ATP-dependent RNA helicase domain of the ZC3H41 protein from Trypanosoma brucei: expression, purification and crystallization

In this study, a structural approach was used to unveil the molecular function of ZC3H41, an essential yet enigmatic protein in the life cycle of T. brucei. Here, the expression, purification and crystallization of this protein are reported.

Abstract

A fragment of the Trypanosoma brucei ZC3H41 protein encompassing the ATP-dependent RNA helicase domain was successfully subcloned for expression in a bacterial system (Escherichia coli). Following expression, the protein was purified and crystallized using the vapor-diffusion method. The protein crystals were optimized at a 1:1 protein:reservoir solution ratio using PPGBA 2000. The optimized crystals diffracted to a d _min of 3.15 Å. The collected data revealed preliminary structural information regarding this newly discovered protein.

1. Introduction  

ATP-dependent RNA helicases play an essential biological role in cellular RNA metabolism (Cordin et al., 2006 ▸). In general, helicases are classified into five superfamilies (SF1–SF5), the two largest of which are SF1 and SF2 (Gorbalenya & Koonin, 1993 ▸). These two superfamilies encompass a large number of DNA and RNA helicases from all kingdoms of life, including parasites. SF2 helicases are highly conserved and share multiple structural motifs. The Asp-Glu-Ala-Asp (DEAD) motif, which is commonly present in the active site of SF2 helicases, defines these helicases as DEAD-box proteins. DEAD-box proteins contain a conserved helicase domain that includes two identical domains resembling bacterial recombinase A (RecA; Fairman-Williams et al., 2010 ▸; Singleton et al., 2007 ▸).

Trypanosoma brucei, a eukaryotic parasite endemic to central and western Africa, causes a severe medical condition termed trypanosomiasis (‘sleeping sickness’). This condition may prove fatal if it remains untreated (Atkins, 1742 ▸; Frearson et al., 2010 ▸). Current treatments for the disease have severe, and in some cases life-threatening, side effects (Steverding, 2008 ▸; Krishna & Stich, 2012 ▸; Brun et al., 2001 ▸). Moreover, the course of treatment is difficult and in many instances requires full hospitalization for the entire duration of treatment (Baker & Welburn, 2018 ▸). The growing need to find novel treatments for this disease have paved the way for new scientific discoveries regarding the parasite, its life cycle and the various parasitic components that may be utilized as drug targets for inhibition of this pathogen.

T. brucei ZC3H41 (ZC3H41) is a zinc-finger protein with a putative C-terminal helicase-like domain (Kramer et al., 2010 ▸). This enigmatic protein was identified through co-precipitation assays by its association with RNA and other cellular factors related to mRNA metabolism (Romaniuk et al., 2016 ▸). Previous studies indicate that the expression of ZC3H41 is elevated during cell stress in the procyclic life stage of the parasite (Fritz et al., 2015 ▸; Eliaz et al., 2017 ▸) and showed association of this protein with the cytoplasmic SL-RNA granules released during cell stress (Eliaz et al., 2017 ▸).

Furthermore, ZC3H41 has been proven to have a vital role in parasite survival, as a conditional knockdown of ZC3H41 in procyclic T. brucei exhibited growth arrest upon knockdown induction (Eliaz et al., 2017 ▸). Overall, the accumulated data on ZC3H41 indicate a crucial role in the life cycle of T. brucei.

The domain topology of ZC3H41 (Fig. 1 ▸ a) includes a C3H1-type zinc-finger domain (residues 129–156) and a helicase C domain (residues 327–494) that bares a putative ATP-binding site as annotated in UniProtKB (https://www.uniprot.org/). To better understand the functional attributes of the ZC3H41 protein, we adopted a structural approach to gain insights into the molecular characteristics of ZC3H41. In the course of efforts to crystallize the recombinant, full-length ZC3H41 protein (ZC3H41^FL), we also generated a stable, truncated version of the protein (ZC3H41^ΔN331) that includes the C-terminal helicase C domain (residues 332–517). Here, we report the expression, purification and preliminary X-ray diffraction data collection of the recombinant ZC3H41^ΔN331 fragment.

Figure 1.

ZC3H41 domain topology and purification. (a) Domain topology of the ZC3H41 protein. (b) Elution profile of hisZC3H41^FL and hisZC3H41^NΔ331 on an analytical Superdex 200 SEC column. The elution volume of the hisZC3H41^FL protein is 15.5 ml, compared with 16.5 ml for hisZC3H41^NΔ33. The inset shows an SDS–PAGE analysis of hisZC3H41^FL and hisZC3H41^NΔ331 after the final purification step. Black asterisks represent degradation products. Blue and pink arrowheads represent the bands corresponding to intact hisZC3H41^FL and hisZC3H41^ΔN331, respectively.

2. Materials and methods  

2.1. Macromolecule production  

The full-length sequence of ZC3H41 from T. brucei (ZC3H41^FL; GenBank ID 3664251) was codon-optimized for bacterial expression and synthesized by a contract service provider (BioBasic Inc.). A helicase-domain construct was obtained by the N-terminal truncation of 331 amino acids using the PCR method (ZC3H41^ΔN331). Both constructs were subcloned into pET-28a(+) downstream of a hexahistidine purification tag and a Tobacco etch virus (TEV) protease cleavage site (ENLYFQ↓G) for tag removal (Table 1 ▸). Construct sequences were validated using Sanger sequencing by Macrogen Europe B.V. (Amsterdam, The Netherlands).

Table 1. hisZC3H41^ΔN331 production information.

Source organism	T. brucei
Forward primer (NdeI)	ATCGGCTACATATGCGTTTCGAAAAACAGCAGGA
Reverse primer (NotI)	ATGCGCGGCCGCTTACAGGGTTTTCGGTTTCAGCT
Cloning vector	pET-28a(+)
Expression vector	pET-28a(+)
Expression host	E. coli Rosetta (DE3)
Complete amino-acid sequence of the construct produced†	MGSSHHHHHHSSGRENLYFQGHMSSAVDSLLVRLKIPDLIPKFREMNVDRIISLRKLSEEELRTAVPDDDQRNRIIEAIKNRGSSEKRTQAPAASINPPRSTDDGGRGGMSYPRGAPRGGGPVRGRGRGGTGSAGEGGFHGSGSNNANMFPSRQRVCNHFLNGDCRYGDSCRYSHNKALCQEAAESVPRRNHDATSLSEDFSETCIIPTHRIKYLLANRADRLRSIHSKNRTHNKSFQHIDPTIEKFELVIYGADQQSVQESKKMILACIGVTREEEQKNRVQYTMNELSSNQRAAKFLAACNMKNEGTVRELSEASLRNIISFFRFEKQQDIRHFWVNTNSEHSKLDIIANIVAQLQGVQAIMFCDQKRVVDMSKVASKITRYFNGVSPLFLHRAIPKEERMKMLQTFKDGKPNENGIRERLLVTNEDYAKLARKTIVPYVNLVINYSVPRSEEYYLLQSLVAGRSDTVGVSIVCVFPHEQSLFQELQRNIPFEELEEEGNFKDAAVKLVYDTVDEPLTGEDADPPSNWHEQLKPKTL

^†The N-terminal hexahistidine tag and the subsequent TEV cleavage site are shown in bold. The sequence of the first 331 residues that were removed for crystallization is shown in italics.

Sequenced clones were transformed into Escherichia coli Rosetta (DE3) strain cells (Novagen) using the heat-shock method. Cells were plated on LB medium containing kanamycin and chloramphenicol and incubated overnight at 37°C. A single colony was used to inoculate 2 ml TB (Terrific Broth) medium (24 mg ml⁻¹ yeast extract, 20 mg ml⁻¹ tryptone, 4 ml l⁻¹ glycerol, 0.017 M KH₂PO₄, 0.072 M K₂HPO₄) supplemented with antibiotics at 310 K and 200 rev min⁻¹. Upon reaching an OD₆₀₀ of 0.5–0.8, protein expression was induced with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 291 K for 12–16 h. The cells were then harvested by centrifugation (15 min at 4415g and 277 K). The pellet was resuspended in ice-cold lysis buffer (buffer L): 50 mM HEPES pH 7.5, 500 mM NaCl, 5 mM β-mercaptoethanol, 0.1% Triton X-100, 10% glycerol, 10 mg lysozyme, 10 U ml⁻¹ DNase, 10 U ml⁻¹ RNase, 1 mM phenylmethylsulfonyl fluoride and one cOmplete EDTA-free protease-inhibitor tablet per 50 ml of suspension (Roche).

The cells were lysed using an AVESTIN emulsifier at 282 K. Cell debris was removed by centrifugation at 14 000g for 45 min at 277 K. All subsequent purification steps were performed at 277 K. The pellet was discarded and the cleared lysate was supplemented with 10 mM imidazole prior to loading onto an immobilized metal-affinity chromatography column (IMAC, Ni–NTA agarose, Qiagen) pre-equilibrated with binding buffer (buffer B; 50 mM HEPES pH 7.5, 500 mM NaCl, 5 mM β-mercaptoethanol). Subsequently, the Ni–NTA column was washed with ten column volumes of buffer B supplemented with 20 mM imidazole followed by 50 mM imidazole. Finally, His-tagged ZC3H41^FL and ZC3H41^ΔN331 (hisZC3H41^FL and hisZC3H41^ΔN331, respectively) proteins were eluted from the column using buffer B supplemented with 350 mM imidazole. 15% SDS–PAGE was used to detect the protein and to estimate its purity. The relevant fractions were pooled, concentrated and loaded onto a size-exclusion chromatography (SEC) column (Superdex 200 10/300 GL, GE Healthcare) pre-equilibrated with 30 mM HEPES pH 7.5, 200 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol). The peak fractions were analyzed on 15% SDS–PAGE. Relevant fractions containing either hisZC3H41^FL or hisZC3H41^ΔN331 were pooled and concentrated to 10 mg ml⁻¹ (hisZC3H41^FL) or 14 mg ml⁻¹ (hisZC3H41^ΔN331) using an Amicon Ultra filter (molecular-mass cutoff 10 kDa, Millipore), aliquoted and flash-frozen in liquid nitrogen. The purified proteins were stored at 193 K until use.

2.2. Crystallization  

Initial screening for crystallization conditions was performed using the hanging-drop vapor-diffusion technique (400 nl drop size with a 1:1 protein:reservoir solution ratio) as set up by a Mosquito crystallization robot (TTP Labtech). To crystallize ZC3H41^FL, we screened ∼1000 conditions at 289 K for each protein preparation (the protein preparations varied in their storage buffers, removal of the purification tag, protein concentration and protein:reservoir solution ratio). A total of 12 000 conditions were screened for the crystallization of ZC3H41^FL with no success.

Purified hisZC3H41^ΔN331 at 14 mg ml⁻¹ was used in commercial screens at various protein:reservoir solution ratios ranging from 1:1 to 3:1. The screening plates were incubated at 289 K. Three days after setup, microcrystals of hisZC3H41^ΔN331 were observed in the following conditions: (i) 10%(v/v) polypropylene glycol 400 (MIDASplus condition 7); (ii) 25%(v/v) pentaerythritol propoxylate (5/4 PO/OH), 0.1 M MES pH 6 (MIDASplus condition 9) and (iii) 25%(v/v) PPGBA 2000, 0.2 M imidazole pH 7 (MIDASplus condition 45). The crystals were then optimized by varying the precipitant concentration, buffer pH, protein concentration, drop size and protein:reservoir solution ratio. Optimal crystals were obtained at a 1:1 protein:reservoir solution using 16%(v/v) PPGBA 2000, 0.2 M imidazole pH 7 (Table 2 ▸, Fig. 2 ▸).

Table 2. Crystallization of hisZC3H41^ΔN331 .

Method	Vapor diffusion
Plate type	24-well VDX hanging-drop plate, Hampton Research
Temperature (K)	289
Protein concentration (mg ml−1)	14
Buffer composition of protein solution	30 mM HEPES pH 7.5, 200 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol
Composition of reservoir solution	16%(v/v) PPGBA 2000, 0.2 M imidazole pH 7
Volume and ratio of drop	2 µl (1:1)
Volume of reservoir (µl)	400

Figure 2.

Crystallization of hisZC3H41^NΔ331 protein in screening plates (a) and post-optimization (b). White arrowheads indicate representative crystals that were cooled for X-ray diffraction experiments.

2.3. Data collection and processing  

Harvesting of the hisZC3H41^ΔN331 crystals was performed using either nylon loops (Hampton Research) or MicroMounts (MiTeGen). The crystals were transferred to reservoir solution supplemented with 25%(v/v) glycerol as a cryoprotectant for 10–30 s and immediately flash-cooled in liquid nitrogen. Data were collected on the EMBL beamline P13 at the PETRA III synchrotron (DESY, Hamburg, Germany). Diffraction images were collected using a PILATUS 6M detector with 0.1° oscillation and an exposure time of 0.04 s per image for a total of 3600 images at two κ angles (0° and 10°). The images were processed with the XDS package using the XDSgui interface (Kabsch, 2010 ▸). Data-collection and processing statistics are summarized in Table 3 ▸.

Table 3. Data-collection and processing statistics for hisZC3H41^ΔN331 .

Diffraction source	Beamline P13, DESY
Wavelength (Å)	0.9802
Temperature (K)	100
Detector	PILATUS 6M
Crystal-to-detector distance (mm)	619
Rotation range per image (°)	0.1
Total rotation range (°)	360
Space group	I222
a, b, c (Å)	81.8, 101.4, 154.8
α, β, γ (°)	90, 90, 90
Mosaicity (°)	0.5864
Resolution range (Å)	30–3.15 (3.20–3.15)
Total No. of reflections	431582 (13722)
No. of unique reflections	21470 (960)
Completeness (%)	99.8 (99.4)
Multiplicity	20 (14.3)
〈I/σ(I)〉†	15.19 (1.79)
R meas	0.143 (2.49)
CC1/2	0.999 (0.791)
Overall B factor from Wilson plot‡ (Å2)	98.43

^†〈I/σ(I)〉 falls below 2 at a resolution of 3.25 Å.

^‡The data are moderately anisotropic and thus the Wilson B factor may be misleading.

3. Results and discussion  

We expressed ZC3H41^FL and ZC3H41^ΔN331 in E. coli and purified them to homogeneity. Although ZC3H41^FL survived a freeze–thaw cycle well, crystallization of ZC3H41^FL proved to be challenging. No unusual behavior was observed in the crystallization drops (neither extreme heavy precipitation nor notable undersaturation of the drop). We assume that this inability to crystallize is a result of both disordered regions in the protein and heterogeneity of the sample originating from degradation (Fig. 1 ▸ b). Hence, we focused our efforts on obtaining a more stable construct that had a better likelihood of crystallizing. The helicase C domain construct (hisZC3H41^ΔN331) produced small crystals at a 1:1 protein:reservoir solution ratio using the vapor-diffusion technique. We were successful in optimizing the initial hit and acquired larger crystals (0.06 mm in the longest dimension; Fig. 2 ▸) that diffracted to d _min = 3.15 Å (Fig. 3 ▸, Table 3 ▸). Assuming the presence of two molecules per asymmetric unit gives a calculated V _M value of 2.97 ų Da⁻¹. To obtain experimental phase information, anomalous diffraction experiments will be performed on recombinant selenomethionine-substituted protein crystals. Selenomethionine-substituted protein has already been obtained and crystallized.

Figure 3.

Oscillation image (0.1°) of a hisZC3H41^NΔ331 protein crystal. The crystal-to-detector distance was 619 mm. Diffraction data were observed to 3.05 Å resolution.

Funding Statement

This work was funded by Israel Science Foundation grant 401/18 to Moshe Dessau.