The tRNA recognition mechanism of the minimalist SPOUT methyltransferase, TrmL

Abstract

Unlike other transfer RNAs (tRNA)-modifying enzymes from the SPOUT methyltransferase superfamily, the tRNA (Um34/Cm34) methyltransferase TrmL lacks the usual extension domain for tRNA binding and consists only of a SPOUT domain. Both the catalytic and tRNA recognition mechanisms of this enzyme remain elusive. By using tRNAs purified from an Escherichia coli strain with the TrmL gene deleted, we found that TrmL can independently catalyze the methyl transfer from S-adenosyl-L-methionine to and isoacceptors without the involvement of other tRNA-binding proteins. We have solved the crystal structures of TrmL in apo form and in complex with S-adenosyl-homocysteine and identified the cofactor binding site and a possible active site. Methyltransferase activity and tRNA-binding affinity of TrmL mutants were measured to identify residues important for tRNA binding of TrmL. Our results suggest that TrmL functions as a homodimer by using the conserved C-terminal half of the SPOUT domain for catalysis, whereas residues from the less-conserved N-terminal half of the other subunit participate in tRNA recognition.

Figure 1.

Domain architectures of SPOUT tRNA MTases and the sequence alignment of TrmLs. (A) The common catalytic domain of SPOUT superfamily is represented as SPOUT, the extension domains are represented by secondary structures, and the amino acid length of the respective SPOUT MTase from E. coli are labeled. (B) Structure-based multiple amino acid sequence alignment of TrmLs from model organisms. Ec, E. coli; Hi, H. influenza; Bs, Bacillus subtilis; Tt, T. thermophilus; Pm, Prochlorococcus marinus; Dv, Desulfovibrio vulgaris; Mg, Mycoplasma genitalium. The secondary structure elements of EcTrmL are labeled above the alignment. The basic amino acid residues on the protein surface that are manipulated in this study are marked with a star. The conserved Tyr142 is marked by a pentagon.

Figure 2.

The methyltransferase activity of EcTrmL. (A) The analytical gel filtration analyzed by superdexTM-75, EcTrmL was eluted at 11.63 ml, the locations of the marker proteins are shown above the graph. (B) Purified tRNAs were analyzed by 12% denatured and 6% native PAGE. Ts refers to tRNA transcripts, WT and dTrmL refer to tRNAs purified from E.coli MT102 and JW3581-1, respectively. (C) and (D) graphically show the methyltransferase activity of EcTrmL for s and s, respectively.

Figure 3.

Overall structure of EcTrmL. (A) Ribbon diagram showing the overall structure of EcTrmL in Apo form (left) and in complex with SAH (right). The structures are shown as dimers, with one subunit in green and the other one in cyan. (B) A subunit from EcTrmL, TtTrmH and HiTrmD are superimposed and represented from the same perspective. The common SPOUT domains are in cyan, the extensions are in magenta, SAH and SAM are shown as sticks. (C) Crystal structure of EcTrmL with the surface colored in light gray, the basic Arg, His and Lys residues are in blue and the SAH are shown as spheres.

Figure 4.

SAH and HEPES binding. (A) SAH bound in subunit A. The carbon atom of SAH is shown in white, the backbone of EcTrmL is in green, and all the residues within 4 Å from SAH are shown in stick. (B) SAH binding details in subunit B, the backbone of EcTrmL is shown in cyan. (C) The crystal structures of SAH molecules from subunit A (green) and subunit B (cyan) are superimposed and shown as sticks, with the structure of HEPES in magenta. (D) The chemical structure of the ribose and phosphate of U/CMP, and the HEPES molecule. (E) The structure of a HEPES molecule bound to EcTrmL, with all the residues within 4 Å shown as sticks. The carbon atoms of HEPES and SAH are shown in magenta and white, respectively.

Figure 5.

Glu scanning of the basic amino acid surface residues. (A) The binding affinities of EcTrmLs for tRNA analyzed by the gel mobility shift assay. (B) and (C) show the methyltransferase activities of the various EcTrmL mutants. (D) The SAH-binding affinity as measured by ITC.

Figure 6.

Ala mutation of residues involved in tRNA binding. (A) The binding affinities of EcTrmLs for analyzed by the gel mobility shift assay. (B) The methyltransferase activities of the various EcTrmL mutants.

Figure 7.

The proposed model of tRNA^Leu binding to EcTrmL. (A) The EcTrmL basic amino acid surface residues (magenta) that are identified as being involved in tRNA binding, the dimer structure is shown in cartoon loop with same color used as in Figure 3. (B) The proposed model of EcTrmL bound with tRNA, the backbone of is shown in brown.

Figure 8.

The effect of dimer formation on SAH and tRNA binding and enzymatic activity. (A) The analytical gel filtration of EcTrmL-Y142A was analyzed by under the same conditions as wild-type EcTrmL and was eluted at 13.13 ml. (B) The binding affinity of WT and Y142A for was analyzed by the gel mobility shift assay. (C) The methyltransferase activity of Y142A. (D) The binding affinity for SAH by ITC.