Structural and functional analyses of the archaeal tRNA m²G/m²₂G10 methyltransferase aTrm11 provide mechanistic insights into site specificity of a tRNA methyltransferase that contains common RNA-binding modules

Abstract

N2-methylguanosine is one of the most universal modified nucleosides required for proper function in transfer RNA (tRNA) molecules. In archaeal tRNA species, a specific S-adenosyl-L-methionine (SAM)-dependent tRNA methyltransferase (MTase), aTrm11, catalyzes formation of N2-methylguanosine and N2,N2-dimethylguanosine at position 10. Here, we report the first X-ray crystal structures of aTrm11 from Thermococcus kodakarensis (Tko), of the apo-form, and of its complex with SAM. The structures show that TkoTrm11 consists of three domains: an N-terminal ferredoxinlike domain (NFLD), THUMP domain and Rossmann-fold MTase (RFM) domain. A linker region connects the THUMP-NFLD and RFM domains. One SAM molecule is bound in the pocket of the RFM domain, suggesting that TkoTrm11 uses a catalytic mechanism similar to that of other tRNA MTases containing an RFM domain. Furthermore, the conformation of NFLD and THUMP domains in TkoTrm11 resembles that of other tRNA-modifying enzymes specifically recognizing the tRNA acceptor stem. Our docking model of TkoTrm11-SAM in complex with tRNA, combined with biochemical analyses and pre-existing evidence, provides insights into the substrate tRNA recognition mechanism: The THUMP domain recognizes a 3′-ACCA end, and the linker region and RFM domain recognize the T-stem, acceptor stem and V-loop of tRNA, thereby causing TkoTrm11 to specifically identify its methylation site.

Trm112, a Protein Activator of Methyltransferases Modifying Actors of the Eukaryotic Translational Apparatus

“…An N-terminal THUMP domain (for thiouridine synthases, RNA methyltransferases and pseudouridine synthetases; [26]) formed by an NFLD (N-terminal ferredoxin-like domain) subdomain fused to a core-THUMP subdomain and a C-terminal class I SAM-dependent MTase domain [12,27]. Such modular organization has been confirmed by the recent crystal structure of the archaeal Trm11 ortholog from Thermococcus kodakarensis [28] and is shared with archaeal Trm14 and bacterial TrmN, which are both responsible for m 2 G formation at position 6 on tRNAs [29,30]. In the 4-thiouridine synthase enzyme ThiI, the THUMP domain was shown to interact with the 3’ CCA end [31] and was then proposed to position the substrate nucleotide in the enzyme active site.…”

“…In S. cerevisiae , Trm112 is needed for the formation of m 2 G 10 modification by Trm11 [12], whereas archaeal orthologs studied so far (PAB1283 from Pyrococcus abyssi and aTrm11 from Thermococcus kodakarensis ) are active on their own [28,32]. Initially, the m 2 G 10 modification could only be recapitulated in vitro using the Sc Trm11-Trm112 complex either purified directly from yeast cells [12] or produced using a wheat germ cell-free translation system [33], suggesting that post-translational modifications might be necessary for enzymatic activity.…”

“…Finally, no protein with significant sequence similarity with Trm112 could be identified in Thermococcales and Methanobacteriales from the Euryarchaeota phylum. This is noteworthy as Trm11 orthologs from two Thermococcales archaea ( Pyrococcus abyssi and Thermococcus kodakarensis ) can form m 2 G (and even m 2 2 G) at position 10 of some tRNAs in vitro without the requirement of a protein partner [28,32]. Bioinformatics analysis supports the existence of Trm11 orthologs in all archaea phyla, and hence, most of the time, these proteins co-occur with Trm112, suggesting that with the exception of Thermococcales and Methanobacteriales Trm11, the other archaeal Trm11 orthologs may exist as a complex with Trm112 and may require Trm112 to be active as observed in eukaryotes.…”

To be or not to be modified: Miscellaneous aspects influencing nucleotide modifications in tRNAs

“…Conversely, the archaeal Trm11 requires an intact elbow and accepting region in the tRNA substrate. A docking model of the tRNA/Trm11 complex suggested that substrate recognition and catalysis is achieved by a molecular ruler mechanism in which the distances between the active site and both the 3′‐CCA end and the variable region are precisely defined . A similar tRNA/enzyme model was proposed for the orthologous yeast Trm11 enzyme that acts as a heterodimer with the Trm112 protein.…”

Evolutionary insights into Trm112-methyltransferase holoenzymes involved in translation between archaea and eukaryotes

“…While nothing is known on bacterial Trm112 orthologues, the detection of Mtq2, Trm9 and Trm11 orthologues in archaeal genomes together with the strong similarity between eukaryotic and archaeal translation machineries suggest that archaeal Trm112 might play a role similar to the eukaryotic Trm112 ( 53–55 ). Indeed, Trm11 orthologues (hereafter named aTrm11) from Pyrococcus abyssii and Thermococcus kodakarensis have been biochemically characterized as enzymes methylating guanine nucleotide at position 10 of some tRNAs ( 56 , 57 ). However, these two enzymes not only catalyze the formation of N2-methylguanosine but also of N2,2-dimethylguanosine and do not require Trm112 to be active.…”