Insights into molecular plasticity in protein complexes from Trm9-Trm112 tRNA modifying enzyme crystal structure

Abstract

Most of the factors involved in translation (tRNA, rRNA and proteins) are subject to post-transcriptional and post-translational modifications, which participate in the fine-tuning and tight control of ribosome and protein synthesis processes. In eukaryotes, Trm112 acts as an obligate activating platform for at least four methyltransferases (MTase) involved in the modification of 18S rRNA (Bud23), tRNA (Trm9 and Trm11) and translation termination factor eRF1 (Mtq2). Trm112 is then at a nexus between ribosome synthesis and function. Here, we present a structure-function analysis of the Trm9-Trm112 complex, which is involved in the 5-methoxycarbonylmethyluridine (mcm5U) modification of the tRNA anticodon wobble position and hence promotes translational fidelity. We also compare the known crystal structures of various Trm112-MTase complexes, highlighting the structural plasticity allowing Trm112 to interact through a very similar mode with its MTase partners, although those share less than 20% sequence identity.

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

“…Sequence alignment of Trm112 orthologs from the three domains of life and crystal structures of eukaryotic Trm112 proteins either in an isolated form [18] or in complex with MTase partners (see below, [19,20,21]) have revealed an organization into two domains. The first domain, contributed by residues from the N- and C-terminal extremities of eukaryotic Trm112 proteins, is conserved within the three domains of life.…”

“…In the structure of isolated Sc Trm112 [18], this β-sheet is composed of three β-strands, and the Trm112 C-terminal extremity folds back onto a hydrophobic region of the Zn-knuckle domain. In the crystal structures of Trm112-MTase complexes [19,20,21], this Trm112 C-terminal extremity adopts a radically different conformation and folds as a fourth β-strand (β4), which is engaged in the interaction with the MTase partners (see Section 2.5). The second domain is contributed by residues from the central region of Trm112 eukaryotic proteins and is absent in bacterial, as well as in some archaeal orthologs.…”

“…All structures of eukaryotic Trm112 solved to date are from fungi ( S. cerevisiae [18,20], Yarrowia lipolytica [21]) or from an intracellular parasite ( Encephalitozoon cuniculi [19]), and they all exhibit one zinc atom coordinated by four cysteine residues in the so-called Zn-knuckle domain. These residues belong to two well-conserved motifs (CX 3-4 C and CX 2 C from the N- and C-terminal parts, respectively; where C is for cysteine and X is for any amino acid; Figure 1b).…”

“…The presence of the methyl group is important for an efficient thiolation, as we detected tRNA mcm 5 U 34 product, but not mcm 5 (s 2 )U 34 after in vitro enzymatic reaction of Sc Trm9-Trm112 on tRNAs purified from the trm9∆ yeast strain. Furthermore, several groups observed a drop in cm 5 (s 2 )U 34 formation upon disruption of TRM9 or TRM112 genes [13,14,21]. In S. cerevisiae , this mcm 5 (s 2 )U 34 tRNA modification confers susceptibility to zymocin, a toxin secreted by the yeast Kluyveromyces lactis , which cleaves specifically the modified anticodon loop, thereby inhibiting translation and leading to death [44].…”

tRNA wobble modifications and protein homeostasis

“…Subsequent conversion of cm 5 U to mcm 5 U requires tRNA-methyltransferases Trm9p and Trm112p with S-adenosyl methionine (SAM) as the methyl donor. 22,23 Finally, addition of s 2 U requires ubiquitin-related modifier 1 (Urm1p) along with its activating protein, Uba4p, and other associated gene products including cysteine desulfurase (Nfs1p), thiouridine modification protein (Tum1p), iron-sulfur cluster binding proteins (Cfd1p, Isu1p, Isu2p, Nbp35p, Cia1p), and s 2 U ligase (Ncs2p, Ncs6p). Cysteine is the main source of sulfur for s 2 U synthesis.…”