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ISSN: 2053-230X

Crystal structure of an inorganic pyrophosphatase from Chlamydia trachomatis D/UW-3/Cx

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aDepartment of Chemistry and Biochemistry, Hampton University, 100 East Queen Street, Hampton, VA 23668, USA, bCenter for Global Infectious Disease Research, Seattle Children's Research Institute, 307 Westlake Avenue North, Suite 500, Seattle, WA 98109, USA, cSeattle Structural Genomics Center for Infectious Disease (SSGCID), Seattle, Washington, USA, dBeryllium, 7869 NE Day Road West, Bainbridge Island, WA 98102, USA, eDepartments of Pediatrics, Global Health, and Biomedical Informatics & Medical Education, University of Washington, Seattle, Washington, USA, and fDivision of Allergy and Infectious Diseases, Department of Medicine, University of Washington, Seattle, Washington, USA
*Correspondence e-mail: oluwatoyin.asojo@hamptonu.edu

Edited by I. Tanaka, Hokkaido University, Japan (Received 4 January 2022; accepted 23 February 2022; online 28 February 2022)

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections globally and is one of the most commonly reported infections in the United States. There is a need to develop new therapeutics due to drug resistance and the failure of current treatments to clear persistent infections. Structures of potential C. trachomatis rational drug-discovery targets, including C. trachomatis inorganic pyrophosphatase (CtPPase), have been determined by the Seattle Structural Genomics Center for Infectious Disease. Inorganic pyrophosphatase hydrolyzes inorganic pyrophosphate during metabolism. Furthermore, bacterial inorganic pyrophosphatases have shown promise for therapeutic discovery. Here, a 2.2 Å resolution X-ray structure of CtPPase is reported. The crystal structure of CtPPase reveals shared structural features that may facilitate the repurposing of inhibitors identified for bacterial inorganic pyrophosphatases as starting points for new therapeutics for C. trachomatis.

1. Introduction

Chlamydiae are obligate intracellular bacteria that infect a wide range of eukaryotes, including humans, animals, insects and free-living amoebae. Chlamydia trachomatis is a Gram-negative coccus that causes a commonly known sexually transmitted infection often called chlamydia. Chronic chlamydia infection often leads to genital, ocular and respiratory disease (Lorenzini et al., 2010[Lorenzini, E., Singer, A., Singh, B., Lam, R., Skarina, T., Chirgadze, N. Y., Savchenko, A. & Gupta, R. S. (2010). J. Bacteriol. 192, 2746-2756.]). The Chlamydia genus is phylogenetically distant from other bacteria, and 30% of its proteins are referred to as hypothetical proteins (Barta et al., 2013[Barta, M. L., Hickey, J., Kemege, K. E., Lovell, S., Battaile, K. P. & Hefty, P. S. (2013). Acta Cryst. F69, 1196-1201.]). Genital chlamydia is a major public health concern, with over 1.8 million cases reported to the US Centers for Disease Control and Prevention (CDC) in 2019. Furthermore, chlamydia is the most common bacterial sexually transmitted infection globally and is a leading cause of infertility (van Bergen et al., 2021[Bergen, J. E. A. M. van, Hoenderboom, B. M., David, S., Deug, F., Heijne, J. C. M., van Aar, F., Hoebe, C., Bos, H., Dukers-Muijrers, N., Götz, H. M., Low, N., Morré, S. A., Herrmann, B., van der Sande, M. A. B., de Vries, H. J. C., Ward, H. & van Benthem, B. H. B. (2021). Sex. Transm. Infect. 97, 501-506.]; Dombrowski, 2021[Dombrowski, J. C. (2021). Ann. Intern. Med. 174, ITC145-ITC160.]). The CDC recommends treating chlamydia in adults and adolescents with 100 mg doxycycline orally twice a day for seven days. Alternatively, a single 1 g oral dose of azithromycin or 500 mg levofloxacin can be administered. However, reinfection is common with all antibiotics, and compliance is low for doxycycline (Centers for Disease Control and Prevention, 2021[Centers for Disease Control and Prevention (2021). Sexually Transmitted Infections Treatment Guidelines, 2021. https://www.cdc.gov/std/treatment-guidelines/default.htm.]). Efforts to identify new treatment strategies for chlamydia at the Seattle Structural Genomics Center for Infectious Disease (SSGCID) include structural studies of C. trachomatis proteins as the first steps towards rational drug discovery. C. trachomatis inorganic pyrophosphatase (CtPPase) was one of the investigated proteins because inorganic pyrophos­phatases from other bacteria have shown promise as potentially selective targets (Pang et al., 2016[Pang, A. H., Garzan, A., Larsen, M. J., McQuade, T. J., Garneau-Tsodikova, S. & Tsodikov, O. V. (2016). ACS Chem. Biol. 11, 3084-3092.]; Lv et al., 2014[Lv, W., Banerjee, B., Molland, K. L., Seleem, M. N., Ghafoor, A., Hamed, M. I., Wan, B., Franzblau, S. G., Mesecar, A. D. & Cushman, M. (2014). Bioorg. Med. Chem. 22, 406-418.]). The production, crystallization and high-resolution structure of CtPPase are presented here.

2. Materials and methods

2.1. Macromolecule production

Cloning, expression and purification were conducted as part of the Seattle Structural Genomics Center for Infectious Disease (SSGCID) following standard protocols described previously (Bryan et al., 2011[Bryan, C. M., Bhandari, J., Napuli, A. J., Leibly, D. J., Choi, R., Kelley, A., Van Voorhis, W. C., Edwards, T. E. & Stewart, L. J. (2011). Acta Cryst. F67, 1010-1014.]; Choi et al., 2011[Choi, R., Kelley, A., Leibly, D., Nakazawa Hewitt, S., Napuli, A. & Van Voorhis, W. (2011). Acta Cryst. F67, 998-1005.]; Serbzhinskiy et al., 2015[Serbzhinskiy, D. A., Clifton, M. C., Sankaran, B., Staker, B. L., Edwards, T. E. & Myler, P. J. (2015). Acta Cryst. F71, 594-599.]). The full-length gene for inorganic pyrophosphatase from C. trachomatis (CtPPase; UniProt O84777) encoding amino acids 1–209 was PCR-amplified from gDNA using the primers shown in Table 1[link]. The gene was cloned into the ligation-independent cloning (LIC) expression vector pBG1861 encoding a noncleavable hexahistidine tag (Aslanidis & de Jong, 1990[Aslanidis, C. & de Jong, P. J. (1990). Nucleic Acids Res. 18, 6069-6074.]; Choi et al., 2011[Choi, R., Kelley, A., Leibly, D., Nakazawa Hewitt, S., Napuli, A. & Van Voorhis, W. (2011). Acta Cryst. F67, 998-1005.]). Plasmid DNA was transformed into chemically competent Escherichia coli BL21(DE3)R3 Rosetta cells. The plasmid containing hexahistidine-tagged C. trachomatis inorganic pyrophosphatase (His-CtPPase) was expression-tested and 2 l of culture were grown using auto-induction medium (Studier, 2005[Studier, F. W. (2005). Protein Expr. Purif. 41, 207-234.]). The expression clone ChtrB.01427.a.B1.GE42413 is available at https://www.ssgcid.org/available-materials/expression-clones/.

Table 1
Macromolecule-production information

Source organism Chlamydia trachomatis (strain D/UW-3/Cx)
DNA source Dr Kevin Hybiske (University of Washington, USA)
Forward primer 5′-CTCACCACCACCACCACCATATGTCTAAAACACCATTATCCATAGC-3′
Reverse primer 5′-ATCCTATCTTACTCACTTACATAAAAAGATTGCAATAGTCTTCGT-3′
Expression vector pBG1861
Expression host E. coli BL21(DE3)R3 Rosetta cells
Complete amino-acid sequence of the construct produced MAHHHHHHMSKTPLSIAHPWHGPVLTRDDYESLCCYIEITPADSVKFELDKETGILKVDRPQKFSNFCPCLYGLLPKTYCGDLSGEYSGQQSNRENIKGDGDPLDICVLTEKNITQGNILLQARPIGGIRILDSEEADDKIIAVLEDDLVYGNIEDISECPGTVLDMIQHYFLTYKATPESLIQAKPAKIEIVGLYGKKEAQKVIRLAHEDYCNLFM

His-CtPPase was purified in a two-step protocol consisting of an immobilized metal-affinity chromatography (IMAC) step and size-exclusion chromatography (SEC). All chromatography runs were performed on an ÄKTApurifier 10 (GE Healthcare) using automated IMAC and SEC programs according to previously described procedures (Bryan et al., 2011[Bryan, C. M., Bhandari, J., Napuli, A. J., Leibly, D. J., Choi, R., Kelley, A., Van Voorhis, W. C., Edwards, T. E. & Stewart, L. J. (2011). Acta Cryst. F67, 1010-1014.]). Thawed bacterial pellets were lysed by sonication in 200 ml buffer consisting of 25 mM HEPES pH 7.0, 500 mM NaCl, 5% glycerol, 0.5% CHAPS, 30 mM imidazole, 10 mM MgCl2, 1 mM TCEP, 250 µg ml−1 AEBSF, 0.025% azide. After sonication, the crude lysate was clarified with 20 µl (25 U µl−1) Benzonase and incubated while mixing at room temperature for 45 min. The lysate was then clarified by centrifugation at 10 000 rev min−1 for 1 h using a Sorvall centrifuge (Thermo Scientific). The clarified supernatant was then passed over an Ni–NTA HisTrap FF 5 ml column (GE Healthcare) which was pre-equilibrated with loading buffer consisting of 25 mM HEPES pH 7.0, 500 mM NaCl, 5% glycerol, 30 mM imidazole, 1 mM TCEP, 0.025% azide. The column was washed with 20 column volumes (CV) of loading buffer and was eluted with loading buffer plus 250 mM imidazole in a linear gradient over 7 CV. Peak fractions, as determined by the UV absorbance at 280 nm, were pooled and concentrated to 5 ml. A Superdex 75 SEC column (GE Healthcare) was equilibrated with running buffer consisting of 25 mM HEPES pH 7.0, 500 mM NaCl, 5% glycerol, 2 mM DTT, 0.025% azide. The peak fractions were collected and analyzed for CtPPase using SDS–PAGE. The SEC peak fractions eluted as a single large peak at a molecular mass of ∼80 kDa, suggesting a trimeric enzyme. Peak fractions were pooled and concentrated to 62 mg ml−1 using an Amicon purification system (Millipore). Aliquots of 200 µl were flash-frozen in liquid nitrogen and stored at −80°C until use for crystallization.

2.2. Crystallization

Purified His-CtPPase was screened for crystallization in 96-well sitting-drop plates against the JCSG+ HTS (Rigaku Reagents) and MCSG1 (Anatrace) crystal screens. Equal volumes of protein solution (0.4 µl) and precipitant solution were set up at 287 K against a 80 µl reservoir in sitting-drop vapor-diffusion format. 3 mM inorganic pyrophosphate was added to the protein solution before crystallization experiments. Crystals were obtained using high sodium chloride and polyethylene glycol 3350 conditions (Table 2[link]). A crystal was cryoprotected by exchange into precipitant supplemented with 15%(v/v) ethylene glycol and vitrified directly in liquid nitrogen.

Table 2
Crystallization

Method Vapor diffusion, sitting drop
Plate type 96-well Compact 300, Rigaku
Temperature (K) 287
Protein concentration (mg ml−1) 31
Buffer composition of protein solution 3 mM inorganic pyrophosphate, 25 mM HEPES pH 7.0, 500 mM NaCl, 5% glycerol, 2 mM DTT, 0.025% azide
Composition of reservoir solution 2 M NaCl, 0.1 M Tris pH 8.5, 25%(v/v) PEG 3350
Volume and ratio of drop 0.4 µl protein plus 0.4 µl reservoir
Volume of reservoir (µl) 80
Composition of cryoprotectant solution 2 M NaCl, 0.1 M Tris pH 8.5, 25%(v/v) PEG 3350, 15%(v/v) ethylene glycol

2.3. Data collection and processing

Data were collected at 100 K on beamline 21-ID-F at the Advanced Photon Source, Argonne National Laboratory (see Table 3[link]). Diffraction data (Table 3[link]) were integrated using XDS and were reduced using XSCALE (Kabsch, 2010[Kabsch, W. (2010). Acta Cryst. D66, 125-132.]). Raw X-ray diffraction images are available at the Integrated Resource for Reproducibility in Macromolecular Crystallography at https://www.proteindiffraction.org.

Table 3
Data collection and processing

Values in parentheses are for the outer shell.

Diffraction source Beamline 21-ID-F, APS
Wavelength (Å) 0.97872
Temperature (K) 100
Detector RayoniX MX300HE CCD
Crystal-to-detector distance (mm) 260
Rotation range per image (°) 1
Total rotation range (°) 200
Space group C2221
a, b, c (Å) 77.16, 121.19, 124.50
α, β, γ (°) 90, 90, 90
Mosaicity (°) 0.24
Resolution range (Å) 44.99–2.25 (2.31–2.25)
Total No. of reflections 226034 (16920)
No. of unique reflections 27864 (2010)
Completeness (%) 99.3 (99.2)
Multiplicity 8.1 (8.4)
I/σ(I)〉 25.24 (3.14)
Rr.i.m. 0.046 (0.661)
Overall B factor from Wilson plot (Å2) 56.57
†Estimated Rr.i.m. = Rmerge[N/(N − 1)]1/2, where N is the data multiplicity.

2.4. Structure solution and refinement

The structure was solved by molecular replacement with Phaser (McCoy et al., 2007[McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C. & Read, R. J. (2007). J. Appl. Cryst. 40, 658-674.]) from the CCP4 suite of programs (Collaborative Computational Project, Number 4, 1994[Collaborative Computational Project, Number 4 (1994). Acta Cryst. D50, 760-763.]; Krissinel et al., 2004[Krissinel, E. B., Winn, M. D., Ballard, C. C., Ashton, A. W., Patel, P., Potterton, E. A., McNicholas, S. J., Cowtan, K. D. & Emsley, P. (2004). Acta Cryst. D60, 2250-2255.]; Winn et al., 2011[Winn, M. D., Ballard, C. C., Cowtan, K. D., Dodson, E. J., Emsley, P., Evans, P. R., Keegan, R. M., Krissinel, E. B., Leslie, A. G. W., McCoy, A., McNicholas, S. J., Murshudov, G. N., Pannu, N. S., Potterton, E. A., Powell, H. R., Read, R. J., Vagin, A. & Wilson, K. S. (2011). Acta Cryst. D67, 235-242.]) using PDB entry 5ls0 (Grzechowiak et al., 2019[Grzechowiak, M., Ruszkowski, M., Sliwiak, J., Szpotkowski, K., Sikorski, M. & Jaskolski, M. (2019). Biochem. J. 476, 2297-2319.]) as the search model. The structure was refined using iterative cycles of Phenix (Liebschner et al., 2019[Liebschner, D., Afonine, P. V., Baker, M. L., Bunkóczi, G., Chen, V. B., Croll, T. I., Hintze, B., Hung, L.-W., Jain, S., McCoy, A. J., Moriarty, N. W., Oeffner, R. D., Poon, B. K., Prisant, M. G., Read, R. J., Richardson, J. S., Richardson, D. C., Sammito, M. D., Sobolev, O. V., Stockwell, D. H., Terwilliger, T. C., Urzhumtsev, A. G., Videau, L. L., Williams, C. J. & Adams, P. D. (2019). Acta Cryst. D75, 861-877.]) followed by manual rebuilding of the structure using Coot (Emsley & Cowtan, 2004[Emsley, P. & Cowtan, K. (2004). Acta Cryst. D60, 2126-2132.]; Emsley et al., 2010[Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. (2010). Acta Cryst. D66, 486-501.]). The quality of the structure was checked using MolProbity (Williams et al., 2018[Williams, C. J., Headd, J. J., Moriarty, N. W., Prisant, M. G., Videau, L. L., Deis, L. N., Verma, V., Keedy, D. A., Hintze, B. J., Chen, V. B., Jain, S., Lewis, S. M., Arendall, W. B., Snoeyink, J., Adams, P. D., Lovell, S. C., Richardson, J. S. & Richardson, J. S. (2018). Protein Sci. 27, 293-315.]). All data-reduction and refinement statistics are shown in Table 4[link]. The structure was refined to a resolution of 2.25 Å. Coordinates and structure factors have been deposited in the Protein Data Bank (https://www.rcsb.org) with accession code 6we5.

Table 4
Structure refinement

Values in parentheses are for the outer shell.

Resolution range (Å) 44.99–2.25 (2.31–2.25)
Completeness (%) 99.2
σ Cutoff F > 1.34σ(F)
No. of reflections, working set 27834 (1787)
No. of reflections, test set 2028 (156)
Final Rcryst 0.181 (0.275)
Final Rfree 0.228 (0.374)
No. of non-H atoms
 Protein 4694
 Ion 3
 Ligand 0
 Water 65
 Total 4762
R.m.s. deviations
 Bond lengths (Å) 0.004
 Angles (°) 0.683
Average B factors (Å2)
 Protein 61.8
 Ion 69.5
 Ligand 0.0
 Water 53.0
Ramachandran plot
 Most favored (%) 98.04
 Allowed (%) 1.96

3. Results and discussion

CtPPase is a small β-strand protein containing a core five-stranded oligonucleotide/oligosaccharide-binding (OB) fold. CtPPase has the prototypical family I pyrophosphatase (PPase) topology. Family 1 PPases are ubiquitous in all kingdoms of life (Kajander et al., 2013[Kajander, T., Kellosalo, J. & Goldman, A. (2013). FEBS Lett. 587, 1863-1869.]). The overall topology of CtPPase resembles an open fist (or baseball mitt) with the substrate-binding cavity sitting in the palm, while β-strands form finger-like structures surrounding the active site (Fig. 1[link]a).

[Figure 1]
Figure 1
CtPPase structure. (a) The superposed CtPPase monomers are almost identical, with r.m.s.d.s of ∼0.3 Å for all atoms and ∼0.17 Å for Cα atoms. The monomers are colored from blue (N-terminus) to red (C-terminus). (b) A prototypical family I PPase hexamer was generated from the asymmetric unit trimer (monomers colored green, cyan and magenta) and a symmetry mate (shown in gray). The sodium ion bound in the active site of each monomer is shown as a purple sphere. (c) The CtPPase active-site loop (cyan) is in the open conformation compared with the closed conformation of M. tuberculosis PPase (MtPPase). The pyrophosphate (orange sticks) in the active site is from MtPPase (PDB entry 5kde), while the sodium ion (purple sphere) is from CtPPase (PDB entry 6we5).

The CtPPase structure was refined to 2.25 Å resolution in space group C2221 with three molecules in the asymmetric unit. Surface-area calculations by PISA (Krissinel, 2015[Krissinel, E. (2015). Nucleic Acids Res. 43, W314-W319.]) suggest a hexamer as the most likely biological assembly (Fig. 1[link]b). Hexamers were previously observed as the biological assembly in other well studied family I PPases, notably E. coli PPases (Cooperman et al., 1992[Cooperman, B. S., Baykov, A. A. & Lahti, R. (1992). Trends Biochem. Sci. 17, 262-266.]). The CtPPase hexamer is similar to those of the well studied family I PPases. Electron density modeled as an Na atom was observed in the active site of each monomer. The active site is where the hydrolysis of pyrophosphate into two phosphate ions occurs. Despite the addition of pyrophosphate to the crystallization buffer, no density was observed for pyrophosphate or phosphate ions. Additionally, the flexible active-site loop is in the open conformation indicative of an apo structure without any substrate or product in the active site of CtPPase (Fig. 1[link]c). Future studies will include investigating whether the presence of the N-terminal hexahistidine tag renders CtPPase inactive and unable to hydrolyze pyrophosphate or form the biological hexamer in solution, or whether additional ions or cofactors need to be added to the enzyme before crystallization to generate the structure of the complex with pyrophosphate or phosphate.

Since bacterial inorganic pyrophosphatases have shown promise as potentially selective targets (Pang et al., 2016[Pang, A. H., Garzan, A., Larsen, M. J., McQuade, T. J., Garneau-Tsodikova, S. & Tsodikov, O. V. (2016). ACS Chem. Biol. 11, 3084-3092.]; Lv et al., 2014[Lv, W., Banerjee, B., Molland, K. L., Seleem, M. N., Ghafoor, A., Hamed, M. I., Wan, B., Franzblau, S. G., Mesecar, A. D. & Cushman, M. (2014). Bioorg. Med. Chem. 22, 406-418.]), CtPPase was compared with other structures to determine whether it could be a viable drug target. PDBeFold analysis (http://www.ebi.ac.uk/msd-srv/ssm/; Krissinel & Henrick, 2004[Krissinel, E. & Henrick, K. (2004). Acta Cryst. D60, 2256-2268.]), the DALI server (http://ekhidna2.biocenter.helsinki.fi/dali/; Holm, 2020[Holm, L. (2020). Protein Sci. 29, 128-140.]) and ENDscript analysis (Gouet et al., 2003[Gouet, P., Robert, X. & Courcelle, E. (2003). Nucleic Acids Res. 31, 3320-3323.]; Robert & Gouet, 2014[Robert, X. & Gouet, P. (2014). Nucleic Acids Res. 42, W320-W324.]) were used to identify the closest structural neighbors of CtPPase. These analyses revealed that despite <37% sequence similarity, CtPPase shares significant secondary-structural similarity with several family I PPases, including some that have shown promise as drug targets (see supporting information and Fig. 2[link]). The supporting information includes detailed results of the DALI (Supplementary Fig. S1) and PDBeFold (Supplementary Table S1) analyses. The overall core structure of CtPPase is highly similar to other bacterial PPases except for two major insertions (residues 71–86 and residues 170–180; Figs. 2[link] and 3[link]). These insertions are on the exterior surface of the hexamer and do not participate in the formation of the hexamer or interact with the active site (Fig. 1[link]c).

[Figure 2]
Figure 2
An ENDscript alignment identifies conserved residues in CtPPase and PPases. Multi-sequence alignment of CtPPase with 41 closest PPases obtained by a BLAST search against the PDBAA database. Identical and conserved residues are highlighted in red and yellow, respectively. Alternate residues are highlighted with gray stars. The different secondary-structure elements shown are α-helices (α), 310-helices (η), β-strands (β) and β-turns (TT).
[Figure 3]
Figure 3
Structural comparison of CtPPase with other PPases. (a) Solvent-accessible surface area colored by sequence conservation. Residues clustered in the active-site cleft are identified by the sodium ion present in the crystal structure of CtPPase (magenta sphere). (b) Coil diagram calculated by ENDscript. The circumference of the ribbon (sausage) represents the relative structural conservation compared with 41 other PPase structures (the same structures as indicated in Fig. 2[link]). Thinner ribbons represent more conserved regions, while thicker ribbons represent less conserved regions. The ten identical residues cluster within or in proximity to the active site. Identical residues are indicated by red regions on the surface and a red ball-and-stick representation in ribbon diagrams. The sodium ion bound in the active site of each monomer is shown as a purple sphere. (c) Comparison of the active and allosteric sites of CtPPase (cyan) with bacterial PPases (gray) and eukaryotic PPases (Homo sapiens PPase, PDB entry 7btn, green; Plasmodium falciparum PPase, PDB entry 5wru, brown). (d) The same view of the structures without the eukaryotic PPases. The top ten unique bacterial PPases were selected from the ENDscript alignment. All three monomer chains of CtPPase are shown.

A comparison of CtPPase with 41 other PPases deposited in the Protein Data Bank using ENDscript identified 19 identical residues which cluster in the active-site pocket (Figs. 2[link] and 3[link]). The active-site region contains a D-(S/G/N)-D-P-ali-D-ali-ali motif, where ali is C/I/L/M/V (Kankare et al., 1994[Kankare, J., Neal, G. S., Salminen, T., Glumoff, T., Cooperman, B. S., Lahti, R. & Goldman, A. (1994). Protein Eng. 7, 823-830.]). PDBeFold analysis also revealed that, as expected, bacterial PPases were structurally most similar to CtPPase (Supplementary Table S1). The most similar structure was from Thermococcus thioreducens, followed by Acinetobacter baumannii, with root-mean-square differences (r.m.s.d.s) of 1.18 and 1.33 Å over 196 and 162 residues, respectively. The top PPases that showed structural similarity are listed in Supplementary Table S1. Our preliminary analysis revealed structural differences between bacterial and eukaryotic PPases that may possibly be exploited for inhibitor design (Fig. 3[link]c).

A manual search of the entire PDB for structures of PPases from other organisms identified 80 different ligand-bound PPase structures. The majority of ligands were metals, ions, substrate or substrate mimics. Most ligands were bound in the active site. However, there were four structures with ligands bound outside the active site: two structures of Mycobacterium tuberculosis PPase (MtPPase), PDB entries 5kde and 5kd7 (Pang et al., 2016[Pang, A. H., Garzan, A., Larsen, M. J., McQuade, T. J., Garneau-Tsodikova, S. & Tsodikov, O. V. (2016). ACS Chem. Biol. 11, 3084-3092.]), and two structures of Burkholderia pseudo­mallei PPase (BpPPase), PDB entries 3ej2 and 3ej0 (Van Voorhis et al., 2009[Van Voorhis, W. C., Hol, W. G. J., Myler, P. J. & Stewart, L. J. (2009). PLoS Comput. Biol. 5, e1000530.]). The MtPPase ligands are low-micromolar IC50 allosteric inhibitors (Pang et al., 2016[Pang, A. H., Garzan, A., Larsen, M. J., McQuade, T. J., Garneau-Tsodikova, S. & Tsodikov, O. V. (2016). ACS Chem. Biol. 11, 3084-3092.]). The BpPPase ligands were discovered from fragment-based screens at the Seattle Structural Genomics Center for Infectious Disease. Superposition of the C. trachomatis (CtPPase) structures with MtPPase and BpPPase revealed that the ligands are small organic compounds that are located in a surface binding pocket on the opposite side to the pyrophos­phate binding pocket (Fig. 3[link]c).

The two previous studies on MtPPase and BpPPase suggest the possibility of an allosteric binding site that small-molecule inhibitors of bacterial PPases could target. Comparison of the CtPPase structure with those of MtPPase and BpPPase shows that the loop adjacent to the putative allosteric binding site has moved into the pocket compared with MtPPase and BpPPase, closing off this site. Inspection of the solvent-accessible surface of CtPPase reveals a medium-sized cleft that partially occupies the fragment-binding site of BpPPase (Fig. 4[link]). Future fragment-based screening targeting this cleft may generate allosteric inhibitors of CtPPase.

[Figure 4]
Figure 4
The bacterial PPase allosteric binding site. The putative allosteric binding site of CtPPase identified from superposition of CtPPase (PDB entry 6we5) with MtPPase (PDB entries 5kde and 5kdf) and BpPPase (PDB entries 3ej0 and 3ej2). (a) Coil diagram of CtPPase (red and white) superimposed on the MtPPase structures with allosteric inhibitors (PDB entries 5kde, yellow, and 5kdf, magenta) and BpPPase bound with fragment compounds (PDB entries 3ej0, wheat, and 3ej2, blue). The location of the compounds is indicated with a black oval. The circumference of the coil represents the relative structural conservation compared with 41 other PPase structures (the same structures as indicated in Fig. 2[link]). (b) A solvent-accessible surface diagram of CtPPase calculated with ENDscript reveals a potential binding pocket labeled the allosteric site on the CtPPase surface in proximity to the compounds.

4. Conclusion

We have determined the structure of an inorganic pyrophos­phatase (PPase) from C. trachomatis. The overall structure is a prototypical bacterial PPase with additional amino acids inserted beyond the conserved active site. CtPPase has a pocket in proximity to the previously identified bacterial allosteric binding sites, suggesting the possibility of developing allosteric inhibitors of CtPPase. While the preliminary structural studies are promising, future studies include validating the enzymatic activity of CtPPase and probing the active and allosteric sites of CtPPase with substrates and potential inhibitors.

Acknowledgements

This manuscript was generated as an educational collaboration between Hampton University (a Historically Black College and University) and the SSGCID. The SSGCID consortium is directed by Dr Peter Myler (Principal Investigator) and comprises many different scientists working at multiple centers towards determining the three-dimensional structures of proteins from biodefense organisms and emerg­ing infectious diseases. In particular, we would like to thank the SSGCID cloning, protein production and X-ray crystallography groups at the Center for Global Infectious Disease Research, the University of Washington and UCB.

Funding information

This work was supported by federal funds from the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Department of Health and Human Services under Contract No. HHSN272201700059C from September 1, 2017. (SSGCID was funded under NIAID Contract Nos. HHSN272201200025C from 1 September 2012 through 31 August 2017 and HHSN272200700057C from 28 September 2007 through 27 September 2012.) JM is part of the inaugural Hampton University Chemistry Education and Mentorship Course-based Undergraduate Research (HU-ChEM CURES) funded by the NIGMS (1U01GM138433 to OAA). JM is also a URISE scholar funded by the NIGMS (T34GM136489 to OAA).

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