TRNA:m2G6 methyltransferase TrmN/Trm14

From Proteopedia

Introduction

TrmN and Trm14 build a family of tRNA:m2G6 methylstransferases, that specifically modify a guanosine on position 6 of tRNA. Transfer RNA (tRNA) molecules are pivotal in the translation of the genetic code into protein sequence. tRNAs consist of 54 to 100 nucleotides (mostly 76 nucleotides) and form a 3-dimensional L-shaped structure and more than 100 tRNA modifications are known up to date, where methylations are the most common ones. Modifications of tRNA can affect the tRNA conformation and thereby also the base pairing capabilities of the anticodon, base pairing with the mRNA codon due to modifications at the anticodon and the recognition by aminoacyl tRNA synthetases at the acceptor stem. Although many tRNA modifications are known, often the proteins involved in the modification are unknown. In 2011 Menenez et al. ^[1] identified in the archeum M. jannaschii an enzyme called Trm14 which is responsible for the m2G modification of tRNA(Cys). Independly Roovers et al. ^[2] identified the orthologous protein TrmN in the bacterium Thermus thermophilus, which is capable of modifying tRNA(Phe).

Structures

For this family four structures are existing of which one 3tma is the crystal structure of TrmN from the bacterium Thermus thermophilus and three 3tlj, 3tl4 and 3tm5 are the crystal structures of Trm14 from the archaeum Pyrococcus furiosus ^{[3] [4]}. Additionally this protein exist also in eukaryotes but their structure is missing. The protein is functional as and consist of two domains. A C-terminal (displayed green) and an N-terminal (displayed red). The N-terminal part was described in literature as two domain, a (core-)THUMP domain and an N-terminal ferrodoxin like domain (NFLD). The crystal structures suggest, that these two domain are actually subdomains of the holo-THUMP domain. An interesting feature is the insertion of a beta-sheet in the THUMP domain of Trm14 compared to TrmN. This beta-sheet is only conserved in Archaea and the replacement of this sheet by two glycines did not have an influence on the tRNA binding and methylation actitity in vitro. Although the THUMP domain is a domain known for tRNA binding, the isolated domain is not capable of binding to tRNA. The methyltransferase domain is binding S-adenosylmethionine as ligand and the structures of Trm14 in complex with SAM, SAH (the reaction educt) and sinefungin (SFG, an inhibitor) are known. The adenosine moieties of the ligands SAH, SAM and SFG are bound to Trm14 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. Interestingly a loop adjacent to the ligand binding pocket is not seen in the electron density of the SAH and SAM bound state of Trm14 and the the structure of TrmN, which is in the apo form. It is possible that this loop undergoes rigidification during the methylation of the guanosine, since the charge distribution of SFG might mimic the transition between SAM and SAH. All in allthe ribose moiety of the co-factor is hydrogen bonded with both its 2′- and 3′ hydroxyls to the side chain of the highly conserved Glu248 of motif II. The 3′-OH is also involved in a hydrogen bond with His198. The adenine moiety of the co-factor is stacked between the side chains of Met225 and Lys249 of Trm14 using van der Waals interactions. The N6 exocyclic amine of the adenine base forms a hydrogen bond to Asp276, while its N1 is within hydrogen bonding distance to the main chain amine of Ala277. Variation of the catalytic ASN293 to alanine leads to loss of function, which is consistent with other RFMs.

Model of tRNA binding

Modeling of tRNA on the structure of TrmN suggests the binding of tRNA with the main part in a groove between the THUMP and RFM domains and binding of the acceptor stem on top of the ligand binding site of the RFM domain. This binding mode is supported by variation of positively charged residues in the groove between the two domains (Arg33, Arg98, Lys129, Arg130, Arg153 & Arg157)to alanine or glutamate and by the double variation of Lys270 and Arg300 (located in vicinity to the ligand binding site) to alanine or glutamate.

References

1. ↑ Menezes S, Gaston KW, Krivos KL, Apolinario EE, Reich NO, Sowers KR, Limbach PA, Perona JJ. Formation of m2G6 in Methanocaldococcus jannaschii tRNA catalyzed by the novel methyltransferase Trm14. Nucleic Acids Res. 2011 Sep 1;39(17):7641-55. Epub 2011 Jun 21. PMID:21693558 doi:10.1093/nar/gkr475
2. ↑ Roovers M, Oudjama Y, Fislage M, Bujnicki JM, Versees W, Droogmans L. The open reading frame TTC1157 of Thermus thermophilus HB27 encodes the methyltransferase forming N(2)-methylguanosine at position 6 in tRNA. RNA. 2012 Apr;18(4):815-24. Epub 2012 Feb 15. PMID:22337946 doi:10.1261/rna.030411.111
3. ↑ Fislage M, Roovers M, Tuszynska I, Bujnicki JM, Droogmans L, Versees W. Crystal structures of the tRNA:m2G6 methyltransferase Trm14/TrmN from two domains of life. Nucleic Acids Res. 2012 Feb 22. PMID:22362751 doi:10.1093/nar/gks163
4. ↑ Fislage M, Roovers M, Munnich S, Droogmans L, Versees W. Crystallization and preliminary X-ray crystallographic analysis of putative tRNA-modification enzymes from Pyrococcus furiosus and Thermus thermophilus. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011 Nov 1;67(Pt 11):1432-5., Epub 2011 Oct 27. PMID:22102250 doi:10.1107/S1744309111036347