines. (c) Expression of the Q36A and E37A mutants and the truncation Δ(E38–G49) in E. coli.

3.3. The extended loop plays an essential role in stabilizing the overall structure of hexameric rings  

The extended loop has been found to act as a key contact point between the interacting monomers of adjacent hexameric rings (Lim et al., 2015 ▸). In our StHcp2 structure, this loop corresponding to residues Arg31–Leu56 can be defined based on the sequence alignment of Hcp homologues with remarkable similarity (Figs. 1 ▸ c and 2 ▸ b), although the residues Glu36–Gly49 are missing in the flexible loop.

In order to evaluate the role of the extended loop in StHcp2, the Q36A and E37A mutants and the truncation Δ(E38–G49) were created to study their effect on the oligomerization state of wild-type StHcp2 in solution. Unexpectedly, both the Q36A mutant and the Δ(E38–G49) truncation exist as inclusion bodies when overexpressed in E. coli and little recombinant protein could be obtained in the supernatant (Fig. 2 ▸ c), indicating that these mutations may significantly affect the overall fold. Analytical ultracentrifugation showed that an additional peak corresponding to an apparent molecular weight of a monomer (20.4 kDa; ∼4.0%) appeared for the E37A mutant (Fig. 2 ▸ a), as well as two peaks corresponding to tetramers (76.2 kDa; ∼26.5%) and hexamers (131.1 kDa; ∼69.5%) similar to those in wild-type StHcp2. This monomeric status has not previously been reported for known Hcp homologues.

4. Discussion  

Our mutation studies on the extended protruding loop revealed that this loop plays an important role in stabilizing the overall conformation of the StHcp2 monomer as well as the hexameric ring. Structural analysis showed that this loop is very close to the interface of the two subunits (Fig. 2 ▸ b) and participates in the multiple contacts of the hexameric ring in the structures of Hcp homologues (Douzi et al., 2014 ▸; Federico et al., 2015 ▸; Lim et al., 2015 ▸; Jobichen et al., 2010 ▸; Osipiuk et al., 2011 ▸). Here, we find that mutation of Glu37 to alanine (E37A) in the loop may partly destabilize the hexameric conformation into monomers. Meanwhile, the mutation of Gln36 to alanine (Q36A) or truncation of residues 38–49 [Δ(E38-G49)] may destroy the natural conformation and cause the formation of inclusion bodies. A recent study showed that the extended loop in B. pseudomallei Hcp1 (PDB entry 3wx6) shows a significant shift compared with other Hcp homologues, and mediates multiple contacts in the hexameric ring interface (Lim et al., 2015 ▸). Q46A and E47A mutations in this loop caused Hcp to be unable to stack as dodecamers and are likely to disrupt the formation of the tube-like assembly (Lim et al., 2015 ▸). These key residues in the extended loop will restrict the Hcp hexameric ring and may further collapse the tube assembly. Meanwhile, the conservation in these extended loops is quite low (apart from the highly conserved Gly45; Fig. 1 ▸ d), and their conformations in known Hcp structures are variable, which may cause their different roles in stabilizing the Hcp nanotube.

In conclusion, this structure–function study on StHcp2 uncovers the critical role of the extended loop, which plays an essential part in stabilizing the overall conformation of the hexamer subunits. This will help us to further understand the role of StHcp2 in the S. typhimurium T6SS assembly.

Supplementary Material

PDB reference: Hcp2 from S. typhimurium, 5xeu