Completeness for I/σ(I) > 0, with the numbers in parentheses for the high-resolution shell (2.38–2.30 Å).
Crystal structure of Thermotoga maritima SPOUT superfamily RNA methyltransferase Tm1570 in complex with S-adenosyl-L-methionine
Article first published online: 2 SEP 2008
Copyright © 2008 Wiley-Liss, Inc.
Proteins: Structure, Function, and Bioinformatics
Volume 74, Issue 1, pages 245–249, January 2009
How to Cite
Kim, D. J., Kim, H. S., Lee, S. J. and Suh, S. W. (2009), Crystal structure of Thermotoga maritima SPOUT superfamily RNA methyltransferase Tm1570 in complex with S-adenosyl-L-methionine. Proteins, 74: 245–249. doi: 10.1002/prot.22249
- Issue published online: 19 NOV 2008
- Article first published online: 2 SEP 2008
- Accepted manuscript online: 2 SEP 2008 12:00AM EST
- Manuscript Accepted: 6 AUG 2008
- Manuscript Received: 8 JUN 2008
- Basic Research Promotion Grant of Korea Research Foundation funded by the Korean Government (MOEST). Grant Number: KRF-2007-313-C00617
- Thermotoga maritima;
- RNA methyltransferase;
- SPOUT superfamily;
RNAs involved in translation and post-transcriptional processing contain a large number of modified nucleosides.1 In tRNAs, over 80 different modifications have been observed,2 and many of these modifications are important for tRNA function. Modifications within the anticodon loop are necessary for proper codon recognition,3 while those in the D- and T-loops facilitate folding and stabilization of the tRNA.4 Ribosomal RNA modifications are clustered around the catalytic center and other crucial regions of the ribosome, further underscoring their importance in translation.5
The “SPOUT” [SpoU-TrmD] superfamily methyltransferases (MTases) are a large class of S-adenosyl-L-methionine (AdoMet)-dependent RNA MTases that exhibit a Rossmannoid α/β-fold with a deep trefoil knot.6 Several members of the SPOUT superfamily have been characterized functionally7 and they have been found to be involved in post-transcriptional RNA modification by catalyzing methylation of the 2′-OH group of ribose,8–10 the N-1 atom of guanosine 37 in tRNA,11, 12 or the N-3 atom of uridine 1498 in 16S rRNA.13 Despite a wealth of sequence data on the SPOUT superfamily MTases in the database, structural data are limited to only a few COGs (Clusters of Orthologous Groups) of the SPOUT superfamily.7 Therefore, further structural characterization of the SPOUT superfamily members are required.
Thermotoga maritima Tm1570 is a member of the SPOUT MTase superfamily,7 belonging to the COG4752 family, which comprises a few bacterial proteins with erratic phyletic distribution, mostly from extremophiles (e.g., T. maritima and Synthropus acidotrophicus) but also from pathogens such as Fusibacterium nucleatum.7 COG4752 also has been dubbed SpoU family, implying a functional link with 2′-O-ribose MTases.14 However, the molecular function of Tm1570 remains to be established. In this study, we have solved the crystal structure of the T. maritima Tm1570 MTase in complex with AdoMet at 2.00 Å resolution. This work represents the first crystal structure of a member of the COG4752 family, thus contributing to expand our knowledge on the structural features of the SPOUT MTase superfamily.
MATERIALS AND METHODS
Protein expression and purification
The Tm1570 gene from T. maritima was cloned into the expression vector pET-28b(+) (Novagen), adding a hexa-histidine-containing 20-residue tag to the N-terminus of Tm1570. The recombinant protein was overexpressed in Escherichia coli Rosetta(DE3) cells using Terrific-Broth culture medium. Protein expression was induced by 0.5 mM isopropyl 1-thio-β-D-galactopyranoside and the cells were incubated for additional 18 h at 15°C following growth to mid-log phase at 37°C. The cells were lysed by sonication in a lysis buffer (50 mM Tris-HCl at pH 7.9, 500 mM NaCl, and 10% (v/v) glycerol) containing 50 mM imidazole. The crude lysate was centrifuged at ∼3000g for 60 min. The supernatant was applied to an affinity chromatography column of nickel-nitrilotriacetic acid-agrose (Qiagen). The protein was eluted with the lysis buffer containing 500 mM imidazole and the eluted sample was diluted fivefold with buffer A (50 mM Tris-HCl at pH 7.2, 5% (v/v) glycerol, and 10 mM β-mercaptoethanol). The diluted sample was applied to a Source 15Q ion-exchange column (GE Healthcare Bio-Science), which was previously equilibrated with buffer A. The protein was eluted with a linear gradient of 0–1.0 M NaCl in buffer A. The next step was gel filtration on a HiLoad 16/60 Superdex-200 prep-grade column (GE Healthcare Bio-Science), employing an elution buffer of 20 mM Tris-HCl at pH 7.2 and 100 mM NaCl. Fractions containing Tm1570 were concentrated to 15 mg mL−1 using an Amicon Ultra-15 centrifugal filter unit (Millipore).
Crystallization, X-ray data collection, and structure determination
Crystals were grown by the hanging-drop vapor diffusion method at 24°C by mixing equal volumes (2 μL each) of the protein solution (at 15 mg mL−1 concentration in 20 mM Tris-HCl at pH 7.2 and 100 mM NaCl) and the reservoir solution. We obtained tetragonal bipyramidal crystals using a reservoir solution consisting of 100 mM Tris-HCl at pH 7.0, 200 mM calcium acetate, and 20% (v/v) PEG 3000. The crystals grew within a few days to approximate dimensions of 0.1 mm × 0.1 mm × 0.3 mm. We soaked the native crystals for 30 min in the above reservoir solution containing 10 mM K2PtCl4 for multiwavelength anomalous diffraction (MAD) phasing.
A platinum derivative crystal was frozen in the cold nitrogen gas stream at 100 K using a cryoprotectant solution, which consisted of 30% (v/v) glycerol added to the reservoir solution. X-ray diffraction data were collected at 100 K on a Bruker CCD area detector system at the BL-6B experimental station of Pohang Light Source, Korea. For each image, the crystal was rotated by 1° and the raw data were processed using the program suit HKL2000.15 The platinum derivative crystal belongs to the space group C2, with unit cell parameters of a = 80.14 Å, b = 63.53 Å, c = 54.73 Å, and β = 128.96° (for peak data). Native data were collected similarly and the cell parameters are a = 79.65 Å, b = 63.40 Å, c = 54.52 Å, and β = 129.03°. There is one Tm1570 monomer per asymmetric unit, giving a solvent fraction of 44.8%. Table I summarizes the data collection statistics.
|A. Data collection and phasing|
|Space group: C2; Unit cell parameters: a = 80.14 Å, b = 63.53 Å, c = 54.73 Å, β = 128.96° (for peak data)|
|Data set||Pt λ1 (peak)||Pt λ2 (inflection)||Pt λ3 (remote)|
|X-ray source||Pohang Light Source (BL-6B)||Pohang Light Source (BL-6B)||Pohang Light Source (BL-6B)|
|X-ray wavelength (Å)||1.07163||1.07204||1.06261|
|Resolution range (Å)||20–2.30||20–2.30||20–2.30|
|Completeness (%)a||98.9 (96.2)a||98.7 (93.5)a||98.5 (93.0)a|
|Rmerge (%)b||0.93 (25.9)b||8.5 (21.6)b||8.3 (22.3)b|
|Riso (%)c||4.1 (7.8)c||6.0 (9.8)c|
|f ′/f ″ (e−)||−2.7/6.7||−9.6/6.5||−2.2/3.7|
|Figure of meritd for MAD phasing: 0.32 / 0.72 for 20–2.30 Å data (before/after density modification)|
|Data set||AdoMet complex|
|X-ray source||Pohang Light Source (BL-6B)|
|X-ray wavelength (Å)||1.0000|
|Resolution range (Å)||20–2.00|
|Completeness (%)e||98.4 (91.2)e|
|Rmerge (%)e||7.1 (24.7)e|
|No. of protein atoms (B-factor, Å2)||1515 (23.7)|
|No. of solvent atoms (B-factor, Å2)||128 (33.3)|
|No. of ligand atoms (B-factor, Å2)||27 (22.3)|
|R.m.s.deviation from ideal geometry|
|Bond lengths (Å)||0.012|
|Bond angles (°)||1.42|
|Most favored (%)||90.5|
|Additional allowed (%)||9.5|
|Generously allowed (%)||0|
We have solved the crystal structure of Tm1570 by MAD phasing at 2.30 Å resolution (Table I). Two platinum sites were located with the program SOLVE16 and they were used to calculate the phases with RESOLVE.17 Phasing statistics are summarized in Table I. The MAD-phased electron density map was of high quality and was readily interpreted by the automatic model building procedure of RESOLVE. The initial model accounted for ∼80% of the backbone of the polypeptide chain with much of the sequence assigned. Subsequent manual model building was done using the program COOT.18 The model was refined with the program REFMAC,19 including the bulk solvent correction. Ten percent of the data were randomly set aside as the test data for the calculation of Rfree.20 Table I summarizes the refinement statistics. The refined model has excellent stereochemistry (Table I), as evaluated by the program PROCHECK.21
Atomic coordinates and structure factors have been deposited in the RCSB Protein Data Bank (accession code 3DCM).
RESULTS AND DISCUSSION
We have determined the crystal structure of Tm1570 complexed with AdoMet. The refined model includes 188 residues, 128 water molecules, and one AdoMet molecule. The R and Rfree factors were 19.8% and 23.5%, respectively, for the 20–2.00 Å data (Table I). The C-terminal four residues of Tm1570 (Ala189-Asp192), as well as the 20-residue N-terminal fusion tag, are disordered in the crystal and have not been included in the model.
The monomer of Tm1570 comprises a central five-stranded, parallel β-sheet (β4-β5-β1-β2-β3) [Fig. 1(A,B)] that is surrounded by seven helices. It is folded into the classical Rossmannoid α/β-fold,6 resembling structures of the core MTase domain in other SPOUT superfamily members. It contains a deep trefoil knot in its C-terminal part [Fig. 1(B)], which is a common feature of the SPOUT superfamily MTases.
Tm1570 exists as a dimer in the crystal [Fig. 1(C)], with a buried surface area of 1490 Å2 per monomer, which corresponds to ∼15% of the monomer surface area (9930 Å2). The dimer interface is mediated through van der Waals packing of a number of hydrophobic residues (Leu29, Ile161, Phe167, Leu170, and Ile178), as well as a hydrogen network formed by polar side chains of Asp33, Thr37, Ser171, and Arg173.
Although AdoMet was not included in the crystallization medium, the Fo – Fc omit electron density map showed a AdoMet molecule bound at the active site of Tm1570 [Fig. 1(A,C)]. The adenosine moiety of AdoMet makes numerous interactions with the knotted loop regions [Fig. 1(D)], while the methionine moiety of AdoMet makes fewer interactions with the protein. Accordingly, the electron density of the aliphatic portion of the methionine moiety is a little weak [Fig. 1(E)]. The methyl group of AdoMet points towards the positively charged cleft [Fig. 1(F)], which is likely to be involved in binding a negatively charged RNA substrate. The AdoMet-binding loop on the knot is stabilized by interactions with the other monomer, indicating that dimerization is important for the MTase activity as in other SPOUT superfamily members.7
A structural similarity search using the DALI server22 revealed RNA MTases as the closest structural homologs of Tm1570. The highest Z-score is obtained with Thermus thermophilus (Tth) tRNA(Gm18) methyltransferase (TrmH)9 [PDB code 1V2X; a root mean square (rms) deviation of 2.4 Å for 139 equivalent Cα positions, a Z-score of 14.7, and a sequence identity of 19%]. TrmH catalyzes methylation of the 2′-OH of guanosine 18 in tRNA. Tm1570 exhibits a ‘perpendicular’ dimerization mode.7 TrmH forms a similar ‘perpendicular’ dimers, whereas TrmD forms ‘antiparallel’ dimers.7 However, a significant difference in the dimerization pattern between Tm1570 and Tth TrmH is indicated by comparing the dimers of Tm1570 and Tth TrmH. The rms deviation between Tm1570 and Tth TrmH dimers (8.2 Å for 202 equivalent Cα positions) is considerably higher than that between monomers of Tm1570 and Tth TrmH. The structural overlap of the dimers also indicates that the key active site residues of Tm1570 are Arg36 and Ser171, corresponding to Arg41 and Ser150 of Tth TrmH, respectively. Identification of the active site residues of Tm1570 by a sequence alignment would have been very difficult, if not impossible, due to a low level of sequence similarity and a long insertion between β1 and α1 in Tm1570 (Ile10-Asn28).
The next highest Z-score is obtained with methyltransferase AviRb from Streptomyces viridochromogenes23 [PDB code 1X7O; a rms deviation of 2.6 Å for 140 equivalent Cα positions, a Z-score of 14.5, and a sequence identity of 19%]. AviRb methylates the 2′-O atom of U2479 of the 23 S ribosomal RNA in gram-positive bacteria and thus mediates resistance to the oligosaccharide (orthosomycin) antibiotic avilamycin.23S. viridochromogenes AviRb methyltransferase exhibits a ‘perpendicular’ dimerization mode. Other RNA MTases such as HI076624 and Tth RrmA6 also show structural similarity to Tm1570.
Unlike many members of the SPOUT MTase superfamily that possess a unique and specialized RNA-binding extension outside the conserved AdoMet-binding domain,7 Tm1570 is composed of a single compact MTase domain. A distinct feature of Tm1570 is the presence of an extended loop between β1 and α1, which spans Ile10-Asn28 [Fig. 1(A)]. This unique insertion in Tm1570 covers the entrance to the active site, with Lys15 and Lys17 contributing to the positively charged surface patch at the entrance possibly to recognize the negatively charged substrate [Fig. 1(F)]. In addition, the side chain of Lys17 forms a hydrogen bond with the main chain carbonyl oxygen of Tyr74 of the other monomer, contributing to the formation of an entrance to the methyl group of the bound AdoMet [Fig. 1(F)]. Similarities in the overall structure and the location of the active site residues of Tm1570 to TrmH and rRNA MTases suggest that Tm1570 likely functions as an RNA 2′-O methyltransferase.
We thank the staff at beamline BL-6B of Pohang Light Source, Korea for assistance during X-ray data collection experiments.