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Published in final edited form as: Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 Oct 31;64(Pt 11):1083–1086. doi: 10.1107/S1744309108034039

Expression, purification, crystallization and preliminary X-ray studies of the TAN1 ortholog from Methanothermobacter thermautotrophicus

Ana P G Silva a, Robert T Byrne a, Maria Chechik a, Callum Smits a, David G Waterman b, Alfred A Antson a
PMCID: PMC2581707  EMSID: UKMS5036  PMID: 18997348

Abstract

MTH909 is the Methanothermobacter thermautotrophicus ortholog of Saccharomyces cerevisiae TAN1, which is required for N4-acetylcytidine formation in tRNA. The protein consists of an N-terminal near ferredoxin-like domain and a C-terminal THUMP domain. Unlike most other proteins containing the THUMP domain, TAN1 lacks any catalytic domains and has been proposed to form a complex with a catalytic protein capable of making base modifications. MTH909 has been cloned, over-expressed and purified. The molecule exists as a monomer in solution. X-ray data from a native crystal, belonging to the space group P6122 (P6522) with the unit cell dimensions of a = 69.9 Å and c = 408.5 Å, have been collected to 2.85 Å resolution.

Synopsis

MTH909, the Methanothermobacter thermautotrophicus ortholog of Saccharomyces cerevisiae TAN1, has been over-expressed, purified and crystallized. X-ray data from a crystal belonging to the space group P6122 (P6522) have been collected to 2.85 Å resolution.

Keywords: THUMP domain, TAN1, RNA-binding

1. Introduction

Following transcription by RNA polymerases, the bases of RNA may be subject to post-transcriptional modification which results in the presence of a diverse range of non-canonical bases in mature RNAs ( ▶). Of the 107 currently known modifications, 91 have been shown to exist in transfer RNA making it by far the most diversely modified RNA ( ▶). The roles of these base modifications are varied, but they commonly play roles in improving the fidelity of translation ( ▶) and structural stability of tRNA ( ▶).

Numerous sequence analyses and experimental studies have made it possible to identify different classes of RNA-modifying enzymes ( ▶; ▶). A number of these enzymes display a degree of modularity such that a catalytic core, which catalyses the modification, is fused to an RNA-binding domain which binds the substrate. One such domain found in all three domains of life is the THUMP domain which is named after the enzymes in which it has been identified: 4-thiouridine synthetases, methylases and pseudouridine synthases ( ▶). Recently structures of three proteins containing the THUMP domain have been reported: Pyrococcus horikoshii ortholog of 4-thiouridine synthetase (ThiI) ( ▶), Bacillus anthracis ThiI ( ▶) and human pseudouridine synthase Pus10 ( ▶). In each case the THUMP domain is positioned at the N-terminal of the protein. In ThiI however there is an additional N-terminal domain containing 60 residues, denominated the near ferredoxin-like domain (NFLD), which is suggested to have a role in RNA binding ( ▶). At present there is no accurate structural information available for the complex formed between the THUMP domain and RNA. Biochemical studies on the Pyrococcus abyssi methyltransferase showed that the standalone THUMP domain has a far weaker affinity for tRNA than the full-length enzyme ( ▶). Nevertheless, interactions between the THUMP domain and tRNA may contribute to targeting the tRNA substrate towards the catalytic domain as in other RNA-modifying enzymes ( ▶; ▶).

TAN1 (for tRNA acetylation) from Saccharomyces cerevisiae has been shown in vivo to have a role in the formation of the modified base N4-acetylcytidine (ac4C) at position 12 of tRNALeu and tRNASer. Interestingly, in vitro studies showed that TAN1 alone was unable to catalyse formation of the modification on its own yet was able to bind tRNA, an observation consistent with its lack of similarity with other acetyltransferases and the presence of the NFLD and THUMP domain ( ▶). Combined, these facts suggest that TAN1 is part of a larger RNA-modifying complex containing a separate catalytic module. Like S. cerevisiae TAN1, its M. thermautotrophicus ortholog MTH909 contains both the NFLD and THUMP domains (13 % sequence identity between the aligned regions), although the length of the polypeptide chain of the archaeal protein is somewhat shorter. MTH909 has a sequence identity of 14% or less with closest homologs for which three-dimensional structures are available,

We present here the results of a preliminary structural investigation of MTH909, which resulted in collection of an essentially complete X-ray data set to 2.85 Å resolution for crystals of native protein.

2. Materials and Methods

2.1 Cloning and expression

The full-length mth909 gene was amplified by PCR from the genomic DNA of M. thermautotrophicus (strain Delta H) using primers containing Nde I and Hind III sites. The insert was then cloned into pET28a (Merck Biosciences) using the same restriction enzyme sites such that the recombinant protein would contain an N-terminal His-tag with a thrombin cleavage site. E. coli Rosetta (DE3) pLysS cells (Merck Biosciences) were transformed with the plasmid and single colonies from a Luria-Bertani (LB) agar plate containing 30 μg l-1 kanamycin were used to inoculate a small LB-kanamycin culture, which was grown overnight at 310 K. 5 ml aliquots of the overnight culture were used the following day to inoculate 500 ml LB-kanamycin cultures. Cultures were grown at 310 K until an OD600 of 0.6-0.8 at which point protein expression was induced by the addition of isopropyl β-D-1-thiolgalactopyranoside (IPTG) to a final concentration of 1 mM. The temperature was reduced to 289 K and the cultures were shaken at 200 rev min-1 overnight. The cells were harvested by centrifugation at 277 K.

2.2 Purification

Cells were resuspended in buffer A (0.5 M NaCl, 20 mM Hepes pH 8 and 20 mM imidazole) supplemented with a cocktail of protease inhibitors (Complete EDTA-free tablets, Roche). Cells were disrupted using a French press (1500 psi), centrifuged at 15 000 rpm for 1 hour (Sorvall SS34) at 277 K and the supernatant was cleared with a 0.45 μm filter (Millipore). The filtrate was loaded onto a 5 ml HisTrap Q HP column (GE Healthcare) equilibrated with buffer A for Ni2+-affinity purification and bound protein was eluted with an increasing proportion of buffer B (0.5 M NaCl, 20 mM Hepes pH 8 and 500 mM imidazole). Fractions containing MTH909 were pooled, buffer exchanged into 50 mM NaCl and 20 mM Hepes pH 8 and then concentrated using VivaScience 5 kDa MWCO concentrators. The N-terminal His-tag of the protein was removed using thrombin (BD Biosciences) in the reaction buffer (0.15 M NaCl, 25 mM CaCl2 and 20 mM Tris pH 8.5) with a MTH909 concentration of 1.6 mg ml-1 and 0.1 mU of thrombin per 1 μg of MTH909. Protein concentration was determined by absorption at 280 nm using an extinction coefficient of 0.378 (ExPASy Proteomics Server, http://ca.expasy.org/). The reaction was left for 24 hours at room temperature in a rotator at 7 rpm and confirmed to be complete by SDS-PAGE. The reaction mixture was loaded on to a 5 ml HisTrap Q HP column equilibrated with buffer C (250 mM NaCl and 20 mM Tris pH 8.5); buffer D (250 mM NaCl, 20 mM Tris pH 8.5 and 500 mM imidazole) was used for the gradient. SDS-PAGE showed that MTH909 eluted in the flow-through fractions (consistent with the removal of the His-tag) and these fractions were pooled and buffer exchanged in to buffer E (50 mM NaCl and 20 mM Tris pH 7.5). Pooled protein was loaded onto a Mono-Q column (GE Healthcare) pre-equilibrated with buffer E. Bound protein was eluted with an increasing proportion of buffer F (1 M NaCl and 20 mM Tris pH 7.5). Fractions containing MTH909 were buffer exchanged into buffer E, concentrated to 10 mg ml-1 and used for crystallization. The purity of MTH909 was analyzed by SDS-PAGE ( ▶).

Figure 1.

Figure 1

Figure 1

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Purification and characterisation of MTH909. A) SDS-PAGE of purified MTH909 before and after thrombin cleavage step. Molecular weights of the markers are in kDa.) B) Analysis of the cleaved MTH909 oligomerization state by combination of size-exclusion chromatography with MALLS. The thin line corresponds to the absorbance monitored at 280 nm. The thick line shows the molecular weight calculated for the eluted species.

2.3 Size-exclusion chromatography and multi-angle laser light scattering

Chromatography was conducted on a Superdex 75 10/300 column (GE Healthcare, bed and void volumes of 24 ml and 8 ml, respectively) at a flow rate of 0.5 ml min-1 in 250 mM NaCl and 20 mM Tris pH 7.5, and the eluting species were monitored at 280 nm. Light scattering data were recorded on an in-line Dawn Heleos II laser-light scattering instrument (Wyatt Technology) and the concentrations of the eluting species were measured using an in-line Optilab rEX refractometer (Wyatt Technology). A refractive index increment (dn/dc) estimate of 0.184 ml g-1 was used for protein ( ▶).

2.4 Crystallization

Initial crystallization screening was carried out in sitting-drop 96-well plates using a Mosquito Nanolitre Pipetting robot (TTP Labtech) and the Index (Hampton Research), PACT ( ▶) and Clear Strategy Screen I and II ( ▶) (Molecular Dimensions) crystallization screens. For crystallization, equal volumes of the protein and reservoir solutions (150 nl + 150 nl) were mixed and equilibrated against 80 μl of the reservoir solution at 293 K. Conditions where small crystals grew were optimized using the same robotic procedure and the resulting crystals were tested using a Rigaku RU-H3R generator with rotating anode equipped with Osmic multilayer optics and a MAR Research mar345 imaging plate detector. The best crystal was frozen in liquid nitrogen after soaking in a cryoprotectant solution containing 0.15 M MgCl2, 0.1 M Tris pH 8, 15% v/v glycerol and 26% w/v PEG 6000.

2.5 Data Collection and processing

X-ray data were collected at ESRF station ID23-1 from a single crystal. Data collection was performed at 100 K using an oscillation range of 0.5° per image with a total crystal rotation of 180°. The diffraction images were indexed and integrated with DENZO and scaled with SCALEPACK ( ▶). Initial attempts to determine the structure by molecular replacement were made using BALBES ( ▶). These were followed by MOLREP ( ▶) and PHASER ( ▶) using the aligned regions of B. anthracis ThiI (PDB code 2c5s), P. horikoshii ThiI (1vbk) and human Pus10 (PDB code 2v9k) as search models.

3. Results and discussion

3.1 Expression and purification

MTH909 was successfully cloned and over-expressed in a soluble form. The protein was purified to homogeneity by a three-step procedure involving two rounds of Ni2+-affinity chromatography separated by cleavage of the His-tag and followed by a final step of anion-exchange chromatography ( ▶). The molecular weight of the protein was determined by mass spectrometry to be 20 596 Da, in agreement with the calculated value of 20 577 Da for the full-length protein containing the GSH residues of the thrombin cleavage site.

3.2 Oligomerization state of MTH909

MTH909 eluted largely as a single peak during the size exclusion chromatography ( ▶). According to MALLS measurements the major peak contains species with an average molecular weight of 20.9 ± 0.4 kDa, close to the theoretical molecular weight of 20.6 kDa for the monomer of MTH909. The eluted species in the very minor peak have an average molecular weight of 46.6 ± 4.7 kDa, which correspond to either dimers of MTH909 or impurities ( ▶). These data show that under the tested conditions MTH909 exists almost exclusively as a monomer in solution.

3.3 Crystallization

Initial crystallization trials resulted in crystals obtained under four different conditions. Two of these conditions where the crystals were very little were further optimized. Crystals grown with 0.1 M Tris pH 8.5, 2 M Ammonium Sulfate (Index condition 6) did not reproduce while optimization of crystals grown with 0.1 M Hepes pH 7.5, 0.2 M MgCl2.6H2O, 25% w/v PEG3350 (Index condition 84) led to crystals with poor diffraction quality (4.5 Å resolution). The two other conditions each produced single crystals that were big enough for data collection. The crystal grown with 0.1 M Bis-Tris pH 6.5 and 2 M Ammonium Sulfate (Index condition 4) diffracted to 3.5 Å but the diffraction pattern was “split”, indicating that it was not a single crystal. The crystal grown with 0.2 M MgCl2, 0.1 M Tris pH 8 and 20% w/v PEG6000 (PACT condition D10) had dimensions of 0.1 × 0.1 × 0.1 mm ( ▶) and diffracted to 3.2 Å in-house. From this crystal essentially complete data to 2.85 Å were collected at the ESRF beamline ID23-1 ( ▶).

Figure 2.

Figure 2

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The best crystal of MTH909 from which the X-ray data have been collected.

Figure 3.

Figure 3

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The diffraction image obtained at the ESRF station ID23-1. Diffraction data were collected at the wavelength of 0.9184 Å with the crystal to detector distance of 488 mm. The resolution at the edges of the image is 3.0 Å.

3.4 X-ray data collection and preliminary analysis

Analysis of merging statistics and systematic absences indicated that crystals belong to the spacegroup P6122 (or P6522) with the unit cell dimensions a = b = 69.9 and c = 408.5 Å. Cumulative intensity distributions indicated no twinning. Data collection and processing statistics are presented in ▶. Three molecules in the asymmetric unit would result in a solvent content of 46.8 % ( ▶). The self-rotation function did not reveal any significant peaks apart from those resulting from the crystallographic symmetry suggesting that the potential non-crystallographic rotation axes could be parallel to the crystallographic ones. Attempts to solve the structure by molecular replacement using the known structures of THUMP-domain containing proteins proved unsuccessful, most probably because of the very low sequence identity between MTH909 and the search models (sequence identities of 14% or less).

Table 1.

X-ray data statistics for the crystal of native MTH909. Values in parentheses represent data for the highest resolution shell

Data collection and Processing Statistics
X-ray source ID23-1, ESRF
Wavelength (Å) 0.9184
Temperature (K) 100
Space Group P6122 or P6522
Unit-cell parameters (Å) a=b=69.9, c=408.5
Resolution range (Å) 25 - 2.85 (2.95-2.85)
No. of unique reflections 14804 (1296)
Rmerge† (%) 10.0 (55.6)
Completeness (%) 98.6 (89.3)
Redundancy 9.8 (5.9)
Average I / σ(I) 19.3 (2.4)
Wilson B (Å2) 37
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†

Rmerge = Σ | Ii - <I> | / Σ <I>, where I is the measured intensity of each reflection and <I> is the intensity averaged over multiple observations of symmetry-related reflections.

In conclusion, MTH909 was successfully purified, crystallized and X-ray data from native crystal were collected to 2.85 Å resolution. Future attempts to solve the structure will be focused on crystallizing Se-Met MTH909 or preparing heavy atom derivatives. Comparison of the structure of MTH909 with structures of other proteins containing the THUMP domain will bring further understanding of its RNA-binding ability.

Acknowledgements

We would like to thank Sam Hart for help during the data collection at the ESRF station ID23-1, as well as ESRF (Grenoble) for provision of data collection facilities. This work was supported by Fundação para a Ciência e a Tecnologia, Portugal (studentship SFRH/BD/17372/2004 to APGS), by the BBSRC (PhD studentship to RTB) and by the Wellcome Trust (fellowship 081916 to AAA).

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