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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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Crystal structures of PfTrm14 and TTCTrmN. (A and C) show the structure of PfTrm14 in cartoon representation (A) and as a topology diagram (C); (B and D) show the structure of TTCTrmN in cartoon representation (B) and as a topology diagram (D). β-strands are shown in red, yellow and blue for the core-THUMP, NFLD and RFM (sub)domains, respectively. α-Helices are shown in dark red, yellow and blue. The β-strand shared by the core-THUMP and NFLD subdomains is shown in orange. The two additional β-strands in the PfTrm14 structure are named βi and βii. The structure of PfTrm14 contains sinefungin bound in the active site, represented in ball and stick.
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gks163-F2: Crystal structures of PfTrm14 and TTCTrmN. (A and C) show the structure of PfTrm14 in cartoon representation (A) and as a topology diagram (C); (B and D) show the structure of TTCTrmN in cartoon representation (B) and as a topology diagram (D). β-strands are shown in red, yellow and blue for the core-THUMP, NFLD and RFM (sub)domains, respectively. α-Helices are shown in dark red, yellow and blue. The β-strand shared by the core-THUMP and NFLD subdomains is shown in orange. The two additional β-strands in the PfTrm14 structure are named βi and βii. The structure of PfTrm14 contains sinefungin bound in the active site, represented in ball and stick.

Mentions: Overall, the structures of PfTrm14 and TTCTrmN are very similar (Figures 1 and 2) with an rmsd of 2.0 Å, calculated over all main chain atoms (286 residues). Globally, PfTrm14 and TTCTrmN adopt a cylindrical shape with an approximate size of 66.0 × 35 × 35 Å3 (Figure 2). The structures consist of two globular domains. An N-terminal THUMP domain spanning approximately the residues 1–183 in PfTrm14 and 1–151 in TTCTrmN, and a C-terminal RFM domain spanning approximately the residues 193–367 in PfTrm14 and the residues 160–335 in TTCTrmN. Both domains are connected via a long linker containing several positively charged amino acids. The orientation of the THUMP and RFM domains toward each other is similar in PfTrm14 and TTCTrmN.Figure 1.


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)

Crystal structures of PfTrm14 and TTCTrmN. (A and C) show the structure of PfTrm14 in cartoon representation (A) and as a topology diagram (C); (B and D) show the structure of TTCTrmN in cartoon representation (B) and as a topology diagram (D). β-strands are shown in red, yellow and blue for the core-THUMP, NFLD and RFM (sub)domains, respectively. α-Helices are shown in dark red, yellow and blue. The β-strand shared by the core-THUMP and NFLD subdomains is shown in orange. The two additional β-strands in the PfTrm14 structure are named βi and βii. The structure of PfTrm14 contains sinefungin bound in the active site, represented in ball and stick.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks163-F2: Crystal structures of PfTrm14 and TTCTrmN. (A and C) show the structure of PfTrm14 in cartoon representation (A) and as a topology diagram (C); (B and D) show the structure of TTCTrmN in cartoon representation (B) and as a topology diagram (D). β-strands are shown in red, yellow and blue for the core-THUMP, NFLD and RFM (sub)domains, respectively. α-Helices are shown in dark red, yellow and blue. The β-strand shared by the core-THUMP and NFLD subdomains is shown in orange. The two additional β-strands in the PfTrm14 structure are named βi and βii. The structure of PfTrm14 contains sinefungin bound in the active site, represented in ball and stick.
Mentions: Overall, the structures of PfTrm14 and TTCTrmN are very similar (Figures 1 and 2) with an rmsd of 2.0 Å, calculated over all main chain atoms (286 residues). Globally, PfTrm14 and TTCTrmN adopt a cylindrical shape with an approximate size of 66.0 × 35 × 35 Å3 (Figure 2). The structures consist of two globular domains. An N-terminal THUMP domain spanning approximately the residues 1–183 in PfTrm14 and 1–151 in TTCTrmN, and a C-terminal RFM domain spanning approximately the residues 193–367 in PfTrm14 and the residues 160–335 in TTCTrmN. Both domains are connected via a long linker containing several positively charged amino acids. The orientation of the THUMP and RFM domains toward each other is similar in PfTrm14 and TTCTrmN.Figure 1.

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.

Show MeSH
Related in: MedlinePlus