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Crystal structures of the tRNA:m2G6 methyltransferase Trm14/TrmN from two domains of life.

Fislage M, Roovers M, Tuszynska I, Bujnicki JM, Droogmans L, Versées W - Nucleic Acids Res. (2012)

Bottom Line: These results represent the first crystallographic structure analysis of proteins containing both THUMP and RFM domain, and hence provide further insight in the contribution of the THUMP domain in tRNA recognition and catalysis.Electrostatics and conservation calculations suggest a main tRNA binding surface in a groove between the THUMP domain and the MTase domain.This is further supported by a docking model of TrmN in complex with tRNA(Phe) of T. thermophilus and via site-directed mutagenesis.

View Article: PubMed Central - PubMed

Affiliation: VIB Department of Structural Biology, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussel, Belgium.

ABSTRACT
Methyltransferases (MTases) form a major class of tRNA-modifying enzymes needed for the proper functioning of tRNA. Recently, RNA MTases from the TrmN/Trm14 family that are present in Archaea, Bacteria and Eukaryota have been shown to specifically modify tRNA(Phe) at guanosine 6 in the tRNA acceptor stem. Here, we report the first X-ray crystal structures of the tRNA m(2)G6 (N(2)-methylguanosine) MTase (TTC)TrmN from Thermus thermophilus and its ortholog (Pf)Trm14 from Pyrococcus furiosus. Structures of (Pf)Trm14 were solved in complex with the methyl donor S-adenosyl-l-methionine (SAM or AdoMet), as well as the reaction product S-adenosyl-homocysteine (SAH or AdoHcy) and the inhibitor sinefungin. (TTC)TrmN and (Pf)Trm14 consist of an N-terminal THUMP domain fused to a catalytic Rossmann-fold MTase (RFM) domain. These results represent the first crystallographic structure analysis of proteins containing both THUMP and RFM domain, and hence provide further insight in the contribution of the THUMP domain in tRNA recognition and catalysis. Electrostatics and conservation calculations suggest a main tRNA binding surface in a groove between the THUMP domain and the MTase domain. This is further supported by a docking model of TrmN in complex with tRNA(Phe) of T. thermophilus and via site-directed mutagenesis.

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Active site of PfTrm14 bound to (A) SAH, (B) SAM and (C) SFG. The 2Fo − Fc omit density map is shown for the ligands. The map is contoured at 2 σ within 1.6 Å of the ligand. The ligands are shown with their C atoms colored green, the amino acid residues are shown with their C atoms colored pink (interacting with adenine), orange (interacting with ribose) or cyan (interacting with the homocysteine part). Hydrogen bonds are indicated by dashed lines.
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gks163-F3: Active site of PfTrm14 bound to (A) SAH, (B) SAM and (C) SFG. The 2Fo − Fc omit density map is shown for the ligands. The map is contoured at 2 σ within 1.6 Å of the ligand. The ligands are shown with their C atoms colored green, the amino acid residues are shown with their C atoms colored pink (interacting with adenine), orange (interacting with ribose) or cyan (interacting with the homocysteine part). Hydrogen bonds are indicated by dashed lines.

Mentions: The adenosine moieties of the ligands SAH, SAM and SFG are bound to PfTrm14 in a pocket located at the first β–α–β–α–β motif of the Rossmann-fold and the cross-over towards the second β–α–β motif, as is often seen in other nucleotide binding proteins (63). The pocket for the methionine moiety is formed by the loop region connecting the THUMP domain to the RFM domain together with the first two α-helices of the RFM domain. Omit maps of the binding pocket of PfTrm14 (Figure 3) show that the ligand SAM is bound in an extended conformation (dihedral C4′, C5′, S, Cγ = −70.7°) which is common for SAM-dependent MTases (4,62). The reaction product SAH is also bound in an extended conformation but with a dihedral angle of −34.6°. The dihedral angle of the bound inhibitor SFG is −101.1°.Figure 3.


Crystal structures of the tRNA:m2G6 methyltransferase Trm14/TrmN from two domains of life.

Fislage M, Roovers M, Tuszynska I, Bujnicki JM, Droogmans L, Versées W - Nucleic Acids Res. (2012)

Active site of PfTrm14 bound to (A) SAH, (B) SAM and (C) SFG. The 2Fo − Fc omit density map is shown for the ligands. The map is contoured at 2 σ within 1.6 Å of the ligand. The ligands are shown with their C atoms colored green, the amino acid residues are shown with their C atoms colored pink (interacting with adenine), orange (interacting with ribose) or cyan (interacting with the homocysteine part). Hydrogen bonds are indicated by dashed lines.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3367198&req=5

gks163-F3: Active site of PfTrm14 bound to (A) SAH, (B) SAM and (C) SFG. The 2Fo − Fc omit density map is shown for the ligands. The map is contoured at 2 σ within 1.6 Å of the ligand. The ligands are shown with their C atoms colored green, the amino acid residues are shown with their C atoms colored pink (interacting with adenine), orange (interacting with ribose) or cyan (interacting with the homocysteine part). Hydrogen bonds are indicated by dashed lines.
Mentions: The adenosine moieties of the ligands SAH, SAM and SFG are bound to PfTrm14 in a pocket located at the first β–α–β–α–β motif of the Rossmann-fold and the cross-over towards the second β–α–β motif, as is often seen in other nucleotide binding proteins (63). The pocket for the methionine moiety is formed by the loop region connecting the THUMP domain to the RFM domain together with the first two α-helices of the RFM domain. Omit maps of the binding pocket of PfTrm14 (Figure 3) show that the ligand SAM is bound in an extended conformation (dihedral C4′, C5′, S, Cγ = −70.7°) which is common for SAM-dependent MTases (4,62). The reaction product SAH is also bound in an extended conformation but with a dihedral angle of −34.6°. The dihedral angle of the bound inhibitor SFG is −101.1°.Figure 3.

Bottom Line: These results represent the first crystallographic structure analysis of proteins containing both THUMP and RFM domain, and hence provide further insight in the contribution of the THUMP domain in tRNA recognition and catalysis.Electrostatics and conservation calculations suggest a main tRNA binding surface in a groove between the THUMP domain and the MTase domain.This is further supported by a docking model of TrmN in complex with tRNA(Phe) of T. thermophilus and via site-directed mutagenesis.

View Article: PubMed Central - PubMed

Affiliation: VIB Department of Structural Biology, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussel, Belgium.

ABSTRACT
Methyltransferases (MTases) form a major class of tRNA-modifying enzymes needed for the proper functioning of tRNA. Recently, RNA MTases from the TrmN/Trm14 family that are present in Archaea, Bacteria and Eukaryota have been shown to specifically modify tRNA(Phe) at guanosine 6 in the tRNA acceptor stem. Here, we report the first X-ray crystal structures of the tRNA m(2)G6 (N(2)-methylguanosine) MTase (TTC)TrmN from Thermus thermophilus and its ortholog (Pf)Trm14 from Pyrococcus furiosus. Structures of (Pf)Trm14 were solved in complex with the methyl donor S-adenosyl-l-methionine (SAM or AdoMet), as well as the reaction product S-adenosyl-homocysteine (SAH or AdoHcy) and the inhibitor sinefungin. (TTC)TrmN and (Pf)Trm14 consist of an N-terminal THUMP domain fused to a catalytic Rossmann-fold MTase (RFM) domain. These results represent the first crystallographic structure analysis of proteins containing both THUMP and RFM domain, and hence provide further insight in the contribution of the THUMP domain in tRNA recognition and catalysis. Electrostatics and conservation calculations suggest a main tRNA binding surface in a groove between the THUMP domain and the MTase domain. This is further supported by a docking model of TrmN in complex with tRNA(Phe) of T. thermophilus and via site-directed mutagenesis.

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