TY - JOUR
AU1 - Dégut,, Clément
AU2 - Roovers,, Martine
AU3 - Barraud,, Pierre
AU4 - Brachet,, Franck
AU5 - Feller,, André
AU6 - Larue,, Valéry
AU7 - Al Refaii,, Abdalla
AU8 - Caillet,, Joël
AU9 - Droogmans,, Louis
AU1 - Tisné,, Carine
AB - Abstract 1-Methyladenosine (m1A) is a modified nucleoside found at positions 9, 14, 22 and 58 of tRNAs, which arises from the transfer of a methyl group onto the N1-atom of adenosine. The yqfN gene of Bacillus subtilis encodes the methyltransferase TrmK (BsTrmK) responsible for the formation of m1A22 in tRNA. Here, we show that BsTrmK displays a broad substrate specificity, and methylates seven out of eight tRNA isoacceptor families of B. subtilis bearing an A22. In addition to a non-Watson–Crick base-pair between the target A22 and a purine at position 13, the formation of m1A22 by BsTrmK requires a full-length tRNA with intact tRNA elbow and anticodon stem. We solved the crystal structure of BsTrmK showing an N-terminal catalytic domain harbouring the typical Rossmann-like fold of Class-I methyltransferases and a C-terminal coiled-coil domain. We used NMR chemical shift mapping to drive the docking of BstRNASer to BsTrmK in complex with its methyl-donor cofactor S-adenosyl-L-methionine (SAM). In this model, validated by methyltransferase activity assays on BsTrmK mutants, both domains of BsTrmK participate in tRNA binding. BsTrmK recognises tRNA with very few structural changes in both partner, the non-Watson–Crick R13–A22 base-pair positioning the A22 N1-atom close to the SAM methyl group. INTRODUCTION Transfer RNAs (tRNAs) contain numerous modified nucleosides formed post-transcriptionally by a variety of enzymes (1). Amongst nucleoside modifications, methylations are the most frequently occurring and position-wise diverse. Their formation is catalysed by methyltransferases (MTases) which most-commonly use S-adenosyl-l-methionine (SAM) as the methyl donor. The tRNA core contains many different modified nucleosides, notably all the 1-methyladenosine (m1A) found in tRNA. The formation of m1A occurs by transfer of the SAM-methyl group onto the N1-atom of adenosine. It is found at positions 9, 14, 22 and 58 of tRNAs (1,2), and is also formed at position 57 as an intermediate in the biosynthesis of 1-methylinosine (3,4). The m1A9, m1A14 and m1A58 nucleosides are involved in non-Watson–Crick base-pairing that are important to assemble the tRNA elbow and to maintain the D- and the T-loops in close contact. Different enzymes are responsible for the m1A formation at the different positions in tRNAs. The m1A at position 9 (m1A9) in human mitochondrial and archaeal tRNAs is formed by the enzyme Trm10 which belongs to the SPOUT family of MTases (5,6). Remarkably, yeast Trm10 forms 1-methylguanosine at position 9 (m1G9) (7) and the Trm10 enzymes from human mitochondria and from several archaea display dual specificity, forming both m1A9 and m1G9. (5,6,8). In human mitochondrial tRNALys, m1A9 prevents the formation of an alternative structure of this tRNA by hampering a Watson–Crick base-pairing between A9 and U64 (9). The presence of m1A14 has only been reported in a limited number of mammalian cytoplasmic tRNAPhe (10) and the gene encoding the corresponding MTase is still unknown. In contrast, the m1A58 modification is found in tRNAs from organisms belonging to all domains of life. The genes and corresponding enzymes responsible for this modification (Trm6-Trm61 in Eukarya and TrmI in Bacteria and Archaea) have been identified and structurally characterised in yeast (11), in human (12), in the bacteria Thermus thermophilus (13–17) and Mycobacterium tuberculosis (18), and in the archaeon Pyrococcus abyssi (4,19). The presence of m1A22 in tRNA is rather scarce and its formation has been much less studied compared to m1A9 and m1A58. For organisms where tRNA sequences are available (1), m1A22 is found in tRNAs of Bacillus subtilis (tRNASer, tRNATyr, both with large variable regions), of Geobacillus stearothermophilus (tRNALeu and tRNATyr), of Mycoplasma capricolum (tRNAGln, tRNACys, tRNAGlu, tRNAHis, tRNALeu, tRNASer and tRNATyr) and of Mycoplasma mycoides (tRNASer). Early studies showed that a SAM-dependent m1A22 MTase activity was present in B. subtilis extracts (20,21). More recently, the yqfN gene of B. subtilis (now trmK) was shown to encode the enzyme TrmK responsible for m1A22 formation in tRNA (22). TrmK belongs to the COG2384 protein family and orthologs are found in Gram-positive and -negative bacteria, without any equivalent found in Eukarya or Archaea. A B. subtilis mutant in which the trmK gene has been inactivated showed no detectable phenotype, neither at growth nor at sporulation level (22). However in the bacteria Streptococcus pneumoniae and M. mycoides, the TrmK ortholog is essential for survival of the organism (23,24). TrmK is well conserved among pathogenic bacteria, such as Vibrio cholerae, Listeria monocytogenes, Staphylococcus aureus, S. pneumoniae, and displays high sequence identity across the family. There are currently three X-ray crystal structures available for COG2384 family members (Protein data bank (PDB): 3LEC, 3GNL, 3KR9) (25) with no further data published on these. Here, we studied the relationship between the structure and the function of the B. subtilis m1A22 MTase TrmK (BsTrmK) with special focus on the enzyme-tRNA recognition. We investigated the properties that establish tRNAs as substrates of BsTrmK, and we report how a single point mutation in tRNA can convert a non-substrate into a substrate. The identified nucleotide playing a central role in BsTrmK substrate definition is involved in base-pairing with A22, the target site of methylation by BsTrmK. We also solved the crystal structure of BsTrmK and used NMR spectroscopy to gain insight into the recognition mode of B. subtilis tRNASer (BstRNASer) by BsTrmK. Based on the NMR data, we constructed a docking model of the BsTrmK/SAM/BstRNASer complex, the validation of which was performed with additional biochemical data. This work provides a clear picture of the relationship between structure and activity for the TrmK protein family. MATERIALS AND METHODS Cloning, expression and purification of BsTrmK for NMR and X-ray studies Recombinant BsTrmK was expressed and purified as previously described (22). The trmK gene was amplified by PCR and cloned into the pCRII blunt vector and then transferred into the pET28b expression vector, allowing T7 expression of an N-terminal His6-tagged recombinant protein. For structural studies, a variant of BsTrmK in which the two cysteine residues were replaced by serine ones (C35S and C152S) was used. Mutagenesis was performed using the QuikChange site-directed mutagenesis kit (Agilent). The presence of the desired mutation in trmK was checked by sequencing. This variant was overexpressed in the E. coli (BL21(DE3) strain). The induction was performed by adding 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) after having grown the bacteria to an optical density of 0.6 at 600 nm. Cells were harvested after incubation at 18°C during 24 h, pelleted and frozen at −80°C until further use. The frozen cells were suspended in 20 ml of a 50 mM Tris/HEPES buffer pH 8.2 containing 500 mM NaCl, 5% glycerol and 1 mM of phenylmethanesulfonylfluoride (PMSF). The suspension was sonicated and the lysate was centrifuged for 30 min at 15 000 g. The resulting supernatant was applied to a 5 ml Nickel Sepharose column (HisTrap, GE Healthcare) previously equilibrated in a 50 mM Tris–Cl buffer pH 8.0 containing 500 mM NaCl and 5% glycerol (equilibration buffer). The resin was then washed with 30 ml of buffer and the protein was eluted with a gradient of the equilibration buffer supplemented with 500 mM imidazole pH 8.0. The N-terminal His6-tag was removed from BsTrmK by thrombin cleavage (25 U thrombin/mg of protein) performed overnight at 4°C. 250 μM PMSF and 1 mM EDTA were then added to the protein sample. For X-ray studies, an additional step of hydrophobic chromatography was performed. The protein in 1 M ammonium sulphate was applied to a Phenyl Sepharose column (HiLoad 26/10 SP Sepharose, GE Healthcare). BsTrmK was eluted by a 1 to 0 M ammonium sulphate reverse gradient. The sample was then concentrated (Millipore Amicon, Molecular weight cut-off 10 kDa) and injected on a size exclusion chromatography column (Superdex-75 26/60, GE Healthcare) equilibrated in a 50 mM Tris–Cl buffer pH 8.0 containing 500 mM NaCl and 2% glycerol. For NMR experiments, the (C35S, C152S) BsTrmK variant was purified as previously described (26). For the assignment procedure of NMR signals, samples of 2H/13C/15N-labeled BsTrmK at 0.7 mM were prepared in a 50 mM sodium phosphate buffer pH 7.0, 500 mM NaCl and 2% glycerol. NMR chemical shift mappings were performed with samples of 2H/13C/15N-labeled BsTrmK concentrated around 0.2 mM in a 50 mM Tris–Cl buffer pH 8.0 containing 100 mM NaCl, 10 mM MgCl2 and 2% glycerol. Production of B. subtilis tRNASer BstRNASer was produced in vitro by T7 transcription and also in vivo in E. coli as described previously (26). To ensure sample homogeneity, prior to purification, in vitro transcribed BstRNASer was extensively dialyzed against water and diluted 10-fold in water, then refolded by heating the sample to 95°C and slowly cooled down to room temperature. 10 mM of MgCl2 was then added. Fragments of BstRNASer were purchased from Dharmacon (GE Healthcare). Analysis of BsTrmK oligomerisation and subsequent MTase activity Pure BsTrmK (5 mg) was loaded on a Superose P12 column equilibrated in a 50 mM Tris–Cl buffer pH 8.0 supplemented with 10 mM MgCl2 and 500 mM NaCl at a flow rate of 24 ml/h. The same sample in the presence of 10 mM DTT was also similarly analysed. The calibration of the column was assessed by loading in the same buffer a mixture of ovalbumine and bovine serum albumine (BSA). The MTase activity of each peak was determined by measuring the amount of 14C transferred to total E. coli tRNA using methyl-14C SAM as methyl donor. The reaction mixture was 200 μl: 70 μg of total E. coli tRNAs, 400 nCi of methyl-14C SAM (50 mCi/mmol), the fraction containing BsTrmK and H2O. Incubation was for 1 h at 37°C. The reaction was stopped by phenol extraction, and tRNA was TCA precipitated and captured on a GF/C Glass microfiber filter (Whatman) for scintillation counting. Cloning of B. subtilis tRNA genes in pUC18 plasmid The cloning of the B. subtilis genes of tRNA Cys, -Gln, -Leu, -His, -Tyr and -Gly were performed as for the cloning of tRNASer (22). The T7 promoter was added immediately upstream of each tRNA gene to promote high levels of transcription. An MvaI restriction site was added downstream of the genes in order to obtain the 3′-CCA extremity of the tRNA transcripts. tRNA sequences are presented in Supplementary Figure S1. Measurements of the MTase activity of BsTrmK using 32P-radiolabelled B. subtilis tRNAs The measurements of MTase activity were conducted on the wild-type (WT) BsTrmK in presence of 10 mM DTT. Radioactive (32P) in vitro transcripts were obtained as previously described (22) using MvaI digested plasmids containing the tRNA genes, or mutated variants, as templates. Mutated tRNA genes were obtained by site directed mutagenesis (Agilent). [α-32P]-ATP (3000 Ci/mmol) was purchased from Perkin Elmer and T7 RNA polymerase from Promega. Radioactive transcripts were purified by 10% polyacrylamide gel electrophoresis. The transcripts (300 000 cpm) were incubated for 15 min at 37°C in a 300 μl reaction mixture containing 0.2 mM SAM, 50 mM MOPS pH6.5, 1 mM MgCl2, 10 mM DTT and various amounts of BsTrmK. They were subsequently ethanol precipitated and digested with nuclease P1. The resulting nucleotides were analysed by thin layer chromatography (TLC) on cellulose plates followed by autoradiography as described previously (4). Plates were visualised by autoradiography. Steady-state kinetic analysis of BsTrmK [Methyl-3H]-SAM (15 Ci/mmol, Perkin Elmer) was mixed with non-radioactive SAM (Sigma) to achieve a specific radioactivity of about 500 cpm/pmol. The methylation kinetic assays were performed in 50 mM Tris–Cl pH 8.0 with 5 mM MgCl2 at 37°C. Aliquots (300 μl) were removed after different incubation times and transferred into 5 ml of 5% (w/v) trichloroacetic acid (TCA) at room temperature for 10 min in order to quench the reaction and to precipitate the tRNA. The precipitates were collected by filtration using GF/C filters (Whatman). The filters were washed with ethanol, dried, and the radioactivity was measured by liquid scintillation counting for 2 min, resulting in a counting error below 4%. Data were corrected by subtracting the background radioactivity determined from a control without enzyme. For the determination of the KM and Vmax of BsTrmK for tRNASer, the reaction mixtures contained BsTrmK (100 nM), tRNASer (10–2000 nM) and 3H-AdoMet (90 μM). Initial rates (vi) for each substrate concentration (tRNASer) were determined from the slope of linear fittings of time course data points (5, 10, 15, 20, 30 min). Enzyme parameters were obtained by non-linear least square fitting using Equation (1) for Michaelis–Menten kinetics. Confidence limits of the parameters at 90% were estimated by Monte-Carlo sampling using the MC-Fit program (27). \begin{eqnarray*}{v_i} = \frac{{{V_{\max }}}}{{2{E_0}}}\left( {{K_{\rm M}} + {S_0} + {E_0} - \sqrt {{{({K_{\rm M}} + {S_0} + {E_0})}^2} - 4{S_0}{E_0}} } \right)\end{eqnarray*} (1) Preparation of BsTrmK mutants and MTase assays All the TrmK mutants were generated using the QuikChange mutagenesis kit (Agilent) and purified by affinity chromatography on Ni2+ loaded Chelating Sepharose as described above. The MTase assays were performed in 50 mM MOPS-Na pH 6.5, 1 mM MgCl2, 10 mM DTT, 50 μg unfractionated tRNA from the B. subtilis (delta trmK) strain (22), 25 nCi [methyl-14C]-SAM (58 mCi/mmol, Perkin Elmer) and 0.2 μg enzyme in a total volume of 300 μl. Incubation was for 30 min at 37°C. The reaction was stopped by phenol extraction and the tRNA was precipitated by transferring the aqueous phase in 5 ml of 5% TCA. The precipitates were collected by filtration using GF/C filters (Whatman). The filters were washed with ethanol, dried, and the radioactivity was measured by liquid scintillation counting. The experiments were performed in triplicates. Crystallisation and X-ray structure resolution of BsTrmK Crystallisation of BsTrmK was performed at 4°C using the sitting-drop vapor-diffusion method. Protein samples were prepared at 3 mg/ml in 50 mM Tris–Cl buffer (pH 8.0) containing 500 mM NaCl and 2% glycerol. Drops of 1.6 μl were prepared by a Cybi-Disk robot mixing equal volume of protein and reservoir (100 μl). Crystals of BsTrmK were first obtained in condition A10 of the Hampton Research Crystal Screen kit. After optimisation, diffracting crystals were finally obtained in 0.2 M ammonium acetate, 0.1 M sodium acetate trihydrate pH 5, 30% (w/v) polyethylene glycol 4000 and 8% 2-methyl-2,4-pentanediol using microseeding from needle clusters. Crystals were harvested and flash-frozen in liquid nitrogen. Diffraction data were collected at beamline ID14-1 of the European Synchroton Radiation Facility (Grenoble, France). X-ray diffraction data were processed using XDS and scaled with SCALA. The structure was solved by molecular replacement with PHASER using an average model of the structures of sp1610 (Streptococcus pneumoniae ortholog of BsTrmK, PDB code 3KR9), LMOf2365_1472 (Listeria Monocytogens, PDB code 3GNL) and of SAG1203 (Streptococcus agalactiae, PDB code: 3LEC). In the resulting model, non-conserved residues were mutated into alanine and model building was first performed with ARP/WARP using the warpNtrace automated procedure. Restrained refinements of the structure were performed with the program phenix.refine in the Phenix suite (28). Model and map visualizations for manual reconstructions were performed with the program Coot (29). The BsTrmK structure has been deposited to the Protein Data Bank under the accession code 6Q56. Normal mode analysis of BsTrmK was conducted with the program ProDy (30) and visualized using VMD (31). Isothermal titration microcalorimetry (ITC) ITC experiments were carried out at 18°C in a MicroCal® ITC200 microcalorimeter with BsTrmK extensively dialyzed against 50 mM Tris–Cl pH 8.0, 100 mM NaCl, 10 mM MgCl2, 2% glycerol. Twenty four injections of 1.6 μl of S-adenosyl-l-homocysteine (SAH) at 800 μM, dissolved in the buffer of BsTrmK dialysis, were made into the cell containing BsTrmK (58 μM). Injections were spaced by 180 s. The ITC data were analysed with the software ORIGIN® using a single set of sites model. NMR chemical shift mapping NMR spectra were recorded at 18°C on Bruker spectrometers (either 950, 800 or 600 MHz) equipped with TCI-5 mm cryoprobes. Backbone assignments were performed as previously described (32). Chemical shift mappings of the interaction between SAH and BsTrmK and BstRNASer and BsTrmK previously bound to SAH were obtained using 2H/13C/15N-labeled BsTrmK (0.14 mM) mixed with 4 equivalents of SAH and two equivalents of BstRNASer in a 3 mm NMR tube. The NMR chemical shift mapping was conducted in a 50 mM Tris–Cl buffer pH 8.0 containing 100 mM NaCl, 10 mM MgCl2 and 2% glycerol. For each mixture, one TROSY experiment (33) was recorded. TOPSPIN, mddNMR (34) and Sparky software were used to process and analyse NMR data. Chemical shift differences Δ(H,N) were derived from 1H and 15N shift differences : Δ(H,N) = √ [(Δ15N WN)2 + (Δ1H WH)2], where Δ = δ complex - δ free (difference of chemical shift between the complex and the free states) and WH = 1 and WN = 1/5. Molecular docking of BsTrmK/SAM/tRNASer BstRNASer model was built using the T. thermophilus tRNASer crystal structure (PDB: 1SER) as template except for the variable region that was not defined in this structure. To model the variable region of BstRNASer, the Human tRNASec crystal structure (PDB: 3A3A) was used. It was manually mutated to the BstRNAser sequence using Coot (29). Model geometry was then idealised with 300 iterations of rna_idealise from the Rosetta package, and subsequently minimised using the Rosetta force field through rna_minimise (35). Docking of B. subtilis tRNASer on BsTrmK/SAM structure was performed using HADDOCK 2.2 (36,37). HADDOCK program is particularly well-suited to deal with data from NMR chemical shift mapping through the possibility to introduce ambiguous restraints (i.e. restraints that are applied between residues and not between atoms). Restraints were generated based on NMR chemical shift mapping upon BstRNASer binding. Residues from BsTrmK with NMR chemical shift variations of their amide group superior to 0.03 ppm upon tRNASer binding (8, 9, 13, 37, 46, 48, 49, 57, 67, 78, 80, 82, 83, 88, 93, 99, 101, 119, 123, 124, 144, 145, 147, 149) were defined as active residues whereas surrounding residues of the surface (3, 7, 10, 11 14, 27, 28, 29, 30, 31, 32, 51, 52, 53, 54, 55, 58, 59, 60, 67, 75, 94, 95, 96, 97, 98, 10, 146, 150, 151, 153) were defined as passive. The A22 nucleotide, the target of BsTrmK for methylation, was defined as active residue for the BstRNASer whereas, the nucleotides 12 to 23 of the D-arm were defined as passive ones. A 1.8 to 3.2 Å distance constraint was applied between the N1-atom of A22 and the SAM-methyl group. The HADDOCK modeling program allows any of the atoms from each residue designated as an ambiguous interaction restraint to interact with the nucleotides 12–23 of the tRNASer D-arm with the constraint that N1 of A22 is placed near the methyl-group of SAM. Ten thousand structures of the complex were generated during the rigid-body energy minimization, and the 400 best solutions based on the intermolecular energy were used for the semiflexible refinement and were subsequently refined in the explicit solvent iteration. Semi-flexible zones (automatically determined by HADDOCK) and full-flexible ones defined on BsTrmK loops (residues 3–10, 49–56, 93–99, 121–127, 146–153) were used. We set the Edesol weight to 0 for all stages of the docking as it is recommended for protein–nucleic acids docking. The 400 structures were then sorted into clusters by calculating the RMSDRNA that we define as the RMSD calculated on the BstRNASer phosphate backbone between two models relative to the BsTrmK backbone. In other words, we compare the positioning of the BstRNASer on BsTrmK in the different models by superimposing the models along the BsTrmK backbone. To belong to the same cluster, two models must have a RMSDRNA below 12 Å, this cut-off was selected to differentiate between major orientation differences but allows for small rocking move of the tRNA on the protein surface. HADDOCK score was then used to identify best solutions, to rank models, and to check consistency of each cluster (Supplementary Figure S12). The atomic coordinates of the top-scoring model of BsTrmK/SAM/BstRNASer complex are available as Supplementary data, TrmK_SAM_tRNASer.pdb. RESULTS BsTrmK displays a broad tRNA substrate specificity Amongst the DNA sequences of all the tRNA genes of B. subtilis, eight different tRNA isoacceptor families bear an A at position 22. For only two of these tRNAs, i.e. tRNATyr and tRNASer, the modification pattern has been determined, and both carry the modified nucleoside m1A22 (1). To determine if the remaining B. subtilis tRNAs (tRNA-Cys, -Gln, -Leu, -His, -Glu and -Gly) could undergo m1A22 formation catalysed by BsTrmK, the gene of one representative of each of the tRNA isoacceptor families (Supplementary Figure S1) was cloned into the pUC18 vector downstream of a T7 promotor to allow in vitro transcription. These tRNAs present a large diversity of sequence, notably at the level of the variable region, e.g. tRNACys with a short variable region and tRNASer with a very long variable region. Transcripts were generated in the presence of α 32P-ATP and used in an in vitro MTase assay with different amounts of purified recombinant BsTrmK (Figure 1). tRNALeu showed a two-fold decreased m1A22 formation (Figure 1G) compared to the other substrate tRNAs, and tRNAGly was not methylated at all by BsTrmK (Figure 1H). Therefore, all B. subtilis tRNAs with an A22, except tRNAGly, could be methylated, with no discrimination between the length of the variable region, indicating that these tRNAs could also be N1-methylated on A22in vivo. This observation would mean that the occurrence of m1A22 is underestimated in tRNA modification databases (1,38), due to a lack of systematic mapping of tRNA modifications in B. subtilis and in Bacteria in general. Figure 1. View largeDownload slide BsTrmK methylates a variety of B. subtilis tRNAs. Autoradiograms of chromatograms of P1 hydrolysates of [α32P]ATP-labelled T7-transcripts of B. subtilis tRNAs incubated during 15 minutes with SAM and increasing amounts of purified BsTrmK (from 0 to 600 nM). The sequences of the tRNAs tested as substrates are drawn above and below the autoradiograms. (A) tRNASer, (B) tRNAHis, (C) tRNATyr, (D) tRNACys, (E) tRNAGln, (F) tRNAGlu, (G) tRNALeu, (H) tRNAGly. Figure 1. View largeDownload slide BsTrmK methylates a variety of B. subtilis tRNAs. Autoradiograms of chromatograms of P1 hydrolysates of [α32P]ATP-labelled T7-transcripts of B. subtilis tRNAs incubated during 15 minutes with SAM and increasing amounts of purified BsTrmK (from 0 to 600 nM). The sequences of the tRNAs tested as substrates are drawn above and below the autoradiograms. (A) tRNASer, (B) tRNAHis, (C) tRNATyr, (D) tRNACys, (E) tRNAGln, (F) tRNAGlu, (G) tRNALeu, (H) tRNAGly. BsTrmK requires a purine at position 13 of the tRNA to catalyse m1A22 formation A comparison of the sequences of substrate (tRNASer, -Tyr, -Cys, -Gln, -Leu, -His and -Glu) and non-substrate tRNAs (tRNAGly) highlights two nucleotides, at positions 13 and 26, as potentially implicated in substrate discrimination by BsTrmK. Both positions are occupied by a purine (G or A) in all BsTrmK substrates, and by U13 and C26 in the non-substrate. To mimic the substrates, nucleotides at these positions in the non-substrate tRNAGly were replaced by either G13, A26, or both (Supplementary Figure S2). Adenosine was chosen for position 26, as this is the most frequently appearing nucleoside at this position in BsTrmK substrates. Both single-mutated tRNAGly (U13G and C26A) turned the previous non-substrate into a substrate for BsTrmK, with U13G increasing the amount of methylated A22 the most (Supplementary Figure S2). Compared to the variants bearing a single mutation, the double-mutant (U13G/C26A) proved to be an even better substrate for BsTrmK. The purine at position 13, important for m1A22 formation catalysed by BsTrmK, forms a non-Watson–Crick base pair, namely a Hoogsteen-Sugar base-pair, with the target nucleotide A22 (as seen for instance in the structure of T. thermophilus tRNASer (39), PDB: 1SER). To further investigate the importance of the nucleotide at position 13 for m1A22 formation, three variants of tRNASer (G at position 13, long variable region) and tRNAHis (A at position 13, short variable region) were produced with varying nucleotides at position 13, i.e. A, U or C for tRNASer, and G, U or C for tRNAHis, and tested as BsTrmK substrates (Figure 2A and B). In agreement with the result obtained with tRNAGly, a pyrimidine at position 13 (U or C) reduced the substrate potential of tRNASer and tRNAHis for BsTrmK. However this reduction is more pronounced for tRNASer indicating that other still unidentified elements are important for TrmK/tRNA recognition. Overall, the presence of purines at positions 13 and 26 renders tRNA a better substrate for BsTrmK. Figure 2. View largeDownload slide BsTrmK MTase assays with B. subtilis tRNAs mutated at position 13, and with tRNAHis altered in the T-loop or in the anticodon stem-loop. [α32P]ATP-labelled T7-transcripts of mutated tRNASer (A) or tRNAHis (B–D) of B. subtilis were incubated during 15 min with SAM and increasing amounts of purified BsTrmK. P1 hydrolysates were separated by thin layer chromatography. After autoradiography the radioactivity of the spots corresponding to pA and pm1A was measured by scintillation counting. The fraction of m1A obtained was plotted versus the corresponding BsTrmK concentration in the reaction mixture (A) for mutants of tRNASer at position 13, (B) for mutants of tRNAHis at position 13, (C) for variants of tRNAHis in the T-loop, (D) for variants of tRNAHis in the anticodon stem-loop. (E) Summary on the tRNASer structure of the determinants required for BsTrmK MTase activity: the length of the variable region (in green) is not important, the base-pair A22–N13 (in red) involves a purine at position 13, an intact 3D structure is necessary notably at the level of the T-loop (in orange) and anticodon stem (in orange). Figure 2. View largeDownload slide BsTrmK MTase assays with B. subtilis tRNAs mutated at position 13, and with tRNAHis altered in the T-loop or in the anticodon stem-loop. [α32P]ATP-labelled T7-transcripts of mutated tRNASer (A) or tRNAHis (B–D) of B. subtilis were incubated during 15 min with SAM and increasing amounts of purified BsTrmK. P1 hydrolysates were separated by thin layer chromatography. After autoradiography the radioactivity of the spots corresponding to pA and pm1A was measured by scintillation counting. The fraction of m1A obtained was plotted versus the corresponding BsTrmK concentration in the reaction mixture (A) for mutants of tRNASer at position 13, (B) for mutants of tRNAHis at position 13, (C) for variants of tRNAHis in the T-loop, (D) for variants of tRNAHis in the anticodon stem-loop. (E) Summary on the tRNASer structure of the determinants required for BsTrmK MTase activity: the length of the variable region (in green) is not important, the base-pair A22–N13 (in red) involves a purine at position 13, an intact 3D structure is necessary notably at the level of the T-loop (in orange) and anticodon stem (in orange). BsTrmK requires the intact three-dimensional structure of tRNA to catalyse m1A22 formation The tRNA molecule adopts a well-known L-shape 3D structure, in which the corner of the L, known as the tRNA elbow (40), is made up of tertiary interactions between the D- and T-loops (41). This network of interactions involves base-pairings of T54 with A58, G18 with Ψ55 and G19 with C56, and a further stacking of the purine at position 57 between base-pairs G18–Ψ55 and G19–C56. To determine if BsTrmK recognises a canonical tRNA L-shape structure, these crucial interactions between the D- and T-loops were abolished by introducing the following mutations in tRNAHis: U54C, U55G/C56G or U54C/U55G/C56G (Figure 2C). The triple-mutant of tRNAHis (U54C/U55G/C56G) showed very low substrate potential compared to WT tRNAHis, suggesting that BsTrmK recognises a canonical L-shaped structure for tRNA binding. In a similar manner, we tested the substrate potential of an RNA transcript covering the D-arm sequence or the D-arm sequence plus the anticodon stem-loop of BstRNASer. None of these transcripts were substrates for BsTrmK (data not shown). Next, the importance of the anticodon stem-loop for tRNA recognition by BsTrmK was evaluated. In order to determine whether BsTrmK interacts with tRNA anticodon loops or anticodon stems, five mutated tRNAHis constructs by altering either the loop or the stem were generated. In the loop, variants with a point mutation (U33G), an insertion of a G near position 36 (+G36a), and deletion of G37 (ΔG37) were produced. In the stem, a variant with three additional GC base pairs (C–G, G–C and C–G between A29–U41 and G30–C40) (ac stem +3 bp), and a variant of tRNAHis in which the entire anticodon stem-loop was removed (Δ(ac stem–loop), Supplementary Figure 1) were generated. None of the alterations in the anticodon-loop resulted in reduced m1A22 formation (Figure 2D). However, the Δ(ac stem–loop) construct showed a severe reduction in m1A22 formation (Figure 2D), suggesting that BsTrmK interacts with the anticodon stem of tRNA. This interaction most probably occurs through non-nucleotide-specific interactions since the (ac stem +3bp) construct remains modified and since the sequences of the anticodon stems are not conserved across BsTrmK substrates. Altogether, these data show that BsTrmK displays a broad tRNA substrate specificity. BsTrmK methylates full-length tRNAs with a purine at position 13 and an intact tRNA elbow structure. It also requires an anticodon stem that is probably recognized in a non-sequence-specific manner. BsTrmK is active as a monomer We previously showed that tetramerisation of bacterial m1A58 tRNA MTase (TrmI) was crucial for tRNA recognition (14,15). This prompted us to investigate the functional oligomeric state of BsTrmK by size exclusion chromatography (SEC) by measuring the MTase activity of each eluted fraction along the chromatogram (Supplementary Figure S3). On the SEC column, BsTrmK eluted both as monomers and higher oligomeric species. The activity measurements of each eluted fraction showed that BsTrmK is functional as a monomer. The higher oligomeric states of BsTrmK are formed via disulphide bonds involving the two cysteines in BsTrmK sequence at positions 35 and 152. Such bonds can be broken by addition of a reducing-agent, and addition of DTT to the MTase reaction buffer resulted in a dramatic increase of the enzymatic activity (Supplementary Figure S3), confirming that BsTrmK functions as a monomer in solution. Crystallisation and structure determination of BsTrmK No crystals of BsTrmK could be obtained either in the presence or absence of DTT. To facilitate crystallisation, the cysteines 35 and 152 were mutated into serine residues. The MTase activity of this mutant was equal to WT BsTrmK (Supplementary Figure S4). Therefore, all following structural studies were performed with this construct. Unlike WT BsTrmK, we obtained crystals of this double-mutant, solved its crystal structure by molecular replacement and refined it to a resolution of 1.98 Å with a final Rfree factor of 24% (Supplementary Table S1). Four molecules are present in the asymmetric unit (ASU) with a solvent percentage of 38.6% (Supplementary Figure S5A). BsTrmK displays an L-shaped structure (Figure 3) which allows two 2-fold symmetry related molecules to face each other in such a way that it hides the catalytic pocket of each protein. The four molecules in the ASU show little conformational difference between them (RMSD of 0.25 Å over all Cα atoms). The superimposition of the BsTrmK structure with its orthologs (PDB: 3KU1, 3GNL, 3KR9) gave an RMSD of 1.5 Å over around 220 Cα atoms, consistent with the high sequence identity of these proteins. Figure 3. View largeDownload slide Crystal structure of BsTrmK. SAM was modelled in the catalytic center of BsTrmK by superposing the BsTrmK structure with that of its ortholog from S. pneumoniae (PDB code 3KU1). It is represented as sticks. (A) Ribbon representation of BsTrmK, α-helices are coloured in red whereas β-strands are in yellow. (B) Location on the structure of BsTrmK of conserved residues, the sequence of B. subtilis TrmK was aligned with those of the representative members of the COG2384 family, (C) Representation of electrostatic surface potentials of TrmK, Positive charges are in blue whereas negative ones are in red with the maximum color saturation corresponding to −5 kT/e (red) and +5 kT/e (blue). The figure was prepared with the APBS PyMOL plug-in (52) and pdb2pqr webserver (53). (D) Normal mode analysis of BsTrmK showing the mobility of the last part of the C-terminal domain. The mean mobility of residues in the six first non-trivial computed normal modes is plotted as a function of the BsTrmK residue number. Figure 3. View largeDownload slide Crystal structure of BsTrmK. SAM was modelled in the catalytic center of BsTrmK by superposing the BsTrmK structure with that of its ortholog from S. pneumoniae (PDB code 3KU1). It is represented as sticks. (A) Ribbon representation of BsTrmK, α-helices are coloured in red whereas β-strands are in yellow. (B) Location on the structure of BsTrmK of conserved residues, the sequence of B. subtilis TrmK was aligned with those of the representative members of the COG2384 family, (C) Representation of electrostatic surface potentials of TrmK, Positive charges are in blue whereas negative ones are in red with the maximum color saturation corresponding to −5 kT/e (red) and +5 kT/e (blue). The figure was prepared with the APBS PyMOL plug-in (52) and pdb2pqr webserver (53). (D) Normal mode analysis of BsTrmK showing the mobility of the last part of the C-terminal domain. The mean mobility of residues in the six first non-trivial computed normal modes is plotted as a function of the BsTrmK residue number. BsTrmK consists of an N-terminal Class I MTase domain linked to a C-terminal coiled-coil domain The BsTrmK protein contains two domains (Figure 3A). The N-terminal catalytic domain, that harbours the binding site of the SAM cofactor, adopts a typical Rossmann-like fold of Class I MTases (RFM; Class-I MTases) with a twisted central β-sheet flanked by six α-helices. In the β-sheet, the five first β-strands are parallel and the last two are antiparallel. The catalytic core shows a high level of sequence conservation across COG2384 proteins (Figure 3B and Supplementary Figure S6). A DALI search (42) in the PDB with the catalytic domain of BsTrmK identified, apart from COG2384 proteins, a ribosomal protein L11 MTase (PDB: 3GRZ, Z-score 19, RMSD 2.1 Å over 147 Cα), the m2G6 tRNA MTase Trm14 (PDB: 3TM4, Z score 17.4, RMSD 2.4 Å over 152 Cα), the m1A58 tRNA MTase TrmI (PDB: 2PWY, Z-score 15.8, RMSD 2.4 Å over 151 Cα) and the m1G37 tRNA MTase Trm5 (PDB: 2ZZM, Z-score 14.5, RMSD 2.9 Å over 147 Cα). These proteins are all Class-I type RFMs, confirming that BsTrmK belongs to this class of proteins, but no obvious information for catalysis or substrate recognition could be drawn from these similarities. The C-terminal domain of BsTrmK contains four α-helices, the two largest of which form a coiled-coil motif. The domain displays low sequence homology across COG2384 proteins (Figure 3B and Supplementary Figure S6), and a DALI search did not reveal any ideal protein-fold matches. However, two proteins with coiled-coil domain were identified with low Z-scores: the seryl-tRNA synthetase (PDB: 1SRY, Z-score 6.0, RMSD 1.1 Å over 50 Cα) and the ribosomal protein L19 (PDB: 1S72, Z-score 6.1, RMSD 2.0 Å over 53 Cα). In these proteins, the encompassed coiled-coil motif binds an RNA, suggesting that in BsTrmK the C-terminal domain could also play such a role. The N- and C-terminal domain of BsTrmK are held together by numerous contacts, and a normal mode analysis of the BsTrmK structure confirms a lack of free movement between the two domains by identifying only the very end of the coiled-coil motif as flexible (Figure 3D). The interaction between BsTrmK and the cofactor product SAH was studied by ITC. The interaction is enthalpy-driven (ΔH ≈ −17 kcal/mol) with a single binding site and a dissociation constant (KD) of 1.7 μM (Supplementary Figure S7). Although SAM or SAH were present in crystallisation trials, crystals were of apo BsTrmK, i.e. no ligand bound. The electron density is well-defined across the BsTrmK structure and particularly for residues constituting the SAM-binding site, even in the absence of the cofactor. Only two residues, K151 and Y153, near the active site, show poor electron density suggesting mobility (Supplementary Figure S5B). SAM was modelled into the active site of BsTrmK, guided by the crystal structure of SAM-bound TrmK from S. pneumoniae (PDB: 3KU1). In the BsTrmK/SAM model, the SAM cofactor is bound at the centre of the catalytic domain in a pocket with residues harbouring negative electrostatic potentials conserved amongst COG2384 proteins (Figure 3B). The methyl group is pointing towards a region of positively charged and well conserved residues, likely important for tRNA binding (Figure 3C). To verify the model, three well-conserved residues (Supplementary Figure S6) directly interacting with SAM, i.e. R9, D78 and M96 were mutated to alanine, and the activity of these BsTrmK variants were tested. Each point-mutation resulted in a dramatic loss of BsTrmK MTase activity, confirming the importance of these residues in SAM binding. Indeed, BsTrmK R9A is totally inactive, and BsTrmK M96A and BsTrmK D78A mutants have almost completely lost MTase activity (Supplementary Figure S8). To further characterise the cofactor binding site, an NMR chemical shift mapping (Supplementary Figure S9) of the SAH binding on BsTrmK was performed. The NMR chemical shift mapping monitors the binding of SAH on the signals of the 15N-labelled amide groups of BsTrmK that were previously assigned (32), and chemical shift variations and peak disappearances resulting from SAH interaction can subsequently be used to map the SAH-binding site on BsTrmK structure. This NMR mapping identified, for instance, residues R9, L10, G77, D78, A94 and G95 as involved in SAH binding, in agreement with the modelled SAM in the BsTrmK structure (Supplementary Figure S9C). NMR chemical shift mapping of the binding interface between BsTrmK and BstRNASer Some post-transcriptional modifications were reported to stabilise and help fold tRNA structures. Therefore, we investigated if modifications in BstRNASer could make this a better substrate for BsTrmK, compared to the unmodified BstRNA. No homologue of TrmK is present in E. coli, enabling us to produce the tRNASer in this host, bearing all modifications formed by the E. coli enzymes i.e. Gm18, D19, T54 and Ψ55 (Figure 4A), but lacking m1A22. The B. subtilis tRNASer was overexpressed and purified in E. coli as previously described (26). The catalytic efficiency of BsTrmK towards this tRNASer was then compared to that measured with an in vitro transcribed tRNASer lacking modifications (Figure 4B). This analysis showed that the tRNASer produced in E. coli is less efficiently modified than the unmodified tRNASer. The catalytic efficiencies, Vmax/KM, for modified tRNASer reached 44% of that obtained for the unmodified tRNASer. The presence of post-transcriptional modifications in tRNA is likely to restrict m1A22 formation by BsTrmK. The in vitro transcribed tRNASer was thus chosen for structural investigations. Figure 4. View largeDownload slide Interaction of BstRNASer with BsTrmK/SAH deciphered by NMR. (A) The presence of post-transcriptional modifications in BsTrmK substrate alters the MTase activity of BsTrmK. Sequence of B. subtilis tRNASer produced in vivo in E. coli showing post-transcriptional modifications incorporated by E. coli modifying enzymes. (B) Kinetic and enzymatic parameters of BsTrmK measured for BstRNASer bearing (tRNASerin vivo) or not bearing (tRNASerin vitro) post-transcriptional modifications. Confidence intervals at 90% are indicated in parenthesis. An unmodified BstRNASer was chosen to study the interaction of BstRNASer with BsTrmK given that BstRNASer prepared in vitro is a better substrate. (C) Selected regions from the superposition of three 2D 1H–15N TROSY experiments, showing amide groups of BsTrmK, alone in black, with the SAH in red and with SAH and BstRNASer in green (D) NMR chemical shift mapping of BstRNASer binding reported onto the molecular surface of BsTrmK. Pink residues are those that disappeared upon addition of tRNA, residues that experience, upon tRNA binding, NMR chemical shift variations between 0.02 and 0.04 ppm are in orange and larger than 0.04 ppm are in red. Residues in grey are residues for which NMR chemical shift variations could not be measured either because it is a proline residue or a residue for which the NMR signal has disappeared upon SAH addition. Figure 4. View largeDownload slide Interaction of BstRNASer with BsTrmK/SAH deciphered by NMR. (A) The presence of post-transcriptional modifications in BsTrmK substrate alters the MTase activity of BsTrmK. Sequence of B. subtilis tRNASer produced in vivo in E. coli showing post-transcriptional modifications incorporated by E. coli modifying enzymes. (B) Kinetic and enzymatic parameters of BsTrmK measured for BstRNASer bearing (tRNASerin vivo) or not bearing (tRNASerin vitro) post-transcriptional modifications. Confidence intervals at 90% are indicated in parenthesis. An unmodified BstRNASer was chosen to study the interaction of BstRNASer with BsTrmK given that BstRNASer prepared in vitro is a better substrate. (C) Selected regions from the superposition of three 2D 1H–15N TROSY experiments, showing amide groups of BsTrmK, alone in black, with the SAH in red and with SAH and BstRNASer in green (D) NMR chemical shift mapping of BstRNASer binding reported onto the molecular surface of BsTrmK. Pink residues are those that disappeared upon addition of tRNA, residues that experience, upon tRNA binding, NMR chemical shift variations between 0.02 and 0.04 ppm are in orange and larger than 0.04 ppm are in red. Residues in grey are residues for which NMR chemical shift variations could not be measured either because it is a proline residue or a residue for which the NMR signal has disappeared upon SAH addition. Attempts to obtain co-crystals of unmodified BstRNASer with BsTrmK and SAH were unsuccessful, and we therefore turned to NMR spectroscopy to decipher the interaction of BsTrmK with BstRNA in solution. No perturbation of BsTrmK resonances was observed for the couple BstRNASer/BsTrmK in the absence of SAH or for BsTrmK-SAH in the presence of an RNA mimicking the D-arm, or the D-arm plus the anticodon stem of BstRNASer (data not shown). The NMR chemical shift mapping was performed by recording a (1H-15N) TROSY experiment on 2H–15N–13C-labeled BsTrmK bound to unlabeled SAH and unlabeled BstRNASer (Figure 4C, Supplementary Figure S10). The mapping monitors the binding of SAH and subsequently the binding of tRNASer by recording NMR chemical shift variations of the amide groups of BsTrmK. Binding of BstRNASer to BsTrmK-SAH caused major perturbations on NMR signals of BsTrmK, i.e. disappearance of 2 peaks corresponding to residues V13 and L57 near the SAH binding pocket, and NMR chemical shift variations of 32 peaks of the N-terminal domain (Supplementary Figure S10B). The residues that experienced significant chemical shift variations upon binding of BstRNASer, for instance G49, G98, I83, I124, D149 and L156 in Figure 4C, outline the binding surface of BstRNASer (residues in red and orange, Figure 4C and D). Residues that do not show chemical shift variations upon binding to BstRNASer correspond to residues that do not take part to this interaction, e.g. Y167 (residues in blue, Figure 4C and D). Residues which disappear upon SAH addition are excluded from further monitoring (residues in grey, Figure 4C and D). The tRNA binding surface locates on a face of BsTrmK also covering the cofactor binding pocket. This surface contains a number of positively charged patches (Figure 3C) ideal for RNA binding. No significant NMR chemical shift variations or disappearances of peaks could be observed within the C-terminal domain of BsTrmK (Figure 4D), indicating that this domain would not participate in tRNA binding. This is surprising as it is commonly thought that such domains in modification enzymes (i.e. domains surrounding the catalytic domain) are involved in RNA-binding, and other proteins with coiled-coil domains use this fold for RNA binding. Since the NMR chemical shift mapping uses the amide group resonances as probes, we cannot exclude that some residues with long side-chains like Arginine or Lysine could interact with BstRNASer in a way that would not be noticed by the backbone NMR chemical shift perturbation analysis. If this is the case, the binding surface of the C-terminal domain is limited to only a few residues, as it would otherwise have been detected by NMR. To investigate if the C-terminal domain of BsTrmK is dispensable for BsTrmK MTase activity, a BsTrmK variant lacking most of the C-terminal coiled-coil motif was designed. This construct of BsTrmK is still well-folded as evidenced by 2D-NMR and able to bind SAH but exhibits no MTase activity (Supplementary Figure S11), suggesting that the coiled-coil motif of the C-terminal domain does, in fact, play a role in tRNA binding. Lastly, the structure of BstRNASer upon binding of BsTrmK was investigated by NMR chemical shift mapping with a 15N-labeled tRNA (Supplementary Figure S12). The overlaps encountered in the NMR spectra did not allow us to assign all imino groups of BstRNASer, notably no assignment is available in the D-stem. However, since the assignment of the variable region is nearly complete, the titration data confirm that this region does not take part in the binding of BsTrmK. Upon binding of BsTrmK, most of the peaks do not experience any chemical shift variation indicating that the global folding of BstRNASer is conserved. Disappearances of peaks are observed at the top of the anticodon stem and at the junction between the acceptor and the T-stem for imino groups that are assigned. tRNA is bound via interactions with both domains of BsTrmK A model of the BsTrmK/SAM/BstRNASer complex was generated with the high-ambiguity-driven biomolecular docking program HADDOCK (36) using the data obtained from NMR as ambiguous interaction restraints (see Materials and Methods). Input data for HADDOCK calculations used BsTrmK residues with significant chemical shift perturbations as residue-level ambiguous interaction restraints, and further included the nucleotides 12 to 23 in the D-arm of tRNASer, assuming that nucleotides around A22 could interact with BsTrmK. A distance restraint between the N1-atom of A22 and the methyl of SAM was also added. Six clusters of models were extracted from HADDOCK calculations (Supplementary Figure S13), each exhibiting a very different relative orientation of the protein on the tRNA (Supplementary Figure S14). This reflects the feature that we do not have any restraint for the interaction of the C-terminal domain of BsTrmK with BstRNASer. The best model according to the HADDOCK scoring function is part of a cluster made of 102 structures with a mean RMSDRNA of 4.2 Å containing most of the best-scoring structures. The 5 other clusters present best-scoring structures with much lower scores. In clusters 5 and 6, there is no interaction between the C-terminal domain of BsTrmK and the tRNA, which is not compatible with our data. In cluster 2, the contacts are located at the level of the anticodon loop, in cluster 3 at the level of the variable stem and in cluster 4 at the level of the D-loop and the T-stem. According to our data, clusters 2 and 3 are not valid. Cluster 4 is only represented by five structures and harbours a much lower binding energy than the best-scoring structure of cluster 1. The top-scoring model of the cluster 1 was thus chosen as the best model of the BsTrmK/SAM/BstRNASer complex (the atomic coordinates of the model are available as Supplementary data, TrmK_SAM_tRNASer.pdb). In this model (Figure 5A), both domains of BsTrmK interact with BstRNASer, mainly through interactions with the phosphodiester backbone of the tRNA. The catalytic domain shares, to a large extend, a larger interaction surface with the tRNA, compared to that of the C-terminal domain. Almost all nucleotides of the D-arm (G13, A14, G15, U16, G19, G23 and G24) and two nucleotides of the anticodon stem (U39 and G41) interact with the catalytic domain of BsTrmK whereas its C-terminal domain only interacts with nucleotides U20 and C56. Indeed, R227 and H200 are involved in the binding of U20 and a surface cluster made by K220, Q216, N217 and Q207 interacts with C56 in the T-loop of BstRNASer. The few interaction points with the tRNA and the nature of the residues involved in interaction (long side-chains) could explain why no interaction was detected by NMR for this domain. In the model, the interaction between the coiled-coil motif of the C-terminal domain and the D- and T-loops helps maintain the position of A22 near the catalytic pocket, explaining why removal of these helices abolished MTase activity. The base-pairing of A22 with G13 allows for positioning the N1-atom of A22 in close proximity to the methyl group of SAM (Figure 5B). Figure 5. View largeDownload slide Model of the BsTrmK/SAM/BstRNASer complex based on the NMR chemical shift mapping and validated by MTase activity of BsTrmK variants mutated at the binding interface with tRNA. (A) Top-scoring structure from HADDOCK docking guided by the NMR chemical shift mapping of the BstRNASer binding on BsTrmK, the D-arm is drawn in orange, the G13-A22 base pair is in red sticks, the SAM on the BsTrmK catalytic pocket is represented as sticks, (B) The base-pair G13-A22 is in close proximity to the SAM-methyl donor in the model (C) MTase activity of BsTrmK variants mutated in the tRNA binding interface (D) Positions of the mutations on BsTrmK/SAM/BstRNASer complex, the BstRNASer is represented with the same color code as that used in Figure 2E, the tRNA elements crucial for BsTrmK activity are in red and orange (E) Profile view of the model. Figure 5. View largeDownload slide Model of the BsTrmK/SAM/BstRNASer complex based on the NMR chemical shift mapping and validated by MTase activity of BsTrmK variants mutated at the binding interface with tRNA. (A) Top-scoring structure from HADDOCK docking guided by the NMR chemical shift mapping of the BstRNASer binding on BsTrmK, the D-arm is drawn in orange, the G13-A22 base pair is in red sticks, the SAM on the BsTrmK catalytic pocket is represented as sticks, (B) The base-pair G13-A22 is in close proximity to the SAM-methyl donor in the model (C) MTase activity of BsTrmK variants mutated in the tRNA binding interface (D) Positions of the mutations on BsTrmK/SAM/BstRNASer complex, the BstRNASer is represented with the same color code as that used in Figure 2E, the tRNA elements crucial for BsTrmK activity are in red and orange (E) Profile view of the model. In the proposed model, the variable region of the tRNA molecule does not interact with BsTrmK, in agreement with the fact that the length of this region does not affect substrate potential. The anticodon stem and the tRNA elbow were shown to be important for BsTrmK MTase activity, and in the model, these two regions interact directly with BsTrmK. The model further suggests an explanation for the preference of a purine at position 13, a pyrimidine at this position would form hydrogen bonds to the Watson–Crick face of A22, which would render the N1-atom non-accessible. To further challenge the model for validation by experimental data, BsTrmK residues in the binding interface were mutated to glutamate or alanine residues and the mutant proteins were tested for MTase activity (Figure 5C). Residues were selected according to the model as: (i) two N-terminal domain residues that are predicted to interact with the tRNA phosphodiester backbone in the anticodon stem (K5E) and in the D-stem (D29A), (ii) C-terminal domain residues predicted to interact with tRNA (H200A, Q207A, K220E, R227A), (iii) A residue not expected to be involved in tRNA binding (E197A) was added as a negative control. All mutant proteins are still folded (Supplementary Figure S15) and showed reduced MTase activity compared to WT BsTrmK, except for the Q207A variant and the E197A one bearing a mutation of a residue outside of the tRNA binding surface. These results indicate that the mutated residues, except Q207 and E197, are important for BsTrmK enzymatic activity, most probably by affecting tRNA binding given their position in BsTrmK structure (Figure 5D and E). In cluster 4, among the mutated residues, only H200 and Q207 make contact with the tRNA, which comforts us in the choice of the best-scoring structure of cluster 1 as the best model. Overall, the obtained model for the BsTrmK/SAM/tRNASer complex is supported by MTase activity data performed on single-point mutants of BsTrmK. In the model, BsTrmK recognises the overall L-shaped structure of tRNA, rather than specific nucleotides, via extensive interactions with the phosphodiester backbone of nucleotides in the D-loop, and point-interactions with the phosphodiester backbone of the T-loop and anticodon stem. This recognition pattern explains how multiple tRNAs, with a variety of sequences, can be accommodated as substrates for BsTrmK. DISCUSSION NMR spectroscopy was a valuable method to get insight into BsTrmK/SAM/BstRNASer interactions X-ray crystallography is the method of choice for obtaining high-resolution structure of tRNA modification enzymes in complex with their tRNA substrates, but the crystallisation of these complexes remains a highly challenging task. Crystallisation is often limited by the flexibility of the tRNAs, the low binding affinity of the partners and the salt concentration needed in many crystallisation assays that weakens the often-electrostatic protein-RNA interaction. Heteronuclear NMR spectroscopy can be a valuable alternative to X-ray crystallography in the absence of diffracting crystals. This method can provide information on the binding interface between the enzyme and the cognate tRNA. In this work, NMR proved a powerful tool for deciphering the tRNA binding patch of BsTrmK. To our knowledge, this is the first time that NMR has been used to obtain information on the binding interface between a modification enzyme and its full-length tRNA substrate. The strategy consisted in solving the crystal structure of BsTrmK and using the measured NMR data to guide the docking of BstRNASer into the protein to build a model of the BsTrmK/SAM/BstRNASer complex. This model was further validated by measuring the MTase activity of BsTrmK variants with single-point mutations located at the proposed protein–RNA interaction surface. The formation of m1A22 by BsTrmK does not require a general base catalyst In the methylation reaction mechanism, the N1-atom of adenine can act as a nucleophile either alone or assisted by a general base catalyst. From a chemical point of view, the N1-position of adenine is a powerful nucleophile which is easily methylated by an electrophile such as methyl-methanesulfonate (43). On the other hand, SAM is a natural electrophile in the cell and it was even proposed that some m1A formation in the cell can occur by direct reaction with SAM for RNAs that have accessible N1-atom of adenosines, especially in environments rich in SAM (43). For BsTrmK, the only residue that is conserved across COG2384 proteins and that could act as a general base catalyst in the reaction mechanism is the aspartate D29. Mutation of D29 to alanine reduced the activity of BsTrmK, but did not fully deactivate the enzyme (Figure 5A), indicating that D29 is not absolutely required for reaction. This result adds to the growing lines of evidence that m1A formation in tRNA does not require a base catalyst and agrees with (i) the previously proposed m1A methylation mechanism for the bacterial m1A58 tRNA MTase TrmI (2,14,17), and (ii) with data published on the dual-specific m1G9/m1A9 tRNA MTase Trm10 from Thermoccocus kodakaraensis (8,44). Enzymes modifying the tRNA core use different strategies to get access to the target nucleoside Considering the published tRNA sequences (1,38), the tRNA core contains many different modifications, such as s4U8, m2G10, Gm18, m1A22, m22G26, m7G46, m5C48, m5U54, m1A58, Ψ55. All these modified nucleosides are involved in base-pairing or tertiary interactions that help shape and lock the tRNA elbow structure. Two crystal structures of tRNA-core-modifying enzymes bound to a full-length tRNA are available: the tRNA-guanine transglycosylase (TGT) that catalyses the formation of archaeosine at G15 (D-loop) bound to tRNAVal (45), and the human tRNA m1A58 MTase (Trm6-Trm61) in complex with its tRNALys3 substrate (12). In the structure of TGT/tRNAVal, the bound tRNA shows an alternative conformation named the λ-form, which is drastically different from the canonical L-shape. In the λ-form, all D-arm secondary base-pairs and canonical tertiary interactions are disrupted and the helical structure is reorganized such that the D-arm nucleotides 9 to 22 are unpaired and protrude from the tRNA. A D-variable helix formed by the base-pairing between residues 23–48, 24–47 and 25–46 is stacked on the anticodon stem to form a double-helical structure. In the structure of Trm6-Trm61/tRNALys3, the D- and the T-arms are detached from each other to expose the A58 N1-atom for methylation, but the L-shape of the tRNA is otherwise maintained. The binding is stabilized by the formation of numerous hydrogen bonds with the C56 nucleobase and the sugar-phosphate backbone. For the s4U8, m5U54 and Ψ55 formations, crystal structures of enzymes responsible for each of these modifications in complex with a fragment of tRNA are available. These structures show a flip of the nucleoside to be modified into the catalytic pocket of the enzyme that is stabilized by numerous hydrogen bonds between the protein and the sugar-phosphate moieties of the nucleotides surrounding the target base (46–48). In all these complexes, the nucleoside to be modified is buried in full-length tRNA structure and is base-paired. To get access to the nucleoside, the enzyme has to both disrupt the base-pairing and partially unfold the tRNA. For nucleosides located nearer the surface of tRNAs like A9, no such conformational changes would be expected. According to a docking model of the Sulfolobus acidocaldarius m1A9 MTase and tRNA, the canonical L-shape of the tRNA is nearly perfectly retained, with only a small flip of the D-stem (49). Like other m1A modified bases, A22 of BsTrmK substrates takes part in non-Watson–Crick base-pairing by binding to either a G or an A at position 13. The NMR data obtained on the BstRNASer show that the variable hairpin is still formed upon binding of BsTrmK indicating that it is not folded in the λ-form. In the tRNA L-shape structure used for the docking, the A22 N1-atom, when base-paired with a G or an A, is not buried deep, and is accessible for modification without need for much structural rearrangement of the tRNA molecule. Therefore, the target atom seems to be rendered accessible by the specific R13–A22 base-pairing that is very stable (50), and allows the placement of the N1-atom of the adenine 22 accessible in the major groove of the tRNA. The major demonstration of the crucial role of the R13-A22 pairing is supported by the fact that replacing pyrimidine 13 of a non-substrate tRNA by a purine renders the tRNA substrate of BsTrmK. In the non-substrate tRNAGly, A22 is involved in a Watson–Crick base pair with U13, which hides the N1-atom of A22 inside the AU base-pair and thus inside the tRNA structure. According to the model proposed in the present work, BsTrmK can bind the tRNA with no to very little deformation of both partners, the G13–A22 base pair allowing the placement of the N1-atom of A22 in close contact with the methyl group of SAM in the active site of the enzyme. However, we cannot definitely rule out that an induced-fit process occurs after binding to stabilize the complex. Disappearance of peaks in the NMR spectra for residues at the top of the anticodon stem and at the junction between the acceptor and the T-stem are observed upon binding of BsTrmK. These base-pairs are located apart from the G13-A22 pair. For instance, a bending at the interface between the acceptor and the T-stems could explain that the imino groups of these base-pairs become more exchangeable with the solvent and disappear from the NMR spectra. This bending would likely allow the acceptor stem to interact with TrmK. Further experiments with a co-crystal between TrmK and a tRNA would be needed to test this hypothesis. Future investigations are needed to establish the real occurrence of m1A22 in tRNAs The presence of m1A22 is not frequently reported in tRNA database and has to date only been identified in some bacteria. In our opinion, this may largely be due to the lack of systematic modification mapping in tRNAs. In vitro, BsTrmK modifies any provided tRNA bearing a purine at position 13, indicating that many tRNAs carrying an A at position 22 could be modified in B. subtilis in vivo. The seemingly low occurrence of this modification therefore reflects either the low availability of B. subtilis tRNA sequences for which the complete identification of modifications has not been performed or that BsTrmK specificity is different in vivo like previously observed for the yeast m1G9 forming enzyme Trm10 (51). Systematic mapping of B. subtilis tRNA modifications could in the future give an answer to this question and confirm whether the specificity observed in this study in vitro is retained in vivo. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS We thank the TGIR-RMN-THC FR3050 at Gif-sur-Yvette for NMR measurement time and Nelly Morellet for helpful discussion and technical advice. The X-ray data were collected on the ID14-eh1 beamline at the European Synchrotron Radiation Facility (ESRF, Grenoble, France). We are grateful to the Local Contact at the ESRF for assisting during data collection. Abdalla Al Refaii was supported by a Khaled Al-As’ad fellowship delivered by the Université libre de Bruxelles. The Fonds D. et A. Van Buuren and the Fonds J. Brachet (ULB) are gratefully acknowledged for financial support. We also thank Kevin Englebert, Jessica Berlier and Marie Morel for taking part to the early stages of this work. FUNDING CNRS, Paris Descartes University, Labex Dynamo Funding for open access charge: CNRS. Conflict of interest statement. None declared. REFERENCES 1. Boccaletto P. , Machnicka M.A. , Purta E. , Piatkowski P. , Baginski B. , Wirecki T.K. , de Crecy-Lagard V. , Ross R. , Limbach P.A. , Kotter A. et al. . MODOMICS: a database of RNA modification pathways. 2017 update . Nucleic Acids Res. 2018 ; 46 : D303 – D307 . Google Scholar Crossref Search ADS PubMed 2. Oerum S. , Degut C. , Barraud P. , Tisne C. m1A Post-Transcriptional modification in tRNAs . Biomolecules . 2017 ; 7 : E20 . Google Scholar Crossref Search ADS PubMed 3. Grosjean H. , Constantinesco F. , Foiret D. , Benachenhou N. A novel enzymatic pathway leading to 1-methylinosine modification in Haloferax volcanii tRNA . Nucleic Acids Res. 1995 ; 23 : 4312 – 4319 . Google Scholar Crossref Search ADS PubMed 4. Roovers M. , Wouters J. , Bujnicki J.M. , Tricot C. , Stalon V. , Grosjean H. , Droogmans L. A primordial RNA modification enzyme: the case of tRNA (m1A) methyltransferase . Nucleic Acids Res. 2004 ; 32 : 465 – 476 . Google Scholar Crossref Search ADS PubMed 5. Kempenaers M. , Roovers M. , Oudjama Y. , Tkaczuk K.L. , Bujnicki J.M. , Droogmans L. New archaeal methyltransferases forming 1-methyladenosine or 1-methyladenosine and 1-methylguanosine at position 9 of tRNA . Nucleic Acids Res. 2010 ; 38 : 6533 – 6543 . Google Scholar Crossref Search ADS PubMed 6. Vilardo E. , Nachbagauer C. , Buzet A. , Taschner A. , Holzmann J. , Rossmanith W. A subcomplex of human mitochondrial RNase P is a bifunctional methyltransferase–extensive moonlighting in mitochondrial tRNA biogenesis . Nucleic Acids Res. 2012 ; 40 : 11583 – 11593 . Google Scholar Crossref Search ADS PubMed 7. Jackman J.E. , Montange R.K. , Malik H.S. , Phizicky E.M. Identification of the yeast gene encoding the tRNA m1G methyltransferase responsible for modification at position 9 . RNA . 2003 ; 9 : 574 – 585 . Google Scholar Crossref Search ADS PubMed 8. Singh R.K. , Feller A. , Roovers M. , Van Elder D. , Wauters L. , Droogmans L. , Versees W. Structural and biochemical analysis of the dual-specificity Trm10 enzyme from Thermococcus kodakaraensis prompts reconsideration of its catalytic mechanism . RNA . 2018 ; 24 : 1080 – 1092 . Google Scholar Crossref Search ADS PubMed 9. Helm M. , Giege R. , Florentz C. A Watson–Crick base-pair-disrupting methyl group (m1A9) is sufficient for cloverleaf folding of human mitochondrial tRNALys . Biochemistry . 1999 ; 38 : 13338 – 13346 . Google Scholar Crossref Search ADS PubMed 10. Sprinzl M. , Vassilenko K.S. Compilation of tRNA sequences and sequences of tRNA genes . Nucleic Acids Res. 2005 ; 33 : D139 – D140 . Google Scholar Crossref Search ADS PubMed 11. Wang M. , Zhu Y. , Wang C. , Fan X. , Jiang X. , Ebrahimi M. , Qiao Z. , Niu L. , Teng M. , Li X. Crystal structure of the two-subunit tRNA m(1)A58 methyltransferase TRM6-TRM61 from Saccharomyces cerevisiae . Sci. Rep. 2016 ; 6 : 32562 . Google Scholar Crossref Search ADS PubMed 12. Finer-Moore J. , Czudnochowski N. , O’Connell J.D. 3rd , Wang A.L. , Stroud R.M. Crystal structure of the human tRNA m(1)A58 Methyltransferase-tRNA(3)(Lys) complex: refolding of substrate tRNA allows access to the methylation target . J. Mol. Biol. 2015 ; 427 : 3862 – 3876 . Google Scholar Crossref Search ADS PubMed 13. Droogmans L. , Roovers M. , Bujnicki J.M. , Tricot C. , Hartsch T. , Stalon V. , Grosjean H. Cloning and characterization of tRNA (m1A58) methyltransferase (TrmI) from Thermus thermophilus HB27, a protein required for cell growth at extreme temperatures . Nucleic Acids Res. 2003 ; 31 : 2148 – 2156 . Google Scholar Crossref Search ADS PubMed 14. Barraud P. , Golinelli-Pimpaneau B. , Atmanene C. , Sanglier S. , Van Dorsselaer A. , Droogmans L. , Dardel F. , Tisne C. Crystal structure of Thermus thermophilus tRNA m1A58 methyltransferase and biophysical characterization of its interaction with tRNA . J. Mol. Biol. 2008 ; 377 : 535 – 550 . Google Scholar Crossref Search ADS PubMed 15. Guelorget A. , Barraud P. , Tisne C. , Golinelli-Pimpaneau B. Structural comparison of tRNA m(1)A58 methyltransferases revealed different molecular strategies to maintain their oligomeric architecture under extreme conditions . BMC Struct. Biol. 2011 ; 11 : 48 . Google Scholar Crossref Search ADS PubMed 16. Takuma H. , Ushio N. , Minoji M. , Kazayama A. , Shigi N. , Hirata A. , Tomikawa C. , Ochi A. , Hori H. Substrate tRNA recognition mechanism of eubacterial tRNA (m1A58) methyltransferase (TrmI) . J. Biol. Chem. 2015 ; 290 : 5912 – 5925 . Google Scholar Crossref Search ADS PubMed 17. Degut C. , Ponchon L. , Folly-Klan M. , Barraud P. , Tisne C. The m1A(58) modification in eubacterial tRNA: An overview of tRNA recognition and mechanism of catalysis by TrmI . Biophys. Chem. 2016 ; 210 : 27 – 34 . Google Scholar Crossref Search ADS PubMed 18. Varshney U. , Ramesh V. , Madabushi A. , Gaur R. , Subramanya H.S. , RajBhandary U.L. Mycobacterium tuberculosis Rv2118c codes for a single-component homotetrameric m1A58 tRNA methyltransferase . Nucleic Acids Res. 2004 ; 32 : 1018 – 1027 . Google Scholar Crossref Search ADS PubMed 19. Guelorget A. , Roovers M. , Guerineau V. , Barbey C. , Li X. , Golinelli-Pimpaneau B. Insights into the hyperthermostability and unusual region-specificity of archaeal Pyrococcus abyssi tRNA m1A57/58 methyltransferase . Nucleic Acids Res. 2010 ; 38 : 6206 – 6218 . Google Scholar Crossref Search ADS PubMed 20. Raettig R. , Kersten H. , Weissenbach J. , Dirheimer G. Methylation of an adenosine in the D-loop of specific transfer RNAs from yeast by a procaryotic tRNA (adenine-1) methyltransferase . Nucleic Acids Res. 1977 ; 4 : 1769 – 1782 . Google Scholar Crossref Search ADS PubMed 21. Kersten H. , Raettig R. , Weissenbach J. , Dirheimer G. Recognition of individual procaryotic and eucaryotic transfer-ribonucleic acids by B subtilis adenine-1-methyltransferase specific for the dihydrouridine loop . Nucleic Acids Res. 1978 ; 5 : 3033 – 3042 . Google Scholar Crossref Search ADS PubMed 22. Roovers M. , Kaminska K.H. , Tkaczuk K.L. , Gigot D. , Droogmans L. , Bujnicki J.M. The YqfN protein of Bacillus subtilis is the tRNA: m1A22 methyltransferase (TrmK) . Nucleic Acids Res. 2008 ; 36 : 3252 – 3262 . Google Scholar Crossref Search ADS PubMed 23. Thanassi J.A. , Hartman-Neumann S.L. , Dougherty T.J. , Dougherty B.A. , Pucci M.J. Identification of 113 conserved essential genes using a high-throughput gene disruption system in Streptococcus pneumoniae . Nucleic Acids Res. 2002 ; 30 : 3152 – 3162 . Google Scholar Crossref Search ADS PubMed 24. Hutchison C.A. 3rd , Chuang R.Y. , Noskov V.N. , Assad-Garcia N. , Deerinck T.J. , Ellisman M.H. , Gill J. , Kannan K. , Karas B.J. , Ma L. et al. . Design and synthesis of a minimal bacterial genome . Science . 2016 ; 351 : aad6253 . Google Scholar Crossref Search ADS PubMed 25. Ta H.M. , Kim K.K. Crystal structure of Streptococcus pneumoniae Sp1610, a putative tRNA methyltransferase, in complex with S-adenosyl-L-methionine . Protein Sci. 2010 ; 19 : 617 – 624 . Google Scholar PubMed 26. Degut C. , Monod A. , Brachet F. , Crepin T. , Tisne C. In Vitro/In vivo production of tRNA for X-Ray studies . Methods Mol. Biol. 2016 ; 1320 : 37 – 57 . Google Scholar Crossref Search ADS PubMed 27. Dardel F. MC-Fit: using Monte-Carlo methods to get accurate confidence limits on enzyme parameters . Comput. Appl. Biosci. 1994 ; 10 : 273 – 275 . Google Scholar PubMed 28. Adams P.D. , Afonine P.V. , Bunkoczi G. , Chen V.B. , Davis I.W. , Echols N. , Headd J.J. , Hung L.W. , Kapral G.J. , Grosse-Kunstleve R.W. et al. . PHENIX: a comprehensive Python-based system for macromolecular structure solution . Acta Crystallogr. D Biol. Crystallogr. 2010 ; 66 : 213 – 221 . Google Scholar Crossref Search ADS PubMed 29. Emsley P. , Lohkamp B. , Scott W.G. , Cowtan K. Features and development of Coot . Acta Crystallogr. D Biol. Crystallogr. 2010 ; 66 : 486 – 501 . Google Scholar Crossref Search ADS PubMed 30. Bakan A. , Meireles L.M. , Bahar I. ProDy: protein dynamics inferred from theory and experiments . Bioinformatics . 2011 ; 27 : 1575 – 1577 . Google Scholar Crossref Search ADS PubMed 31. Humphrey W. , Dalke A. , Schulten K. VMD: visual molecular dynamics . J. Mol. Graph. 1996 ; 14 : 33 – 38 . Google Scholar Crossref Search ADS PubMed 32. Degut C. , Barraud P. , Larue V. , Tisne C. Backbone resonance assignments of the m1A22 tRNA methyltransferase TrmK from Bacillus subtilis . Biomol. NMR Assign. 2016 ; 10 : 253 – 257 . Google Scholar Crossref Search ADS PubMed 33. Pervushin K.V. , Wider G. , Wuthrich K. Single Transition-to-single transition polarization transfer (ST2-PT) in [15N,1H]-TROSY . J. Biomol. NMR . 1998 ; 12 : 345 – 348 . Google Scholar Crossref Search ADS PubMed 34. Orekhov V.Y. , Jaravine V.A. Analysis of non-uniformly sampled spectra with multi-dimensional decomposition . Prog. Nucl. Magn. Reson. Spectrosc. 2011 ; 59 : 271 – 292 . Google Scholar Crossref Search ADS PubMed 35. Das R. , Karanicolas J. , Baker D. Atomic accuracy in predicting and designing noncanonical RNA structure . Nat. Methods . 2010 ; 7 : 291 – 294 . Google Scholar Crossref Search ADS PubMed 36. Dominguez C. , Boelens R. , Bonvin A.M. HADDOCK: a protein-protein docking approach based on biochemical or biophysical information . J. Am. Chem. Soc. 2003 ; 125 : 1731 – 1737 . Google Scholar Crossref Search ADS PubMed 37. de Vries S.J. , van Dijk A.D. , Krzeminski M. , van Dijk M. , Thureau A. , Hsu V. , Wassenaar T. , Bonvin A.M. HADDOCK versus HADDOCK: new features and performance of HADDOCK2.0 on the CAPRI targets . Proteins . 2007 ; 69 : 726 – 733 . Google Scholar Crossref Search ADS PubMed 38. Juhling F. , Morl M. , Hartmann R.K. , Sprinzl M. , Stadler P.F. , Putz J. tRNAdb 2009: compilation of tRNA sequences and tRNA genes . Nucleic Acids Res. 2009 ; 37 : D159 – D162 . Google Scholar Crossref Search ADS PubMed 39. Biou V. , Yaremchuk A. , Tukalo M. , Cusack S. The 2.9 A crystal structure of T. thermophilus seryl-tRNA synthetase complexed with tRNA(Ser) . Science . 1994 ; 263 : 1404 – 1410 . Google Scholar Crossref Search ADS PubMed 40. Zhang J. , Ferre-D’Amare A.R. The tRNA elbow in structure, recognition and evolution . Life (Basel) . 2016 ; 6 : E3 . Google Scholar PubMed 41. Giege R. , Juhling F. , Putz J. , Stadler P. , Sauter C. , Florentz C. Structure of transfer RNAs: similarity and variability . Wiley Interdiscip. Rev. RNA . 2012 ; 3 : 37 – 61 . Google Scholar Crossref Search ADS PubMed 42. Holm L. , Sander C. Touring protein fold space with Dali/FSSP . Nucleic Acids Res. 1998 ; 26 : 316 – 319 . Google Scholar Crossref Search ADS PubMed 43. Reichle V.F. , Kaiser S. , Heiss M. , Hagelskamp F. , Borland K. , Kellner S. Surpassing limits of static RNA modification analysis with dynamic NAIL-MS . Methods . 2019 ; 156 : 91 – 101 . Google Scholar Crossref Search ADS PubMed 44. Krishnamohan A. , Dodbele S. , Jackman J.E. Insights into catalytic and tRNA recognition mechanism of the Dual-Specific tRNA methyltransferase from Thermococcus kodakarensis . Genes (Basel) . 2019 ; 10 : E100 . Google Scholar Crossref Search ADS PubMed 45. Ishitani R. , Nureki O. , Nameki N. , Okada N. , Nishimura S. , Yokoyama S. Alternative tertiary structure of tRNA for recognition by a posttranscriptional modification enzyme . Cell . 2003 ; 113 : 383 – 394 . Google Scholar Crossref Search ADS PubMed 46. Alian A. , Lee T.T. , Griner S.L. , Stroud R.M. , Finer-Moore J. Structure of a TrmA-RNA complex: a consensus RNA fold contributes to substrate selectivity and catalysis in m5U methyltransferases . Proc. Natl. Acad. Sci. U.S.A. 2008 ; 105 : 6876 – 6881 . Google Scholar Crossref Search ADS PubMed 47. Hoang C. , Ferre-D’Amare A.R. Cocrystal structure of a tRNA Psi55 pseudouridine synthase: nucleotide flipping by an RNA-modifying enzyme . Cell . 2001 ; 107 : 929 – 939 . Google Scholar Crossref Search ADS PubMed 48. Pan H. , Agarwalla S. , Moustakas D.T. , Finer-Moore J. , Stroud R.M. Structure of tRNA pseudouridine synthase TruB and its RNA complex: RNA recognition through a combination of rigid docking and induced fit . Proc. Natl. Acad. Sci. U.S.A. 2003 ; 100 : 12648 – 12653 . Google Scholar Crossref Search ADS PubMed 49. Van Laer B. , Roovers M. , Wauters L. , Kasprzak J.M. , Dyzma M. , Deyaert E. , Kumar Singh R. , Feller A. , Bujnicki J.M. , Droogmans L. et al. . Structural and functional insights into tRNA binding and adenosine N1-methylation by an archaeal Trm10 homologue . Nucleic Acids Res. 2016 ; 44 : 940 – 953 . Google Scholar Crossref Search ADS PubMed 50. Mladek A. , Sharma P. , Mitra A. , Bhattacharyya D. , Sponer J. , Sponer J.E. Trans Hoogsteen/sugar edge base pairing in RNA. Structures, energies, and stabilities from quantum chemical calculations . J. Phys. Chem. B . 2009 ; 113 : 1743 – 1755 . Google Scholar Crossref Search ADS PubMed 51. Swinehart W.E. , Henderson J.C. , Jackman J.E. Unexpected expansion of tRNA substrate recognition by the yeast m1G9 methyltransferase Trm10 . RNA . 2013 ; 19 : 1137 – 1146 . Google Scholar Crossref Search ADS PubMed 52. Baker N.A. , Sept D. , Joseph S. , Holst M.J. , McCammon J.A. Electrostatics of nanosystems: application to microtubules and the ribosome . Proc. Natl. Acad. Sci. U.S.A. 2001 ; 98 : 10037 – 10041 . Google Scholar Crossref Search ADS PubMed 53. Dolinsky T.J. , Nielsen J.E. , McCammon J.A. , Baker N.A. PDB2PQR: an automated pipeline for the setup of Poisson-Boltzmann electrostatics calculations . Nucleic Acids Res. 2004 ; 32 : W665 – W667 . Google Scholar Crossref Search ADS PubMed Author notes The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors. © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. 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TI - Structural characterization of B. subtilis m1A22 tRNA methyltransferase TrmK: insights into tRNA recognition
JF - Nucleic Acids Research
DO - 10.1093/nar/gkz230
DA - 2019-05-21
UR - https://www.deepdyve.com/lp/oxford-university-press/structural-characterization-of-b-subtilis-m1a22-trna-methyltransferase-xTenQ7Risu
SP - 4736
VL - 47
IS - 9
DP - DeepDyve
ER -