Structural characterization of B. subtilis m1A22 tRNA methyltransferase TrmK: insights into tRNA recognition

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.

Figure 1.

_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) tRNA^Ser, (B) tRNA^His, (C) tRNA^Tyr, (D) tRNA^Cys, (E) tRNA^Gln, (F) tRNA^Glu, (G) tRNA^Leu, (H) tRNA^Gly.

Figure 2.

_BsTrmK MTase assays with B. subtilis tRNAs mutated at position 13, and with tRNA^His altered in the T-loop or in the anticodon stem-loop. [α³²P]ATP-labelled T7-transcripts of mutated tRNA^Ser (A) or tRNA^His (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 tRNA^Ser at position 13, (B) for mutants of tRNA^His at position 13, (C) for variants of tRNA^His in the T-loop, (D) for variants of tRNA^His in the anticodon stem-loop. (E) Summary on the tRNA^Ser 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 3.

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 4.

Interaction of _BstRNA^Ser 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 tRNA^Ser 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 _BstRNA^Ser bearing (tRNA^Serin vivo) or not bearing (tRNA^Serin vitro) post-transcriptional modifications. Confidence intervals at 90% are indicated in parenthesis. An unmodified _BstRNA^Ser was chosen to study the interaction of _BstRNA^Ser with _BsTrmK given that _BstRNA^Ser 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 _BstRNA^Ser in green (D) NMR chemical shift mapping of _BstRNA^Ser 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 5.

Model of the _BsTrmK/SAM/_BstRNA^Ser 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 _BstRNA^Ser 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/_BstRNA^Ser complex, the _BstRNA^Ser 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.