Mechanism of activation of methyltransferases involved in translation by the Trm112 ‘hub’ protein

Cloning, mutagenesis, expression and purification of proteins

Genes encoding Encephalitozoon cuniculi (Ec)-Mtq2 (UniProtKB entry: Q8SRR4), eRF1 and eRF3 were cloned from Ec-GB-M1 genomic DNA (generous gift from Prof. Vivares, Université Blaise Pascal, Clermont-Ferrand, France) into pET9 plasmids with a C-terminal hexahistidine tag. In parallel, a DNA sequence was designed to encode Ec- Trm112 (UniProtKB entry: Q8SUP0) and Mtq2 with a C-terminus His-tag. This fragment was obtained by de novo gene synthesis (GenScript Corporation, Piscataway, NJ, USA) and was further subcloned into pET21-a between NdeI and XhoI sites. Expression of the His-tagged Mtq2-Trm112 complex was done in E. coli BL21 (DE3) Gold strain (Novagen). The 800 ml of culture in 2xYT medium (BIO101 Inc.) containing ampicillin at 50 µg/ml were incubated at 37°C until an OD600 of about 0.6–0.8 and induced with IPTG 0.5 mM final at 28°C for 4 h. The cells were harvested by centrifugation, resuspended in 40 ml of 20 mM Tris–HCl, pH 7.5, 200 mM NaCl, 5 mM β-mercaptoethanol (buffer A) and stored at −20°C. Cell lysis was achieved by sonication. His-tagged complex was purified on a nickel-nitrilotriacetic acid column (Qiagen Inc.) followed by gel filtration on a SuperdexTM 75 (16/60) column (GE Healthcare) equilibrated in buffer A.

Plasmids used for over-expression of yeast proteins were mutagenized following Quick change mutagenesis protocol (Stratagen) and sequenced. Expression and purification of S. cerevisiae and human proteins were done as described previously (21,22).

Yeast strain construction

Genomic cassette insertion through homologous recombination was used for C-terminus epitope-tagging in yeast according to published protocol (38). Point mutations in Trm9-13Myc encoding gene sequence were also introduced by cassette insertion through homologous recombination (39). Detailed experimental procedure is described in Supplementary Data.

Co-immunoprecipitation and western blot

Soluble protein extracts were prepared by the glass bead method starting from 10⁸ to 10⁹ mid-log growing cells. Cells were washed once in water and pellets were frozen in liquid nitrogen and then stored at −20°C. Cells were thawed and resuspended in ice-cold breaking buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 20 mM MgCl₂, 5 mM EDTA, 1% NP-40) supplemented with Complete EDTA-free anti-protease cocktail (Roche Diagnostics). After addition of acid-washed glass beads, cells were lysed by 5 pulses of vortexing for 30 s separated by cooling intervals on ice. Soluble fractions were collected after centrifugation for 20 min at 4°C at 13 000 rpm. Based on Bradford assay, 500 µg of proteins were incubated with mouse monoclonal 9E10 anti-myc antibody (Santa Cruz Biotechnology) for 1 h on ice. Immune complexes were then incubated for 30 min at 4°C on a rotating wheel after addition of protein G-agarose (Sigma). Agarose beads were washed four times in breaking buffer. The final pellet was resuspended in cracking buffer (40 mM Tris–HCl, pH 6.8, 8 M Urea, 5% SDS, 0.1 mM EDTA, 1% β-mercaptoethanol, 0.04% Bromophenol Blue) (40) and boiled for protein release from beads. Proteins from starting soluble fraction and from final supernatant were resolved on a 15% polyacrylamide gel and transferred onto Protran nitrocellulose membrane (Whatman). Probing was performed using either mouse 9E10 anti-myc or 12CA5 anti-HA (Santa Cruz Biotechnology) as primary antibody (1/500) and sheep anti-mouse HRP-conjugated IgG as secondary antibody (1/3000, GE HealthCare). Immunoblots were developed using ECL (GE HealthCare).

Zymocin killer assay

This assay was performed as previously described (41). Procedure is described in Supplementary Data.

Crystallization and structure solution

Thin needle crystals were grown at 19°C from a 0.1:0.1 µl mixture of 7 mg/ml complex solution with a crystallization solution containing 20% poly ethylene glycol (PEG) 4000, 10% isopropanol, 100 mM HEPES (pH 7.5). For data collection, the crystals were transferred into a cryoprotectant crystallization solution with progressively higher ethylene glycol concentrations up to 30% v/v. The datasets were collected at the Zn-edge on beam line Proxima-1 (SOLEIL, St-Aubin, France). The structure was determined by the SAD method (Single wavelength Anomalous Dispersion) using the anomalous signal from the Zn element (see Supplementary Data for details).

The final model contains all the Mtq2 residues (from Met1 to Ser164) as well as residues 1–32 and 36–125 from Trm112, which are well defined in electron density. In addition, one zinc atom, one SAM molecule and 136 water molecules have been modelled. Statistics for data collection and refinement are summarized in Supplementary Table S1. The atomic coordinates and structure factors have been deposited into the Brookhaven Protein Data Bank under the accession number 3Q87.

In vitro methylation assay

Methylation assays were done as described previously by incubating eRF1, eRF3 and GTP with Mtq2-Trm112 complex in presence of [³H] SAM (22).

SAM-binding to human Mtq2 (Hs-Mtq2)

An equimolar amount of Hs-Trm112 was added to partially purified Hs-Mtq2 (corresponding approximately to 100 pmol) and 0.5 µl of [³H] SAM (78 Ci/mmol, Perkin Elmer) in 50 µl final volume methylation buffer for 15 min at 30°C. Reaction was stopped by filtration through nitrocellulose filters (0.45 µm HAWP02500 Millipore) followed by washing with methylation buffer. Radioactivity on filter then represents an amount of SAM bound to Hs-Mtq2. Controls with Hs-Trm112 alone showed very low background fixation. We checked by western blot that Hs-Trm112 was not increasing binding of Hs-Mtq2 itself to the filter, increasing artifactually SAM-binding.

Crystal structure

To understand the molecular basis responsible for the interaction between Mtq2 and Trm112, we have crystallized the Mtq2-Trm112 complex from the small parasite E. cuniculi (Ec-Mtq2-Trm112, see ‘Cloning, mutagenesis, expression and purification of proteins’ for more details). We took advantage of the presence of a zinc atom bound to Trm112 as observed for the S. cerevisiae protein (22) to solve the structure of this complex by the SAD method using the zinc anomalous signal. The final model has been refined to 2.1 Å resolution and contains one Mtq2-Trm112 heterodimer in the crystal asymmetric unit (Figure 1A, Supplementary Table S1 and Figure S1 for electron density map). Although we did not pre-incubate the Mtq2-Trm112 complex with SAM for the crystallization experiments, an unambiguous electron density corresponding to a SAM molecule is present in its expected binding site, indicating that this ligand was co-purified with the complex. SAM is bound at the C-terminal face of the Mtq2 central β-sheet and interacts exclusively with residues from Mtq2.

Mtq2 adopts the typical class I SAM-dependent MTase fold composed of a central seven-stranded β-sheet (strand order: β3↑β2↑β1↑β4↑β5↑β7↓β6↑) surrounded by three α-helices (α1–α3) on one side and two (α4 and α5) on the other (42). A search for proteins with similar three-dimensional structure [program DALI (43)] identified the C-terminal domain MTase from the PrmC/HemK as the best hit (Z-score = 18.5; RMSD of 2.2 Å over 159 Cα atoms, 19% sequence identity) (14,44). Interestingly, this enzyme performs the same chemical reaction as Mtq2, since it modifies the Gln side chain of the GGQ motif from bacterial release factors RF1 and RF2. The bacterial PrmC MTases are formed by an N-terminal helical domain and a C-terminal MTase domain connected by a short β-hairpin. As anticipated by sequence analysis (5), only the MTase domain is present in Mtq2. In proteins from both kingdoms, these domains are very similar and bind the SAM/S-adenosyl-l-homocysteine (SAH) cofactor in the same way.

The Ec-Trm112 protein is composed of two domains: a zinc-binding domain formed by both N- and C-terminal extremities and a central domain. Its overall structure is very similar to yeast Trm112 [Z-score = 9.3 and global RMSD of 2.9 Å over 102 Cα atoms, 18% sequence identity; Supplementary Figure S2A, (22)]. The zinc-binding domain consists of an α-helix packed onto a four-stranded antiparallel β-sheet (RMSD with the equivalent domain from yeast Trm112 of 1.4 Å over 57 Cα atoms, 30% sequence identity). Similarly, to yeast Trm112, four cysteine residues coordinate the zinc atom. The central domain is a four-helix bundle in the E. cuniculi protein but it is less similar to yeast Trm112 (three-helix bundle) than the zinc-binding domain (RMSD of 3.4 Å over 42 Cα atoms, 5% sequence identity). In this domain, only helices α3 and α4 from Ec-Trm112 match with the equivalent helices from yeast Trm112. Helix α2 from the yeast protein structurally matches with the elongated stretch preceding helix α2 in Ec-Trm112. Helix α3′ from Ec-Trm112 corresponds to the linker connecting helices α3 and α4 in yeast protein. Another important difference between the E. cuniculi and yeast Trm112 structures resides in the orientation of the last ten C-terminal residues that are relatively well conserved. This region was proposed to be part of the homodimer interface in yeast Trm112 and this has since been confirmed by site-directed mutagenesis (data not shown). In the present Mtq2-Trm112 complex, this region cannot adopt the same conformation and folds back onto Trm112 strand β3 to avoid important steric clashes (Supplementary Figure S2B).

Mtq2-Trm112 interface

In the complex, Mtq2 binds mainly to the zinc-binding domain from Trm112 via a β-zipper interaction between Mtq2 strand β3 and Trm112 strand β4 that are associated in a parallel manner (Figure 1A). As a consequence, their β-sheets form a continuous large eleven-stranded β-sheet. Additional contacts are also made by residues from the loop connecting strands β3 to β4 in Mtq2 with helix α1 and the α1-β1 loop from Trm112 (Supplementary Figures S3A–C, S4 and S5). The complete interface buries a total solvent accessible surface area of 2000 Ų and contains 12 hydrogen bonds, clustered in two regions (see Supplementary Table S2). H-bonds at the β-zipper interface are mostly realized between main chain atoms from both partners (Supplementary Figure S3A). Additional H-bonds occur between residues from loop connecting β1 to α2 in Trm112 and from loop β3-β4 in Mtq2 (Supplementary Figure S3B). Non-polar atoms clustered at the centre of this contact area provide two-thirds of the interface (Supplementary Figure S3B–C). This is indeed illustrated by the strictly conserved Ile¹²⁵ (S. cerevisiae numbering) from Trm112 strand β4. Its substitution by an aspartic residue results in complete inactivation without disrupting the Mtq2-Trm112 complex (Table 1). A few contacts also exist between strand β2 as well as the β3–β4 loop in Mtq2 and the loop connecting β1 to α2 from the Ec-Trm112 central domain. Helix α2 in yeast Trm112 matches with the region corresponding to the Ec-Trm112 β1-α2 loop and hence is predicted to be important for Mtq2-Trm112 complex formation (Supplementary Figure S3C). This is supported by experiments showing that the yeast complexes containing yeast Trm112 single point mutants F46D and R53E are less soluble and stable than the wild-type complex and have lost eRF1 MTase activity (Table 1). In addition, superposition of yeast Trm112 onto our structure of the complex suggests that additional contacts could occur between helix α2 from Sc-Trm112 and the N-terminal face of the Mtq2 β-sheet as it faces two loops (connecting α1 to β1 and α2 to β2) that are predicted to be longer in other organisms such as S. cerevisiae than in E. cuniculi. This is supported by the N43R mutant, which has decreased eRF1 MTase activity (Supplementary Figure S3C and Table 1).

Trm112 enhances SAM-binding by Mtq2

Trm112 is needed to solubilize and activate Mtq2 in yeast (22). Similarly, human Mtq2 (Hs-Mtq2) protein performs eRF1 methylation only after incubation with human Trm112 (21). In order to understand the mechanism of Mtq2 activation by Trm112, we used the structure of the complex to examine the effect of site-directed mutations. Our strategy is based upon two important observations. First, Trm112 shields from the solvent a large hydrophobic surface from Mtq2 by using a hydrophobic region that is involved in yeast Trm112 homodimer formation, explaining why Trm112 solubilizes Mtq2 (Supplementary Figure S3D–E), (22). Second, the loop connecting strands β3–β4 in Mtq2, which is largely involved in the interaction with Trm112, is also involved in SAM-binding (Figure 1B). This led us to speculate that although it does not interact directly with SAM, Trm112 may stabilize this loop in a conformation allowing Mtq2 to bind more tightly the SAM cofactor. In particular, the Asp⁶⁹ side chain and Leu⁷⁰ main chain amide groups from this loop are hydrogen-bonded to the adenine moiety of SAM. We have therefore assessed the importance of these two residues for MTase activity by site directed mutagenesis of the corresponding residues in yeast Mtq2 protein (Asp¹⁰⁶/Leu¹⁰⁷). Co-expression of this double mutant (D106A/L107A) with Trm112 yielded soluble complex, indicating that these mutations do not prevent complex formation. Similarly to the D77A mutant (Asp⁵¹ in E. cuniculi), a mutation known to disrupt SAM-binding by MTases, the D106A/L107A, a double mutant is completely unable to methylate eRF1 (Table 1). We next took advantage of the fact that Hs-Mtq2, in contrast with Sc-Mtq2, can be purified in low amounts following over-expression in E. coli in the absence of Hs-Trm112 (21). Although this protein is well folded according to circular dichroism experiments (data not shown), it is unable to methylate eRF1 on its own (21). This could be due to its intrinsically low SAM-binding activity (Figure 1C). Incubation of this protein with an equimolar amount of Hs-Trm112 resulted in restoration of SAM-binding activity (Figure 1C). It is noteworthy that Hs-Trm112 is more efficient when co-expressed with Hs-Mtq2 than upon incubation of the separately purified proteins (Figure 1C). Hence, at least for human proteins, Trm112 strongly stimulates SAM-binding to Mtq2, a prerequisite for enzymatic activity.

MTase active site

Mapping of sequence conservation at the surface of the Mtq2-Trm112 heterodimer clearly identifies a strongly conserved patch centred on the SAM methyl group as an excellent candidate for the Mtq2 active site (Figure 2A). In addition, the absence of longer loops in orthologous proteins within this putative active site led us to speculate that this structure is an excellent template for the design of mutants from yeast Mtq2 that will affect eRF1 MTase activity.

Superposition of the PrmC-RF1 and Mtq2-Trm112 complexes shows that this conserved region corresponds to the bacterial PrmC active site (14). In particular, the Asn-Pro-Pro-Tyr (NPPY) signature found in PrmC but also in DNA N6-adenine and N4-cytosine MTases modifying nitrogens conjugated to planar systems such as an amide group or a nucleotide base (3), matches perfectly the corresponding signature in Mtq2 (Figure 2B). In the complex between PrmC and RF1, this motif forms H-bonds with the Gln side chain of the GGQ motif and positions it ideally for subsequent methylation (14,15). As observed for bacterial PrmC, the Asn¹²² (Asn⁸⁵ in Ec-Mtq2) side chain in this NPPY signature is crucial for eRF1 methylation as its substitution by Ala completely abolishes yeast Mtq2 MTase activity (Table 1). We further focused our attention on two strictly conserved residues (Tyr⁴ and Asp⁹ in Ec-Mtq2, Tyr¹⁵ and Asp²⁰ in yeast) from the N-terminal part of Mtq2 that may participate directly in the coordination of the GGQ motif from eRF1, according to the superposition of the crystal structure of PrmC-RF1 complex onto Mtq2 protein. Both residues are hydrogen-bonded via their side chains and the aromatic ring from Tyr¹⁵ packs onto the methyl sulfonio group of the SAM molecule. In order to analyze their role in eRF1 methylation, we have performed conservative substitution of these residues. The Tyr¹⁵ side chain was replaced by Phe (Y15F), a substitution that should not much affect SAM-binding. Asp²⁰ was replaced by the isosteric Asn side chain (D20N). Both mutants proved to be completely inactive (Table 1).

In addition, this conserved region displays a negative electrostatic potential that very likely compensates for the positive electrostatic potential surrounding the eRF1 GGQ motif (Figure 2C). We generated several charge inversion mutants of negatively charged residues from this highly conserved putative active site: E16K, E19K, D26K/E29K and E212K (for clarity, yeast numbering is used in this section unless specifically stated). We also tested the following substitutions: F22A, R207A and R207E. Activity measurements show that all the strictly conserved residues located along the solvent exposed face of Mtq2 helix α1 (Glu¹⁹, Phe²², Asp²⁶ and Glu²⁹) are crucial for eRF1 methylation, suggesting that this helix is directly involved in substrate recognition (Table 1). This structure-based site-directed mutagenesis approach maps the Mtq2-Trm112 active site to a highly conserved and negatively charged region. This is particularly interesting, since a model of the S. cerevisiae eRF1-eRF3-GTP complex shows that the eRF1 GGQ motif is surrounded by highly conserved and basic residues (see ‘Discussion’ section).

Three additional Mtq2 mutants of solvent-exposed residues affect significantly eRF1 methylation. These are E16K from the loop preceding helix α1 as well as R207A and R207E from strand β6. These residues are located on both sides of helix α1 and hence define a relatively large substrate-binding site. Other mutated positions from Mtq2 (E212K) and Trm112 (A106E, E107K, I118E, Y120E and N123R) affect only partially (50–75%) MTase activity (Table 1). This notably suggests that Trm112 may also participate in substrate binding by this heterodimeric MTase.

The structure of the Mtq2-Trm112 complex can serve as a model for the Trm9-Trm112 complex

Trm112 interacts with and activates three class I SAM-dependent MTases: Mtq2, Trm9 and Trm11. These MTases seem to compete for binding to Trm112, suggesting that they interact with Trm112 in a similar manner (22,27,30–32,36). Hence, our structure of the Mtq2-Trm112 complex may serve as a good model for the Trm9-Trm112 and Trm11-Trm112 complexes and provide a template for functional studies.

To validate this hypothesis, we have studied the Trm9-Trm112 complex from S. cerevisiae by using K. lactis zymocin toxicity as a tool. This toxin specifically cleaves some tRNAs at the wobble uridine (U34) position. In order to be recognized by the zymocin, these tRNAs need to be fully modified and Trm9 is one of the enzymes involved (45). Hence, this toxin kills wild-type but not S. cerevisiae trm9-Δ or trm112-Δ strains. Using bioinformatics (see Supplementary Data for details on Trm9 model generation), we have generated a model for the S. cerevisiae Trm9 3D-structure and superimposed it onto Mtq2 in the Mtq2-Trm112 complex. We used this model to generate two mutant strains (N89K/L91R and F105E) aimed at disrupting the Trm9-Trm112 interface. As a positive control, we also generated a third mutant strain by substituting the strictly conserved aspartic acid residue proposed to be involved in SAM-binding by Ala (D72A) to inactivate Trm9 as previously done in other MTases (46). We then tested the effect of zymocin addition on cultures of wild-type and mutant yeast strains. All three mutants are resistant to zymocin supporting the hypothesis that these Trm9 mutants are inactive (Figure 3A). Next, the ability of these mutants to interact in vivo with Trm112 was assessed by co-immunoprecipitation. Whereas Trm112 co-immunoprecipitates with wild-type Trm9, no interaction is detected with the N89K/L91R or F105E mutants (Figure 3B). These mutants were expressed at similar levels to wild-type Trm9 protein in vivo, confirming that these mutations have no effect on protein stability. We therefore, conclude that these mutations confer the zymocin resistance phenotype by disrupting the Trm9–Trm112 interaction. However, Trm112 still co-immunoprecipitates with the Trm9 D72A mutant. The zymocin resistance phenotype is here due to the lack of SAM-binding necessary for a normal Trm9 activity. The Trm9–Trm112 interaction is obviously weaker with the Trm9 D72A mutant than with wild-type Trm9, suggesting SAM-binding by Trm9 may influence the strength of interaction between Trm9 and Trm112.

Altogether, these experiments strongly support the hypothesis that Trm112 interacts individually with Mtq2, Trm9 and Trm11 and in a similar way. Hence, the structure of the Trm112-Mtq2 complex provides a good model for further mutagenesis studies of the Trm9-Trm112 and Trm11-Trm112 complexes.