Research ArticleBIOCHEMISTRY

Structural insight into the methyltransfer mechanism of the bifunctional Trm5

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Science Advances  01 Dec 2017:
Vol. 3, no. 12, e1700195
DOI: 10.1126/sciadv.1700195
  • Fig. 1 The overall structure of the PaTrm5a-tRNAPhe-SAH ternary complex and the multiple sequence alignment of the representatives from other species.

    (A) Biosynthetic pathways for wyosine derivatives in archaea including P. abyssi. Note that the same enzyme Trm5 performs two methyltransfers and is marked in red. (B) The front and back views of the complex in ribbon rendition. The D1, D2, and D3 domains are colored pale green, yellow orange, and light blue, respectively. The active site SAM is shown as spheres, whereas tRNA is in orange. The linker connecting D1 and D2 is colored blue.

  • Fig. 2 Substrate interactions with the catalytic region in the PaTrm5a-tRNAPhe-SAH ternary complex.

    (A) The recognition pattern of SAH. The residues participating in ligand recognition are depicted in a ball-and-stick model and labeled. The hydrogen bonds are shown by the red dashed lines (distance, <3.6 Å). The distance of the S atom of SAH to N1 of G37 is shown by the yellow dashed line. (B) The recognition of the anticodon bases A35-U39. (C) Time course of the relative methyltransfer activities of PaTrm5a and mutants that are involved in substrate recognition. The measurements were made at 1- and 2-min time points. The activity of wild-type (WT) PaTrm5a at the 2-min time point was normalized to 100%, and the readings at time point zero were used as blanks. Error bars represent SD calculated from at least three measurements. All the 2FoFc maps are contoured at 1σ.

  • Fig. 3 Structural changes upon the formation of complex.

    (A) Structure comparison of the enzyme in complex with tRNA (PDB ID: 5WT1; coloring scheme as in Fig. 1A) with the tRNA-free form (PDB ID: 5HJK; the three domains are colored green, yellow, and purple). The structural changes for Lys319Pro320 and Arg133 before and after the binding of tRNA are indicated by the red circles and enlarged in the lower left corner. The tRNA molecule is depicted in orange. (B) tRNA structural changes compared to yeast tRNAPhe. PatRNAPhe is superimposed onto the canonical, free yeast tRNAPhe (PDB ID: 4TNA). The view angle is rotated 180° with respect to that in (A). (C) A detailed comparison of the structural differences between the MjTrm5b-tRNACys (cyan, PDB ID: 2ZZN) and the PaTrm5a-tRNAPhe (orange, PDB ID: 5WT1) complexes.

  • Fig. 4 The flexibility of the D1 domain.

    (A) The specific interactions involving the G19-C56 base pair at the outer corner of tRNA. The 2FoFc map is contoured at 1σ. (B) EMSA of the WT, DelD1, and single mutants with tRNAPhe. (C) The methyltransfer activity of the DelD1 mutant. (D) SAXS results of the apoprotein, including the simulated SAXS profiles of the tRNA-free MjTrm5b structure (blue, PDB ID: 2YX1) and the tRNA-free PaTrm5a structure (cyan, PDB ID: 5HJJ), along with the experimental SAXS data (pink). The MjTrm5b (blue) and the PaTrm5a structures (cyan) were superimposed onto their respective DR model (pink) using PyMOL. The P(r) distance distribution function is shown in the inset.

  • Fig. 5 Recognition of tRNA truncation mutants by PaTrm5a.

    (A) Cartoon drawings showing the sequences and predicted secondary structures of the full-length and truncation mutants of the PatRNAPhe transcript. (B) The methyltransfer activity assays of tRNA truncation mutants by PaTrm5a.

  • Fig. 6 The structure of the PaTrm5a-tRNAPhe-MTA ternary complex.

    (A) The overlay of the two cocrystal structures. The coloring scheme for the SAH cocrystal structure is as in Fig. 1A, whereas the MTA cocrystal structure is in magenta. The SAH and MTA molecules present at the active site are shown in a ball-and-stick model. (B) The recognition pattern of MTA. The 2FoFc map is contoured at 1σ. The distance of the sulfur atom of MTA to N1 of G37 is indicated by the number near the yellow dashed line. (C) The tRNA-mediated crystal packing. The box on the right is in the close-up view.

  • Fig. 7 Structural basis for the bifunctional methyltransfer activity of PaTrm5a.

    (A) The PaTrm5a-tRNAPhe-SAH ternary complex model containing the modified base. The modified tRNA is omitted for clarity except for the imG-14 base at position 37. The distances of the sulfur atom of SAH to N1 and C7 of imG2 are shown (units in angstroms), along with the distance of Lys324 to N5 of imG-14. (B) Active site comparison of the PaTrm5a in the ternary complex with the MjTrm5b complex in cartoon representation. The residues participating in ligand recognition through hydrogen bonds and hydrophobic contacts are depicted in sticks and eyelashes, respectively. The equivalent hydrophobic residues in the two models are compared, and the two key residues are circled. The tricyclic imG-14 substrate is blocked by the highly conserved NLPK motif in MjTrm5b, and the second methylation reaction is thus prevented. For clarity, the tRNA molecules are omitted.

  • Table 1 Data collection and refinement statistics.
    CrystalsPaTrm5a-tRNAPhe-MTA (5WT3)PaTrm5a-tRNAPhe-SAH (5WT1)
    Data collection
      BeamlinesSSRF-BL19U1SSRF-BL17U1
      Space groupC2221P2
      a, b, c (Å)58.1, 212.3, 130.0102.1, 57.0, 115.8
      α, β, γ (°)90, 90, 9090, 101.5, 90
      Resolution (Å)50–3.20 (3.31–3.20)*50–2.60 (2.69–2.60)
      Rmerge0.079 (0.174)0.077 (0.911)
      I(I)18.9 (8.5)15.6 (1.67)
      Completeness (%)99.6 (98.0)99.5 (99.9)
      Redundancy6.2 (5.9)3.7 (3.8)
    Refinement
      Resolution (Å)50–3.20 (3.45–3.20)40.22–2.60 (2.66–2.60)
      Number of reflections13,48140,281
      Rwork/Rfree§0.246/0.2560.247/0.274
      Number of atoms
        Protein2,2684,966
        tRNA1,5652,895
        Ligand20 (MTA)52 (SAH)
        Water molecules1360
      B factors (Å2)
        Protein107.370.9
        tRNA93.668.2
        Ligand129.1 (MTA)55.0 (SAH)
        Water molecules76.655.7
      Root-mean-squared deviations
        Bond lengths (Å)0.0050.005
        Bond angles (°)0.940.97
        Ramachandran favored (%)96.3698.63
        Allowed3.331.37
        Outliers (%)0.30

    *Values in parentheses are for the highest-resolution shell.

    Rmerge = Σ|(I − 〈I〉)|/σ(I), where I is the observed intensity.

    Rwork = Σhkl||Fo| − |Fc||/Σhkl|Fo|, calculated from working data set.

    §Rfree is calculated from 5.0% of data randomly chosen and not included in refinement.

    Supplementary Materials

    • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/12/e1700195/DC1

      fig. S1. Biosynthesis of wyosine derivatives in eukaryotic tRNAPhes.

      fig. S2. The overall structure of the PaTrm5a-tRNAPhe-SAH ternary complex.

      fig. S3. The asymmetric unit contents in the cocrystals of the PaTrm5a-tRNAPhe-SAH ternary complex.

      fig. S4. The multiple sequence alignment of Trm5 sequences from different model organisms.

      fig. S5. The interaction mode of U33/G34 in the PaTrm5a-tRNAPhe-SAH ternary complex.

      fig. S6. Purity test of tRNA truncation mutants by size exclusion chromatography and Urea-PAGE.

      table S1. Specific interactions between the enzyme and the tRNA substrate.

      table S2. Statistics on SAXS data collection, analysis, and modeling.

    • Supplementary Materials

      This PDF file includes:

      • fig. S1. Biosynthesis of wyosine derivatives in eukaryotic tRNAPhes.
      • fig. S2. The overall structure of the PaTrm5a-tRNAPhe-SAH ternary complex.
      • fig. S3. The asymmetric unit contents in the cocrystals of the PaTrm5a-tRNAPhe-SAH ternary complex.
      • fig. S4. The multiple sequence alignment of Trm5 sequences from different model organisms.
      • fig. S5. The interaction mode of U33/G34 in the PaTrm5a-tRNAPhe-SAH ternary complex.
      • fig. S6. Purity test of tRNA truncation mutants by size exclusion chromatography and Urea-PAGE.
      • table S1. Specific interactions between the enzyme and the tRNA substrate.
      • table S2. Statistics on SAXS data collection, analysis, and modeling.

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