Structures of Trichomonas vaginalis macrophage migratory inhibitory factor

This article is part of a focused issue on empowering education through structural genomics.

The unicellular parasitic protozoan Trichomonas vaginalis causes trichomoniasis, the most prevalent nonviral sexually transmitted disease globally. T. vaginalis evades host immune responses by producing homologs of host proteins, including cytokines such as macrophage migration inhibitory factor. T. vaginalis macrophage migration inhibitory factor (TvMIF) helps to facilitate the survival of T. vaginalis during nutritional stress conditions, increases prostate cell proliferation and invasiveness, and induces inflammation-related cellular pathways, thus mimicking the ability of human MIF to increase inflammation and cell proliferation. The production, crystallization and three structures of N-terminally hexahistidine-tagged TvMIF reveal a prototypical MIF trimer with a topology similar to that of human homologs (hMIF-1 and hMIF-2). The N-terminal tag obscures the expected pyruvate-binding site. The similarity of TvMIF to its human homologs can be exploited for structure-based drug discovery.

1. Introduction

Trichomonas vaginalis is a unicellular parasitic protozoan that is responsible for trichomoniasis, the most prevalent nonviral sexually transmitted disease globally (Edwards et al., 2016). There are ∼156 million new cases of trichomoniasis worldwide each year (Molgora et al., 2023). According to the Centers for Disease Control and Prevention, ∼2.6 million people in the USA have trichomoniasis, and population studies show that the highest incidence is among incarcerated women (https://www.cdc.gov/std/treatment-guidelines/trichomoniasis.htm). Humans are the only T. vaginalis hosts, and trichomoniasis increases susceptibility to HIV, infertility, preterm birth, HPV, and prostate cancer (Tsang et al., 2019; Van Gerwen & Muzny, 2019; Zhang et al., 2022). Despite its clinical significance, the molecular mechanisms underlying the development, immune evasion and host–parasite interactions of T. vaginalis remain poorly understood. T. vaginalis is a priority infectious disease for structural studies by the Seattle Structural Genomics Center for Infectious Disease (SSGCID). T. vaginalis evades host immune responses by producing homologs of host proteins, including cytokines such as macrophage migration inhibitory factor (MIF; Twu et al., 2014). Protozoan parasite MIF homologs mimic their human MIF counterparts (hMIF-1, NCBI Accession No. CAG30406.1; hMIF-2, NCBI Accession No. CAG30317.1), facilitating the modulation of host immune responses and suppressing apoptosis-induced cell death (Twu et al., 2014; Ghosh et al., 2019). The human MIFs (hMIF-1 and hMIF-2) share ∼35% sequence identity, while T. vaginalis macrophage migration inhibitory factor (TvMIF) shares ∼31% sequence identity with hMIF-1 and hMIF2. It has previously been demonstrated that TvMIF elicits antibodies in infected individuals, increases prostate cell proliferation, and invasiveness, and induces inflammation-related cellular pathways, thus mimicking the ability of human MIF to increase inflammation and cell proliferation (Twu et al., 2014). Furthermore, TvMIF has been shown to enhance the survival of Trichomonas during nutritional stress conditions (Chen et al., 2018). Thus, TvMIF facilitates parasite survival during infection and binds to the human CD74 MIF receptor, triggering epithelial cell inflammation and proliferation pathways linked to the progression and pathogenesis of prostate cancer (Twu et al., 2014; Tsang et al., 2019). Here, we present the purification, crystallization and structural and functional analysis of TvMIF as a first step towards uncovering features that mediate its functions.

2. Materials and methods

2.1. Macromolecule production

TvMIF was cloned, expressed, and purified as described previously (Bryan et al., 2011; Choi et al., 2011; Serbzhinskiy et al., 2015). The full-length gene for a putative macrophage migration inhibitory factor from T. vaginalis ATCC PRA-98/G3 (UniProt A2DXT4) encoding amino acids 1–115 was PCR-amplified from gDNA using the primers shown in Table 1. The gene was cloned into the pET-28a expression vector with an N-terminal histidine tag. The plasmid DNA was transformed into chemically competent Escherichia coli BL21(DE3) Rosetta cells. After testing for expression, 2 l of culture was grown using auto-induction medium (Studier, 2005) in a LEX Bioreactor (Epiphyte Three) as described previously (Serbzhinskiy et al., 2015). The expression clone is available for request online at https://www.ssgcid.org/available-materials/expression-clones/.

Table 1
Macromolecule-production information

Source organism	Trichomonas vaginalis ATCC PRA-98/G3
DNA source	CollegeCodon optimized and synthetically generated plasmid from Twist Bioscience
Expression vector	pET-28a, AVA N-terminal tag
Expression host	Escherichia coli BL21(DE3) Rosetta
Complete amino-acid sequence of the construct produced†	MAHHHHHHMGTLEAQTQGPGSMPALVIKTNAKFTEEEKSKATEELGNIVSKVLGKPISYVMVTLEDGVAVRFGGSDEKAAFMSLMSIGGLNRAVNKRASAALTKWFTDHGFQGDRIYIVFNPKSAEDWGFNGDTFA

†The additional N-terminal amino acid residues are in bold.

N-terminally hexahistidine-taggedTvMIF (His-TvMIF) was purified using a previously described two-step protocol consisting of an immobilized metal (Ni²⁺) affinity chromatography (IMAC) step followed by size-exclusion chromatography (SEC) on an ÄKTApurifier 10 (GE Healthcare) using automated IMAC and SEC programs (Serbzhinskiy et al., 2015). Briefly, thawed bacterial pellets (25 g) were lysed by sonication in 200 ml lysis buffer [25 mM HEPES pH 7.0, 500 mM NaCl, 5%(v/v) glycerol, 0.5%(w/v) CHAPS, 30 mM imidazole, 10 mM MgCl₂, 400 µg ml⁻¹ lysozyme, 3 U ml⁻¹ Benzonase]. After sonication, the crude lysate was treated with 20 ml (25 U ml⁻¹) of Benzonase and incubated with mixing for 45 min at room temperature. The lysate was 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 had been pre-equilibrated with wash buffer [25 mM HEPES pH 7.0, 500 mM NaCl, 5%(v/v) glycerol, 30 mM imidazole]. The column was washed with 20 column volumes (CV) of wash buffer and eluted with elution buffer (20 mM HEPES pH 7.0, 500 mM NaCl, 5%(v/v) glycerol, 500 mM imidazole) over a 7 CV linear gradient.

The peak fractions were pooled and concentrated to 5 ml for SEC. The 5 ml protein sample was loaded onto a Superdex 75 26/60 column (GE Biosciences) attached to an ÄKTAprime plus FPLC system (GE Biosciences) that had been equilibrated with SEC buffer (20 mM HEPES pH 7.0, 300 mM NaCl, 5% glycerol, 1 mM TCEP). TvMIF eluted from SEC as a single, symmetrical, monodisperse peak accounting for >90% of the protein product of molecular mass ∼19 kDa, suggesting purification as a monomer (expected monomer molecular mass of 15 kDa). The peak fractions were collected and assessed for purity by SDS–PAGE, which also suggested monomeric protein. The peak fractions were pooled and concentrated to ∼20 mg ml⁻¹ using an Amicon purification system (Millipore). 110 µl aliquots of His-TvMIF were flash-frozen in liquid nitrogen and stored at −80°C until use. His-TvMIF protein is available for request online at https://www.ssgcid.org/available-materials/ssgcid-proteins/.

2.2. Crystallization

Three crystal forms of His-TvMIF are reported, and all crystals were grown in UVXPO MRC (Molecular Dimensions) sitting-drop vapor-diffusion plates using the Berkeley (Pereira et al., 2017; Rigaku Reagents), Index (Hampton Research) and Morpheus (Gorrec, 2009; Molecular Dimensions) crystallization screens as listed in Table 2.

Table 2
Crystallization

Crystal form	PDB entry 8uz4, apo, P41212	PDB entry 8ur4, I4122	PDB entry 8ur2, I41
Temperature (K)	291	291	291
Protein concentration (mg ml^−1)	35.4	35.4	35.4
Buffer composition of protein solution	20 mM HEPES pH 7.0, 300 mM NaCl, 5% glycerol, 1 mM TCEP	20 mM HEPES pH 7.0, 300 mM NaCl, 5% glycerol, 1 mM TCEP, 5 mM sodium 4-hydroxyphenylpyruvate	20 mM HEPES pH 7.0, 300 mM NaCl, 5% glycerol, 1 mM TCEP, 5 mM sodium pyruvate
Composition of reservoir solution	Berkeley D5: 100 mM HEPES free acid/sodium hydroxide pH 7.5, 200 mM ammonium acetate, 25%(w/v) PEG 3350	Index A4: 0.1 M bis-Tris pH 6.5, 2.0 M ammonium sulfate	Morpheus B12: 12.5%(v/v) MPD, 12.5%(v/v) PEG 1000, 12.5%(w/v) PEG 3350, 100 mM Tris–Bicine pH 8.5, 30 mM NaF, 30 mM NaBr, 30 mM NaI
Volume and ratio of drop	0.2 µl, 1:1	0.2 µl, 1:1	0.2 µl, 1:1
Volume of reservoir (µl)	40	40	40
Composition of cryoprotectant solution	80 mM HEPES free acid/sodium hydroxide pH 7.5, 160 mM ammonium acetate, 20%(w/v) PEG 3350, 20%(v/v) PEG 200	2.5 M lithium sulfate, 0.1 M bis-Tris pH 6.5, 2.0 M ammonium sulfate, 20%(w/v) PEG 3350, 20%(v/v) PEG 200	Directly from crystallization buffer

2.3. Data collection and processing

All data sets were collected at 100 K on a Dectris EIGER2 XE 9M detector on beamline 19-ID at NSLS-II, Brookhaven National Laboratory (Table 3). Intensities were integrated using XDS (Kabsch, 1988, 2010) via autoPROC (Vonrhein et al., 2011), and the Laue class analysis and data scaling were performed with AIMLESS (Evans, 2011). Raw X-ray diffraction images have been stored with 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.

Data set	PDB entry 8uz4, apo, P41212	PDB entry 8ur4, I4122	PDB entry 8ur2, I41
Temperature (K)	100	100	100
Space group	P41212	I4122	I41
a, b, c (Å)	80.72, 80.72, 121.15	109.87, 109.87, 125.88	118.39, 118.39, 106.66
α, β, γ (°)	90, 90, 90	90, 90, 90	90, 90, 90
Resolution range (Å)	80.71–2.40 (2.46–2.40)	82.78–2.55 (2.62–2.55)	83.71–1.90 (1.95–1.90)
Total No. of reflections	379848 (29417)	211801 (15124)	787413 (58149)
Completeness (%)	100 (100)	100 (100)	100 (100)
Multiplicity	23.2 (24.6)	16.4 (15.8)	13.6 (13.6)
〈I/σ(I)〉	21.2 (1.6)	14.8 (1.6)	16.6 (1.8)
Rr.i.m.	0.093 (2.75)	0.113 (2.02)	0.078 (1.68)
Rp.i.m.	0.020 (0.55)	0.033 (0.51)	0.021 (0.45)

2.4. Structure solution and refinement

The three structures were determined by molecular replacement with Phaser (McCoy et al., 2007) from the CCP4 suite of programs (Collaborative Computational Project, Number 4, 1994; Krissinel et al., 2004; Winn et al., 2011; Agirre et al., 2023) using PDB entry 1mif (Sun et al., 1996) as the search model. The structure was refined using Phenix (Liebschner et al., 2019). Structure quality was checked with MolProbity (Williams et al., 2018). Data-reduction and refinement statistics are shown in Table 4. Coordinates and structure factors have been deposited with the Worldwide PDB (wwPDB) as entries 8uz4 (P4₁2₁2 form), 8ur4 (I4₁22 form) and 8ur2 (I4₁ form). The accuracy of the ligands and waters was also checked with the CheckMyBlob server (Kowiel et al., 2019; https://checkmyblob.bioreproducibility.org/server/).

Table 4
Structure refinement

Values in parentheses are for the outer shell.

Structure	PDB entry 8uz4, apo, P41212	PDB entry 8ur4, I4122	PDB entry 8ur2, I41
Resolution range (Å)	67.17–2.40 (2.55–2.40)	82.78–2.55 (2.75–2.55)	83.71–1.90 (1.93–1.90)
Completeness (%)	100 (100)	100 (100)	99.8 (99.8)
No. of reflections
Working set	16289 (2517)	12901 (2400)	57681 (2599)
Test set	816 (131)	646 (126)	2990 (145)
Final Rcryst	0.245 (0.397)	0.253 (0.339)	0.192 (0.341)
Final Rfree	0.264 (0.431)	0.280 (0.416)	0.230 (0.383)
No. of non-H atoms
Protein	2588	2576	4713
Ion	0	5	1
Ligand	0	0	6
Water	3	0	167
Total	2591	2581	4887
R.m.s. deviations
Bond lengths (Å)	0.008	0.003	0.011
Angles (°)	1.124	0.501	0.909
Average B factors (Å^2)
Protein	96.3	90.9	54.4
Ion	0	88.5	124.4
Ligand	0	0	60.0
Water	61.9	0	47.5
Ramachandran plot (%)
Favored regions	99	98	99
Additionally allowed	1	2	1
Outliers	0	0	0

3. Results and discussion

The crystallized protein, His-TvMIF, included 21 additional amino-acid residues at the N-terminus corresponding to the purification tag and cleavage site (Table 1). Structures of His-TvMIF were determined in three different space groups (Table 4). The first is a P-centered tetragonal structure (PDB entry 8uz4) with no additional density for ligands, as was expected. It has three monomers in the asymmetric unit corresponding to the prototypical MIF trimer (Fig. 1a). Each monomer was refined with 115 amino acids. Attempts at co-crystallization with sodium 4-hydroxyphenylpyruvate resulted in an apo structure, determined in the tetragonal space group I4₁22 (PDB entry 8ur4), that contains three monomers per asymmetric unit (Fig. 1b).

Figure 1 Quaternary structure of His-TvMIF. All three structures reveal prototypical MIF trimers. (a) The apo structure (PDB entry 8uz4) and (b) an attempt at co-crystallization with sodium 4-hydroxyphenylpyruvate (PDB entry 8ur4) are prototypical MIF trimers. (c) The co-crystal with pyruvate (PDB entry 8ur2) is a dimer of two prototypical MIF trimers.

The third structure was co-crystallized with pyruvate (PDB entry 8ur2) and determined in the tetragonal space group I4₁. This structure is a hexamer or dimer of the prototypical MIF trimer (Fig. 1c). The six monomers include two with 115 amino acids, two with 100 amino acids, one with 102 amino acids and one with 99 amino acids. Analysis of all three structures with the Protein Interfaces, Surfaces and Assembly service (PISA) at the European Bioinformatics Institute (https://www.ebi.ac.uk/pdbe/prot_int/pistart.html) suggests that TvMIF forms a stable prototypical MIF trimer (Krissinel, 2015). All three structures contain prototypical MIF trimers (Fig. 1d) that superpose with human hMIF-1 (PDB entry 1mif; Fig. 2a) and hMIF-2 (PDB entry 3ker; Fig. 2b). The r.m.s.d. for superposing C^α atoms of TvMIF trimers with the hMIF-1 trimer is ∼1.1 Å, while that with hMIF-2 is ∼1.2 Å. PDBeFold (https://www.ebi.ac.uk/msd-srv/ssm/) analysis (Krissinel & Henrick, 2004) using a default threshold of 70% was used to identify the closest structural neighbors of TvMIF as hMIF-1 and MIFs from other infectious protozoa, notably Entamoeba histolytica (PDB entry 6cuq; Seattle Structural Genomics Center for Infectious Disease, unpublished work) and Toxoplasma gondii (PDB entry 4dh4; Sommerville et al., 2013).

Figure 2 Comparison with human MIF homologs. (a) His-TvMIF trimers (shown in gray) superpose well with each other and with hMIF-1 (PDB entry 1mif, shown in blue). (b) They also superpose well with hMIF-2 (PDB entry 3ker). All three structures reveal prototypical MIF trimers. (c) The additional residues in His-TvMIF occupy the location of the tautomerase inhibitor IPP (shown as golden sticks) in hMIF-2; further details of the N-terminus are shown in the enlarged red parentheses.

The hMIF-2 structure has an inhibitor, 4-IPP, bound in the tautomerase inhibitory site in the amino-terminus. The N-terminal residues of His-TvMIF obscure this inhibitor-binding site, which is otherwise accessible in both hMIF-1 and hMIF-2 (Fig. 2c). The obstruction by the N-terminal extension explains why 4-hydroxyphenylpyruvate does not co-crystallize with His-TvMIF since its expected binding site is blocked. The obstruction also explains why pyruvate does not bind in the expected tautomerase site. A similar obstructed N-terminus was observed in the recently reported structure of Onchocerca volvulus MIF (Kimble et al., 2024).

The F_o − F_c omit electron-density maps of PDB entry 8ur2 can be modeled with pyruvate (Supplementary Fig. S1a). The location of the density is different from previously identified MIF pyruvate-binding sites, which are always at the His-tag-obscured N-terminus. We checked whether another molecule from the protein purification or crystallization solution was bound instead of pyruvate. However, pyruvate matches better than MPD or glycerol (Supplementary Fig. S1b). LigPlus analysis reveals that only one amino acid, Asp68, interacts with the pyruvate (Fig. 3). Furthermore, Asp68 is not conserved among MIFs. The location differs from the previously identified hMIF-1 allosteric inhibitor site (PDB entry 6peg; Cirillo et al., 2020; Fig. 3b) and that of 4-IPP in hMIF-2 (PDB entry 3ker; Rajasekaran et al., 2014; Fig. 3c). Further analysis is required to determine whether this newly identified pyruvate-binding site is biologically relevant or merely a crystallization artifact, as suggested by CheckMyBlob.

Figure 3 Ligand-interaction plots generated with LigPlus show different amino acids involved in the binding of pyruvate (Pyr202) by His-TvMIF (PDB entry 8ur2), of IPP (RW1120) by hMIF-2 (PDB entry 3ker) and of an allosteric inhibitor (4fq201) by hMIF-1 (PDB entry 6peg).

It has previously been demonstrated that recombinant TvMIF has tautomerase activity and mimics the ability of human MIF to increase inflammation and cell proliferation (Twu et al., 2014). These studies were performed with carboxyl-terminally hexahistidine-tagged TvMIF, leaving the N-terminus unobstructed (Twu et al., 2014). Future studies will include removal of the N-terminal tag and the generation of co-crystal structures of untagged TvMIF-1 with known MIF inhibitors.

4. Conclusion

The production, crystallization and three structures of N-terminally hexahistidine-tagged TvMIF (His-TvMIF) reveal a prototypical MIF trimer with a topology similar to that of the human homologs (hMIF-1 and hMIF-2). The N-terminal tag obscures the expected pyruvate-binding site. The similarity of TvMIF to its human homologs and to other MIFs (Supplementary Fig. S2) can be exploited for structure-based drug discovery.