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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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(A and B) Electrostatic surface potential mapped on the solvent accessible surface of PfTrm14 (A) and TTCTrmN (B). kb = Boltzmann’s constant, T = temperature, ec = charge of an electron. (C and D) Mapping of conservation scores of amino acids on the surface of PfTrm14 (C) and TTCTrmN (D). The color code of the conservation score is indicated in the legend. The bound SFG (PfTrm14) and modeled SAM (TTCTrmN) in the active site of the RFM domain are shown in ball and stick representation.
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gks163-F4: (A and B) Electrostatic surface potential mapped on the solvent accessible surface of PfTrm14 (A) and TTCTrmN (B). kb = Boltzmann’s constant, T = temperature, ec = charge of an electron. (C and D) Mapping of conservation scores of amino acids on the surface of PfTrm14 (C) and TTCTrmN (D). The color code of the conservation score is indicated in the legend. The bound SFG (PfTrm14) and modeled SAM (TTCTrmN) in the active site of the RFM domain are shown in ball and stick representation.

Mentions: Another obvious difference of the THUMP domain of PfTrm14 in comparison to TTCTrmN and other known THUMP domains concerns an insertion of two anti-parallel β-strands between β-strand 3 and helix 2 of the NFLD subdomain of the THUMP domain of PfTrm14 (Figure 2A and C). This two-stranded anti-parallel β-sheet is extending away from the THUMP domain on the opposite side of the presumed tRNA binding surface and the SAM binding pocket of the RFM domain (see further). Sequence analysis using a BLAST search (61) showed that this insertion is conserved only in Archaea, with over 50 matches found. No comparable insertions were found in THUMP domains of Bacteria and Eukaryota. Interestingly, these two β-strands are made up mainly of conserved positively charged (five lysines or arginines) and aromatic residues (Y51, Y52), resulting in one side of the β-sheet having a positively charged surface (Figure 4A). To investigate a possible role of these two β-strands in tRNA binding and catalysis of the m2G6 modification, we made two different deletion variants of PfTrm14. In a first variant, the peptide region spanning the inserted β-sheet (residues Y51 to E61) was replaced by two glycines that should be sufficient to span the distance between β-strand 3 and helix 2 of the THUMP domain (PfTrm14_2G variant). In a second variant, this region was replaced by the corresponding loop region of TTCTrmN (R41 to G45) (PfTrm14_TTC variant). The G6 MTase activity and tRNA binding affinity of both variants were compared with the wild-type PfTrm14 (Supplementary Figure S3A and B). Interestingly, only small changes in affinity towards tRNA and in the catalytic rate were observed in vitro upon deletion. This might suggest that the β-strands are a conserved ornament of the basic NFLD fold or a relic of a previous activity. Alternatively, this element could be involved in another, yet undiscovered, function of archaeal Trm14.


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)

(A and B) Electrostatic surface potential mapped on the solvent accessible surface of PfTrm14 (A) and TTCTrmN (B). kb = Boltzmann’s constant, T = temperature, ec = charge of an electron. (C and D) Mapping of conservation scores of amino acids on the surface of PfTrm14 (C) and TTCTrmN (D). The color code of the conservation score is indicated in the legend. The bound SFG (PfTrm14) and modeled SAM (TTCTrmN) in the active site of the RFM domain are shown in ball and stick representation.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks163-F4: (A and B) Electrostatic surface potential mapped on the solvent accessible surface of PfTrm14 (A) and TTCTrmN (B). kb = Boltzmann’s constant, T = temperature, ec = charge of an electron. (C and D) Mapping of conservation scores of amino acids on the surface of PfTrm14 (C) and TTCTrmN (D). The color code of the conservation score is indicated in the legend. The bound SFG (PfTrm14) and modeled SAM (TTCTrmN) in the active site of the RFM domain are shown in ball and stick representation.
Mentions: Another obvious difference of the THUMP domain of PfTrm14 in comparison to TTCTrmN and other known THUMP domains concerns an insertion of two anti-parallel β-strands between β-strand 3 and helix 2 of the NFLD subdomain of the THUMP domain of PfTrm14 (Figure 2A and C). This two-stranded anti-parallel β-sheet is extending away from the THUMP domain on the opposite side of the presumed tRNA binding surface and the SAM binding pocket of the RFM domain (see further). Sequence analysis using a BLAST search (61) showed that this insertion is conserved only in Archaea, with over 50 matches found. No comparable insertions were found in THUMP domains of Bacteria and Eukaryota. Interestingly, these two β-strands are made up mainly of conserved positively charged (five lysines or arginines) and aromatic residues (Y51, Y52), resulting in one side of the β-sheet having a positively charged surface (Figure 4A). To investigate a possible role of these two β-strands in tRNA binding and catalysis of the m2G6 modification, we made two different deletion variants of PfTrm14. In a first variant, the peptide region spanning the inserted β-sheet (residues Y51 to E61) was replaced by two glycines that should be sufficient to span the distance between β-strand 3 and helix 2 of the THUMP domain (PfTrm14_2G variant). In a second variant, this region was replaced by the corresponding loop region of TTCTrmN (R41 to G45) (PfTrm14_TTC variant). The G6 MTase activity and tRNA binding affinity of both variants were compared with the wild-type PfTrm14 (Supplementary Figure S3A and B). Interestingly, only small changes in affinity towards tRNA and in the catalytic rate were observed in vitro upon deletion. This might suggest that the β-strands are a conserved ornament of the basic NFLD fold or a relic of a previous activity. Alternatively, this element could be involved in another, yet undiscovered, function of archaeal Trm14.

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