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Differentiating analogous tRNA by fragments of the methyl donor

GEORGES LAHOUD,1 SAKURAKO GOTO-ITO,2,3 KEN-ICHI YOSHIDA,2,3 TAKUHIRO ITO,2 SHIGEYUKI YOKOYAMA,2,3 and YA-MING HOU1,4 1Thomas Jefferson University, Department of Biochemistry and Molecular Biology, Philadelphia, Pennsylvania 19107, USA 2RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro, Tsurumi, Yokohama, Kanagawa 230-0045, Japan 3Department of Biophysics and Biochemistry, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

ABSTRACT Bacterial TrmD and eukaryotic-archaeal Trm5 form a pair of analogous tRNA that catalyze methyl transfer from S-adenosyl methionine (AdoMet) to N1 of G37, using catalytic motifs that share no sequence or structural homology. Here we show that natural and synthetic analogs of AdoMet are unable to distinguish TrmD from Trm5. Instead, fragments of AdoMet, adenosine and methionine, are selectively inhibitory of TrmD rather than Trm5. Detailed structural information of the two in complex with adenosine reveals how Trm5 escapes targeting by adopting an altered structure, whereas TrmD is trapped by targeting due to its rigid structure that stably accommodates the fragment. Free energy analysis exposes energetic disparities between the two enzymes in how they approach the binding of AdoMet versus fragments and provides insights into the design of inhibitors selective for TrmD. Keywords: S-adenosyl methionine (AdoMet); adenosine; methionine; m1G37-tRNA

INTRODUCTION veloped and adapted over time with comparable substrate binding affinities. This presents severe challenges to the con- Analogous enzymes are thought to be independent inven- ventional approach of structure-based design of substrate tions of nature (Galperin et al. 1998), arising from different analogs with sufficient selectivity between analogous en- phylogenetic lineages with unrelated structures to catalyze zymes. To date, no successful substrate analogs have been the same reaction. These enzymes are unlike the commonly reported that target specific members of a pair of analogous found homologous enzymes, which share similar sequence enzymes. and structural folds. The existence of analogous enzymes, Here we address this challenge by evaluating the deter- present in different biological domains, suggests the attrac- minants of the methyl donor AdoMet for selective inhibi- tive possibility of species-specific targeting. For example, tion of the analogous tRNA methyltransferases TrmD and the identification of a bacterial that is structurally Trm5, which catalyze the synthesis of the m1G37 base 39 distinct from its human counterpart would provide a prom- adjacent to the anticodon. TrmD is broadly conserved in all ising target for the development of new antibacterial drugs. bacterial species, whereas Trm5 is found in and Such a strategy would circumvent the difficulty in targeting but is absent from (Bjork et al. 1989, 2001; subtle differences between bacterial and human enzymes of O’Dwyer et al. 2004). While both enzymes recognize homologous origins. However, despite the appeal of this AdoMet as the methyl donor and G37-tRNA as the idea, obstacles remain high with targeting analogous en- acceptor, they share no structural homology. TrmD func- zymes. The key reason is that while these enzymes possess tions as a homodimer and binds AdoMet using a rare trefoil- different catalytic structures from each other, they recog- knot fold (Ahn et al. 2003; Elkins et al. 2003), whereas nize the same substrates and cofactors using tight networks Trm5 functions as a monomer and binds AdoMet using of enzyme-substrate interactions that have been well de- the popular fold known as the (Goto-Ito et al. 2008, 2009). Importantly, crystal structures of these enzymes show that the AdoMet structure when 4 Corresponding author. bound to TrmD exists in an L-shaped bent conformation E-mail [email protected]. Article published online ahead of print. Article and publication date are (Ahn et al. 2003), where the methionine moiety bends over at http://www.rnajournal.org/cgi/doi/10.1261/rna.2706011. the adenosine moiety, whereas the AdoMet structure when

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Lahoud et al. bound to Trm5 exists in an extended open conformation 2005; Renalier et al. 2005) is catalyzed by a single Trm56 (Fig. 1; Goto-Ito et al. 2009). The two enzymes also differ in enzyme in most organisms but by an RNA–protein (RNP) the recognition of G37 in tRNA (Christian and Hou 2007): complex in some archaea, where the in the complex TrmD requires only an extended anticodon stem–loop perform this reaction in conjunction with an RNA guide structure for activity, whereas Trm5 requires the intact made up of C/D boxes. The synthesis of C35 (C, pseudouri- tRNA structure. Furthermore, the rate-limiting step in the dine) in the anticodon of tRNATyr also involves analogous TrmD-catalyzed reaction occurs during the synthesis of enzymes, with Pus7 acting as the single enzyme in eukaryotes m1G37, whereas that for Trm5 occurs after synthesis and with an H/ACA-guided RNP in some archaea (Muller (Christian et al. 2010b). The fundamental structural and et al. 2009). However, detailed structural and kinetic in- kinetic differences between the two enzymes and their split formation is lacking for these alternative pairs. Importantly, along the evolutionary separation of bacteria from archaea the reaction catalyzed by TrmD and Trm5 is rare in that it and eukaryotes make this pair a paradigm to explore alone is required for optimal growth in bacteria (Bjork et al. conceptual strategies for targeting analogous enzymes. 1989; Hagervall et al. 1993; O’Dwyer et al. 2004; Baba et al. tRNA modification enzymes are attractive for targeting, 2006) and in eukaryotes (Bjork et al. 2001). This phenom- because of their role in the adjustment of cell fitness and vi- enon is due to the essential role of m1G37 in the prevention ability. Extensive cellular resources are dedicated to synthe- of translational errors on the ribosome (Hagervall et al. size these enzymes, which are responsible for the over 100 1993). Although m1A58 in the T loop of tRNA has also been modified nucleotides that have been identified in tRNA. shown to be essential for the viability of yeast (Anderson While many nucleotide modifications in tRNA can be et al. 2000), due to its role in maintaining steady-state levels achieved by a specific enzyme, others are achieved by mul- of eukaryotic initiator tRNA (Anderson et al. 1998), it is not tiple enzymes in successive reactions (e.g., wybutosine; Noma uniformly present in human cells (Saikia et al. 2010) and is et al. 2006). In general, inactivation of one modification rare in bacteria, indicating that it is not a broad-based anti- enzyme for a tRNA often has no clear growth defect, microbial target. Also, while m1A58 is synthesized by the TrmI although inactivation of additional enzymes can cause enzyme in bacteria (Roovers et al. 2004; Varshney et al. 2004) synthetic lethality (e.g., Persson et al. 1997; Urbonavicius but by a Trm6–Trm61 complex in eukaryotes (Anderson et al. 2002). The synergistic effects on growth between et al. 1998), the bacterial TrmI shares homology with the tRNA modification enzymes, together with the observation eukaryotic Trm61, indicating an orthologous relationship that hypo-modifications accelerate tRNA degradation between the two enzymes. (Alexandrov et al. 2006; Chernyakov et al. 2008), provide High-resolution crystal structures of TrmD and Trm5 an underpinning for the notion that tRNA modification reveal that both enzymes have an extensive network of enzymes function collectively as a device to regulate cellular hydrogen-bonding (H-bonding) and hydrophobic interac- processes and as a sensor of the cellular metabolic status in tions to recognize AdoMet. While different in spatial both bacteria and eukaryotes (Persson 1993). configuration, each network is elaborate and comprehen- While there are other analogous pairs of tRNA modifi- sive in the stabilization of the N1,N6, and N7 of the adenine cation enzymes, none are as well characterized as TrmD ring, the 29- and 39-OH of the ribose, and the carboxyl and and Trm5. For example, the synthesis of Cm32 and Um32 amino groups of methionine in the methyl donor. These (Cm and Um = ribose 29-O-methylation of C and U) in the stabilizing forces are well visualized in a binary structure of anticodon loop is catalyzed by TrmJ in bacteria with a TrmD with AdoMet and are preserved in a product complex trefoil-knot structure but is catalyzed by Trm7 in eukary- with S-adenosyl homocysteine (AdoHcy) (Ahn et al. 2003; otes with a Rossmann-fold structure (Purta et al. 2006). Elkins et al. 2003). They are also clearly identified in a binary The synthesis of Cm56 in the T loop (Clouet-d’Orval et al. structure of Trm5 with sinefungin and in a ternary structure with AdoMet and tRNA (Goto-Ito et al. 2008, 2009). The extensive stabilizing networks established for AdoMet are consistent with the comparably high affinity of both enzymes for the methyl donor. Indeed, fluorescence binding analysis of a model pair consisting of Escherichia coli TrmD and archaeal Methanococcus jannaschii Trm5 reveals their Kd for AdoMet in the range of 1–3 mM (Christian et al. 2006, 2010b), which explains their similar catalytic efficiencies in steady state and in single turnover assays (Christian et al. 2004, 2010b). AdoMet is the major biological methyl donor in diverse FIGURE 1. Two structures of AdoMet. (A) AdoMet when bound to methyl transfer reactions, due to the attachment of an elec- TrmD (Ahn et al. 2003). (B) AdoMet when bound to Trm5 (Goto-Ito et al. 2009). Drawn based on the coordinates in the structures trophilic methionine methyl group to a positively charged (PDBID: 1UAK and 2ZZN) by PyMol. sulfur atom to provide a thermodynamically favorable

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Analogous tRNA methyltransferases driving force for methyl transfer (Cantoni 1975). The two than in Trm5, thus exhibiting selectivity against TrmD. In a fragments of AdoMet, adenosine and methionine, are small competitive inhibition model, the maximum velocity (Vmax) and soluble and both are amenable to structural modifica- of the reaction and the catalytic turnover kcat (= Vmax/½E, tions through synthetic chemistry, thus emphasizing their where ½E = enzyme concentration) are unchanged, whereas potential for structure-guided drug development programs. the apparent affinity of the substrate to the binding site is Here we explore analogs of AdoMet in search of the deter- decreased, leading to an increase in the apparent Km of the minants that are able to discriminate TrmD from Trm5, substrate. We determined the inhibition constant Ki of an using kinetic analysis as a tool to monitor the synthesis of analog by measuring the apparent Km (AdoMet) as a function m1G37. A clear advantage of kinetic analysis over fluores- of analog concentration and fitting the data to the competi- cence or other types of binding assays is the ability to tive inhibition equation apparent K = K 3ð1+ ½IÞ, where m m Ki examine the contribution of binding free energy and reac- Km is the Michaelis constant of AdoMet and ½I is the con- tivity of all of the substrates, cofactors, and discrete states centration of the analog inhibitor. Because Km (AdoMet) of the enzyme along the reaction coordinate. Also, by differed substantially between TrmD and Trm5 (Fig. 2), we 1 focusing on the m G37 reaction, kinetic analysis can normalized Ki (analog) by Km (AdoMet). Mathematically, identify AdoMet analogs that specifically inhibit the re- Ki/Km is equivalent to IC50/½S+Km (Hamilton-Miller action, rather than those that inhibit other AdoMet-de- 1966) and it is an approximation of IC50/½S at saturating pendent reactions. For example, analogs that target TrmD substrate concentration relative to Km (AdoMet). Analog can be separated by cross-reactivity analysis from those that differentiation of TrmD from Trm5 was evaluated by the target TrmH (Nureki et al. 2004) and TrmL (Benı´tez-Pa´ez ratio of ½Ki/Km (Trm5)/½Ki/Km (TrmD), which was inter- et al. 2010), which adopt the same trefoil-knot structure as preted as the fold sensitivity of TrmD to the analog relative in TrmD but synthesize Gm18 and Um34 on tRNA, to Trm5 (Fig. 2). respectively. E. coli TrmD was assayed at 37°C with an in vitro Using E. coli TrmD and M. jannaschii Trm5 as a pair of transcript of E. coli tRNALeu, while M. jannaschii Trm5 was model enzymes, we find that neither naturally existing nor assayed at 55°C with a transcript of M. jannaschii tRNACys, synthetic analogs of AdoMet are able to distinguish be- using previously established conditions (Elkins et al. 2003; tween the two enzymes; rather, we find that fragments of Christian et al. 2004). These transcripts were chosen for their the methyl donor and their derivatives selectively inhibit optimal sequence contexts for TrmD and Trm5, respectively, TrmD more than Trm5. Crystal structural analysis of each and were each refolded by annealing and verified as tRNA enzyme in complex with the adenosine fragment reveals substrates by demonstrating a capacity for methylation to new insight into the molecular details of how the relatively z70% in extended time courses (Christian et al. 2010b). inflexible structure of TrmD subjects the bacterial enzyme Measurements of inhibition were exhaustively controlled: to targeting and inactivation, whereas the more fluid Enzymes were assayed at concentrations corrected by active- structure of Trm5 enables the eukaryotic-archaeal enzyme site titration and at levels that allowed linear synthesis of to escape targeting. Free energy analysis of binding provides m1G37 over time, while saturation of ½3H-AdoMet and evidence for an energetic penalty in the TrmD binding to tRNA was established at concentrations 10-fold higher than AdoMet versus fragment that is not observed in the Trm5 the previously determined Kd of each substrate (Christian binding. The disparities in binding energies between the et al. 2010b). two enzymes provide insight into their distinct AdoMet binding modes and suggest novel approaches toward the No differentiation by natural and synthetic development of selective antibiotics targeting the bacterial AdoMet analogs enzyme. Together, this work demonstrates that the frag- ment-based drug discovery approach (Congreve et al. 2008; The naturally existing AdoMet analogs sinefungin and Hesterkamp and Whittaker 2008), initially developed AdoHcy were evaluated as inhibitors of methyl transfer to identify small molecules that target individual proteins (Fig. 2). Sinefungin is an antifungal and antiparasitic anti- or , can be successfully exploited to differentiate the biotic produced by Streptomyces incarnates that shares with disparate substrate binding determinants of analogous AdoMet the adenosine fragment but differs by possessing enzymes. an ornithine instead of a methionine fragment, whereas AdoHcy is the product of methyl transfer that lacks the methyl group of AdoMet. Kinetic analysis showed that, RESULTS while both analogs were competitive inhibitors of AdoMet, they exerted similar effects on TrmD and Trm5 without Kinetic analysis of AdoMet analogs discrimination. For example, analysis of sinefungin in- To distinguish TrmD from Trm5, we sought to identify hibition of m1G37 synthesis as a function of AdoMet con- structural analogs of AdoMet that would more effectively centration revealed the same pattern for both enzymes: compete for the binding site of the methyl donor in TrmD While increasing concentration of the inhibitor had no effect

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Lahoud et al.

showed that the Ki/Km value of TrmD (0.8 6 0.5) is smaller than that of Trm5 (1.17 6 0.37) by 1.5-fold. Synthetic analogs with an allyl (AdoPropen) or propargyl (AdoButyn) group in place of the reactive methyl groupofAdoMetweremadebyregio- specific chemical S-alkylation of AdoHcy (Dalhoff et al. 2006b). Both analogs have been shown as substrates for sev- eral DNA methyltransferases (Dalhoff et al. 2006a), including the N6A meth- yltransferase M.TaqI, the C5C methyl- transferase M.HhaI, and the N4C meth- yltransferase M.BcnIB, for transfer of the side chains to diverse DNA sub- strates. While these analogs were not accommodated by E. coli TrmD or M. jannaschii Trm5 for transfer of their side chains to tRNA (data not shown), they were competitive inhibitors of AdoMet, permitting the determination of whether steric bulk in the vicinity of the sulfonium ion might engender in- hibition specificity. Both analogs inhib- ited each enzyme more poorly than sinefungin or AdoHcy with an increased Ki, indicating that neither is particularly tolerant of steric bulk in the vicinity of the sulfonium ion. However, as with sinefungin and AdoHcy, the FIGURE 2. Kinetic parameters for E. coli TrmD and M. jannaschii Trm5. Michaelis constants Ki/Km values for TrmD and Trm5 were Km (AdoMet) and inhibition constants (Ki) for all analogs were measured in steady state and are reported as mean values of at least two independent determinations (6 standard deviation closely similar, although a noticeable values). Ki/Km is calculated from respective values for each enzyme and the ratio ½Ki/Km (Trm5)/ difference was observed with AdoButyn ½Ki/Km (TrmD) is reported. Also see Supplemental Figure S1. with a Trm5/TrmD ratio of 4.5-fold. Presumably, the rigid propargyl substit- on the Vmax or kcat of the reaction, it progressively elevated uent in AdoButyn is more effective for inhibiting TrmD the apparent Km values of AdoMet (Fig. 3A,B), indicating than Trm5. competition of sinefungin for the AdoMet-binding site on both enzymes. Fitting the apparent K (AdoMet) as m Differentiation by fragments of AdoMet a function of sinefungin concentration to a linear equation derived from the competitive inhibition model returned To elucidate the determinants in AdoMet that interact with the Km (AdoMet) for TrmD (5.0 6 0.8 mM) and for Trm5 TrmD and Trm5, we tested adenosine and methionine (0.42 6 0.08 mM) in the absence of the analog (the fragments and their derivatives for competitive inhibition y-intercepts in Fig. 3C,D), both of which are closely similar to of methyl transfer. As expected, all of these fragments the values reported previously (8 mM for E. coli TrmD and showed substantially higher Ki values for TrmD and Trm5 1.0 6 0.1 mM for M. jannaschii Trm5) (Elkins et al. 2003; relative to natural or synthetic analogs of the parental Christian et al. 2006). Extrapolation of the linear data to the methyl donor (Fig. 2), consistent with their smaller and less x-intercepts returned the Ki (sinefungin) for TrmD (0.62 6 complex molecular structures. Importantly, all of the 0.06 mM) and for Trm5 (33 6 2 nM) (Fig. 3C,D). The fragments tested, including adenosine, methionine, and Ki/Km value calculated for TrmD (0.12 6 0.07) and for most of the nucleoside and amino acid derivatives, showed Trm5 (0.08 6 0.02) was closely similar, with the value for selectivity against TrmD by exhibiting substantially lower Trm5 slightly lower than the value for TrmD by 0.7-fold. Ki/Km values to TrmD relative to Trm5. For example, Similar inhibition of the two enzymes was also observed adenosine inhibited TrmD with a Ki/Km of 7 6 1 and for AdoHcy; analysis of Ki (AdoHcy) and Km (AdoMet) inhibited Trm5 with a markedly larger Ki/Km of 153 6 5, a

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Analogous tRNA methyltransferases

FIGURE 3. Sinefungin inhibition of m1G37-tRNA synthesis. (A) Analysis of initial rate of methyl transfer by TrmD (36 nM) as a function of AdoMet concentration (0–25 mM) in the presence of 20 mM E. coli tRNALeu and increasing concentration of sinefungin (0–4 mM). At each 1 concentration of sinefungin, the value kcat (in sec ) was calculated from Vmax/½E, where Vmax was derived from fitting the data of V0 as a function of AdoMet concentration to the Michaelis-Menten equation, while the value of apparent Km (in mM) was derived from the same fit. Values of kcat and apparent Km at each sinefungin concentration are listed. The kcat values remained constant with increasing concentration of sinefungin, while the apparent Km values increased; this indicates that sinefungin was a competitive inhibitor of AdoMet. (B) Analysis of initial rate of methyl transfer by Trm5 (25 nM) as a function of AdoMet concentration (0–5 mM) in the presence of 6 mM M. jannaschii tRNACys and increasing concentration of sinefungin (0–60 nM). (C) The concentration-dependent apparent Km (AdoMet) for TrmD was fit to a linear equation to derive Km (AdoMet) from the y-intercept and Ki (sinefungin) from the x-intercept. (D) The concentration-dependent apparent Km (AdoMet) for Trm5 was fit to a linear equation to derive Km (AdoMet) from the y-intercept and Ki (sinefungin) from the x-intercept. difference of 22-fold. Substitution of the 59-OH of adenosine ment confers an important determinant of interaction for with 59-½2-aminoethyl thio as in 59-½(2-aminoethyl)thio-59- Trm5. deoxy-adenosine (AETA) (Goedecke et al. 2001) selectively The kinetic analysis is relevant for inhibition in vivo. The inhibited TrmD by 17-fold. Substitution of 6-NH2 of intracellular concentration of AdoMet in bacteria (z400 mM) adenosine with 6-chloro as in 6-chloropurine selectively (Javor 1993) is saturating relative to Km (AdoMet) of E. coli inhibited TrmD by 14-fold. Substitution of 6-NH2 with TrmD (5.0 6 0.8 mM, Fig. 2). Similarly, the intracellular 6-carbonyl as in inosine selectivity inhibited TrmD by concentration of AdoMet in archaea and eukaryotes is likely 33-fold. In these nucleoside derivatives, the selectivity against saturating relative to Km (AdoMet) of M. jannaschii Trm5 TrmD is due to preferential increases in the Ki to Trm5 (0.42 6 0.08 mM, Fig. 2), based on the known intracellular relative to TrmD. For example, the Ki (inosine) for Trm5 is concentration of AdoMet in humans (z60 mM; de Ferra higher than the Km (AdoMet) by 3200-fold, as compared to and Baglioni 1983). In both cases, the parameter Ki/Km can the 98-fold difference between the Ki (inosine) and Km be simplified as IC50/½S to allow calculation of the IC50 value (AdoMet) for TrmD. Such preferential increases in the Ki from (Ki/Km) 3 ½S (the intracellular concentration of for Trm5 were also observed for the amino acid derivatives, AdoMet). For example, in the case of adenosine, the IC50 methionine and methylthio-cysteine (MeS-cysteine). The (adenosine) calculated for TrmD (7 3 400 mM) is smaller only exception among the fragment analogs without selec- than IC50 (adenosine) calculated for Trm5 (153 3 60 mM) tivity against TrmD was MeS-adenosine, due to a smaller in- by more than threefold in the respective cellular conditions, crease in Ki for Trm5 relative to TrmD, presumably suggesting that the fragment has the capacity to selectively because the methylthio-substitution in the adenosine frag- inhibit TrmD in vivo. This analysis reveals that, due to the

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Lahoud et al. z6-fold difference in the intracellular AdoMet concentration in bacteria and in humans, analogs that are selective against TrmD must exhibit at least a six- fold difference in Ki/Km of TrmD relative to Trm5 to offset the difference in the intracellular concentrations of AdoMet in vivo. Given that a 10-fold difference in binding affinity is desirable for a selective inhibitor, this requires a 60-fold dif- ference in Ki/Km when considering the cellular concentrations of AdoMet in humans and bacteria.

Kinetic Kd of adenosine to TrmD and Trm5 FIGURE 4. Determination of kinetic Kd (adenosine). (A) Determination of kinetic Kd To independently evaluate the selective (adenosine) for E. coli TrmD. Kinetic analysis was performed in single turnover conditions by rapid mixing of TrmD (0.4 mM) and adenosine (0.08–8 mM) in one syringe with tRNA (10 inhibition by fragments of AdoMet, we mM) and AdoMet (60 mM) in the second syringe (shown in the scheme). The reaction was focused on the adenosine fragment and quenched after one turnover (11 sec) by acid and the amount of product synthesis was determined its affinity to E. coli TrmD determined. Fitting the data of concentration-dependent product synthesis to a quadratic and M. jannaschii Trm5 by measuring binding equation revealed the Kd (adenosine) for TrmD. (B) Determination of kinetic Kd (adenosine) for M. jannaschii Trm5. Kinetic analysis was performed in single turnover the kinetic Kd as a different probe than conditions by rapid mixing of Trm5 (0.4 mM) and adenosine (0.2–17 mM) in one syringe with the steady-state Ki/Km. The measure- tRNA (16 mM) and AdoMet (60 mM) in the second syringe (shown in the scheme). The reaction was quenched after one turnover (8 sec) and the amount of product synthesis was ments of kinetic Kd were performed by rapid mixing of an enzyme-inhibitor determined. Fitting the data of the concentration-dependent product synthesis to a quadratic binding equation revealed the Kd (adenosine) for Trm5. Error bars correspond to standard complex with saturating AdoMet and deviation from three independent measurements. The inset in each graph shows the order of tRNA to allow the formation of enzyme- mixing of reagents and the reaction time. (C) A summary of Kd (AdoMet) and Kd (adenosine) substrate and enzyme-inhibitor com- for E. coli TrmD and M. jannaschii Trm5. The Kd (AdoMet) for Trm5 was previously determined (Christian et al. 2010b). The ratio of Kd (adenosine)/Kd (AdoMet) was calculated plexes in rapid equilibria, so that the for both enzymes to reflect the competitive binding of adenosine for the AdoMet binding site. extent of methyl transfer in one turn- Values in parentheses correspond to the normalized ratios of Trm5 relative to TrmD. Also see over would provide a direct readout of Supplemental Figure S2. the amount of the uninhibited enzyme capable of synthesis of m1G37 (Fig. 4A,B). The time periods permitted for one turnover of E. in the steady-state analysis of ½Ki (adenosine)/½Km (AdoMet) coli TrmD and M. jannaschii Trm5 were respectively 11 and (Fig. 2). 8 sec, based on the single-turnover rate constant of each enzyme (Christian et al. 2010b). As the inhibitor concen- X-ray structural analysis of adenosine-bound TrmD tration increased, the amount of the active enzyme avail- and Trm5 complexes able for methyl transfer progressively decreased. The data of m1G37 synthesis as a function of adenosine concentration To gain structural insight into the selective binding affinity were well fit to a quadratic equation, revealing the kinetic of adenosine to TrmD rather than to Trm5, crystal struc- Kd for TrmD (372 6 37 mM) and for Trm5 (910 6 58 mM) tures of the two enzymes were solved in complex with the (Fig. 4A,B). As a control, the kinetic Kd (AdoMet) for E. fragment (detailed information summarized in Supplemen- coli TrmD was determined (5.3 6 0.4 mM) by a similar tal Table S1). The TrmD structure was solved at 2.25 A˚ assay but without the inhibitor (Supplemental Fig. S2), resolution with the Haemophilus influenzae enzyme, which while the kinetic Kd (AdoMet) for M. jannaschii Trm5 was shares 83% sequence identity and 91% similarity with E. previously determined (0.44 6 0.09 mM) (Christian et al. coli TrmD. As seen in the previous structures of TrmD 2010b). For each enzyme, normalization of Kd (adeno- (Ahn et al. 2003; Elkins et al. 2003), the two active sites in sine) by Kd (AdoMet) was used to evaluate the compe- the dimeric H. influenzae enzyme were symmetrically as- tence of the inhibitor to compete for the AdoMet-bind- sembled in the dimer interface, with each consisting of ing site during the rapid binding equilibria of one methyl residues from the AdoMet-binding motifs in the N-termi- transfer. Analysis of this normalized ratio revealed a nal domain of one monomer and residues from the tRNA- preferential binding of adenosine to TrmD than to Trm5 binding motifs in the C-terminal domain of the second by 29-fold (Fig. 4C), similar to the 22-fold preference monomer. A comparison with the previous AdoMet-bound

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Analogous tRNA methyltransferases structure of TrmD (Ahn et al. 2003) revealed that the two H180, all of which made adjustments to occupy the space adenosine molecules bound in the new structure were in left open by the absence of the amino acid. Overall, virtually overlapping positions as the adenosine moiety of a pairwise superposition of the Ca-atoms of the AdoMet- the previous structure (Fig. 5A). The overlapping occupancy and adenosine-bound complexes revealed identical struc- of adenosine in the adenosine-pocket of the AdoMet- tures (Fig. 5C) with a root-mean-square deviation (RMSD) binding site was also preserved in the AdoHcy-bound of 0.18 A˚ for 217 of the 236 atoms aligned. The confor- structures of TrmD (Ahn et al. 2003; Elkins et al. 2003). mation of the adenosine-bound N-terminal domain was The network of H-bonds that stabilizes AdoMet was essentially unchanged in the two structures (RMSD of 0.13 maintained in its entirety to stabilize adenosine in the A˚ for the 147 atoms aligned), while the movement of the new structure, including those linking adenosine N1,N6, C-terminal domain was slightly more pronounced (RMSD and N7 with, respectively, the amide group of I133, the of 0.28 A˚ for the 66 atoms aligned). The latter movement carbonyl groups of G134 and Y136, and the amide of L138 may be due to the disordering of residues 161–171 in both in the main chain. As in the previous structures, the ribose structures. The superposition also showed that adenosine oxygen of adenosine in the new structure was stabilized by binding to TrmD created few changes to the extensive dimer the main-chain amide of G140 and G141, while the 29-and interface that forms the active site. Thus, the structure of 39-OH groups were stabilized respectively by the main-chain TrmD appears rigid enough that it can use the same active of L87 and side-chain of Y86. However, minor changes were site and overall conformation to accommodate adenosine in observed in the new structure relative to the previous the adenosine pocket of the AdoMet- or AdoHcy-binding structures, which were primarily associated with the confor- site. mation of the residues involved in the stabilization of the The adenosine-bound Trm5 structure was solved at methionine moiety of AdoMet, including S170, D177, and a 3.05 A˚ resolution with the M. jannaschii enzyme. Trm5 consists of three domains: an N-termi- nal D1 domain that contacts the tertiary core (elbow) region of the tRNA L shape in a ternary complex of the enzyme with tRNA and AdoMet (Goto-Ito et al. 2009); a D2 domain that recognizes G37 and the anticodon loop; and a D3 domain that stabilizes AdoMet binding and catalyzes methyl transfer. Due to the lack of a Trm5 structure bound to AdoMet, the new structure was compared to the sinefungin-bound structure of the en- zyme (Goto-Ito et al. 2008). This com- parison revealed that, in contrast to TrmD, significant misalignment of aden- osine with the sinefungin site of Trm5 was observed, primarily due to a 0.6 A˚ translation and 0.5 A˚ downward shift of adenosine toward the methionine pocket (Fig. 5B). The movement of adenosine disturbed the H-bond and hydrophobic FIGURE 5. Structural analysis of adenosine-bound TrmD and Trm5 complexes. (A) network established for the adenosine Superposition of the Ca atoms of adenosine in the adenosine-bound (magenta) complex fragment in the sinefungin-bound struc- onto the structure of the AdoMet-bound complex of H. influenzae TrmD (orange, PDB ID: ture by weakening or abolishing the 1UAK). Adenosine and AdoMet are colored in blue and cyan, respectively. Enzyme residues that form the interacting network with AdoMet are depicted, showing H-bonds (dashed lines) enzyme interactions with the adenine with the methyl donor. These residues are in overlapping positions in the two structures. (B) ring through D251, V252, and I224 and Superposition of the Ca atoms of adenosine in the adenosine-bound (magenta) complex onto the interactions with the ribose through the sinefungin-bound complex of M. jannaschii Trm5 (orange, PDB ID: 2YX1). Adenosine and F203, D223, and L266. More substantial sinefungin are colored in blue and cyan, respectively. Enzyme residues that form the interacting network with sinefungin are depicted, showing H-bonds (dashed lines) with the disruptions were observed in the confor- AdoMet analog. The superposition reveals a shift of adenosine toward the methionine pocket mation of the residues in the D2–D3 by 0.6 A˚ with the sugar flexed away from the active site by 1.35 A˚ . Residues with the most domain responsible for recognition of significant shifts are those that contact G37-tRNA in the D2 domain, including Y177 and R136. G37-tRNA, including N265, P267, and (C) A ribbon diagram showing the superposition of the overall adenosine-bound and AdoMet- bound structures of H. influenzae TrmD. (D) A ribbon diagram showing the superposition of Y177 that played a role in stabilizing the the overall adenosine-bound and sinefungin-bound structures of M. jannaschii Trm5. purine base, R136, R181, and R186 that

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Lahoud et al. stabilized the conformation of the anticodon loop, and E185 TABLE 1. Calculation of binding free energy that was proposed to deprotonate N1 of G37 before methyl transfer (Christian et al. 2010a). The other catalytic residue DG0 (kcal/mol) 6 R145 that presumably stabilized O of G37 during deproton- Chemical TrmD Trm5 ation (Christian et al. 2010a) was in a disordered region. All of these residues are important for the reaction mechanism AdoMet 7.5 9.6 of Trm5, and their displacements from the positions in the Sinefungin 8.8 11.2 AdoHcy 7.6 9.5 AdoMet- or sinefungin-bound complex due to adenosine AdoPropen 6.5 8.8 binding indicated a coordinated dismantling of the active AdoButyn 7.2 8.3 site. Displacement in the D1 domain was also observed (Fig. Adenosine 6.3 6.3 5D), most notably for residues that recognized the backbone AETA 7.1 7.3 structure of the tRNA tertiary core (e.g., K10, R16, and K27). MeS-Adenosine 6.4 8.9 However, because D1 is flexibly linked to the D2–D3 6-ChloroPurine 4.8 5.0 Inosine 4.7 4.3 domain, its displacement in the new structure from the Methionine 2.5 3.3 sinefungin-bound structure might be due to crystal packing MeS-Cysteine 2.1 2.0 effects. An overall pairwise superposition was thus performed 0 0 only with the Ca atoms of the D2–D3 domains in the Free energy of binding DG (kcal/mol) is calculated from DG = RT ln (Km or Ki), where Km and Ki are interpreted as dissociation present structure relative to the sinefungin-bound struc- constants, R is the gas constant 1986 cal K1 mol1, and T is the ture, which revealed a 0.46 A˚ RMSD (based on alignment absolute temperature. Values of Km and Ki are taken from Figure 2, of 236 out of 253 residues), significantly higher than the based on the kinetic determination of E. coli TrmD at 37°C (310 K) ˚ and M. jannaschii Trm5 at 55°C (328 K). This analysis shows that 0.18 A value obtained with TrmD. The sinefungin position adenosine binding accounts for 85% of the binding energy of in the sinefungin-bound structure differed slightly from the AdoMet to TrmD (6.3/7.5 = 84%), but only 65% of the binding AdoMet position in the ternary complex of Trm5–AdoMet– energy of AdoMet to Trm5 (6.3/9.6 = 66%). tRNA. An overall pairwise superposition performed with the Ca atoms of the D2–D3 domains in the present structure same catalytic turnover rate (Christian et al. 2010b), relative to the ternary complex structure revealed an even suggesting that the use of Km and Ki values for free energy higher RMSD value of 0.65 A˚ . calculation was reasonable for both enzymes. In addition, the Km and Kd values of AdoMet are closely similar for both enzymes, and the K /K ratios of adenosine are closely DISCUSSION i m similar to the Kd ratios (Figs. 2, 4). Free energy calculations While analogous enzymes exist in unrelated structures, indicate that TrmD interacts with AdoMet with lower they recognize the same substrates using distinct but well- affinity than does Trm5 (7.5 vs. 9.6 kcal/mol, respec- developed interacting networks to catalyze the same reac- tively), and that the difference in the apparent binding free tion. Identifying substrate analogs that can preferentially energy between the two enzymes remains more or less the bind to one enzyme but not the other has proven to be same in all of the natural and synthetic analogs as well as in a significant challenge. The challenge is clearly illustrated for the MeS-adenosine analog, which is the only fragment that the TrmD and Trm5 pair, which as we show here cannot be lacks selectivity. Interestingly, free energy calculations in- distinguished by several previously well-characterized natu- dicate that TrmD interacts with similar affinity as does ral and synthetic AdoMet analogs. Instead, fragments of Trm5 with fragments or fragment derivatives that show AdoMet, particularly adenosine and its purine derivatives, selectivity. However, additivity analysis of TrmD reveals show discerning and specific inhibition of TrmD. To our that the sum of the free energies of binding the adenosine best knowledge, this work is the first and successful ap- and methionine fragments (½6.3 + ½2.5 = 8.8 kcal/ plication of the fragment-based structure-activity analysis to mol) is smaller than the free energy of binding AdoMet by differentiate the substrate binding determinants of analogous 1.3 kcal/mol, suggesting the existence of an energetic enzymes. penalty in the binding of AdoMet relative to fragments. In contrast, additivity analysis of Trm5 reveals that the sum of the free energies of binding adenosine and methionine Free energy analysis (½6.3 + ½3.3 = 9.6 kcal/mol) is the same as the free To gain insight into the AdoMet binding determinants of energy of binding AdoMet, suggesting that the fragment TrmD and Trm5, the free energy of binding the methyl free energies of interaction for Trm5 are additive. The donor by these enzymes relative to the analogs studied here disparity in free energy additivity of TrmD and Trm5 is was calculated and summarized (Table 1), based on the likely attributable to the unusual bent conformation of experimentally determined Km and Ki values (Fig. 2). AdoMet when bound to TrmD in contrast to the extended Although TrmD and Trm5 differ in the rate-limiting step conformation when bound to Trm5 (Fig. 1). The bent in the control of their catalytic turnover, they display the conformation of AdoMet is also observed in the structures

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Analogous tRNA methyltransferases of sinefungin and AdoHcy when bound to TrmD (Ahn et al. relative to TrmD in structural analysis (Supplemental 2003; Elkins et al. 2003), while the extended conformation is Table S1). preserved in the structure of sinefungin when bound to Combining free energy analysis with structural analysis, Trm5 (Goto-Ito et al. 2008). The consistent bent conforma- we propose a model to explain the selectivity of adenosine tion in AdoMet and in natural analogs when bound to targeting to TrmD. In this model (Fig. 6), the adenosine TrmD suggests that the energetic penalty in the binding of fragment binds to the adenosine pocket of TrmD in an these compounds by TrmD is involved in the entropic costs overlapping position, trapping the enzyme in an inactive associated with stabilizing the bent conformation, preventing state that is likely stabilized by the rigidity of the enzyme the enzyme from full utilization of the binding energy of its structure. In contrast, the fragment binds to the adenosine component fragments. In contrast, the additivity of binding pocket of Trm5 in a misfit position, which is corroborated by Trm5 suggests that the enzyme can capture the full spec- with global structural perturbation in the active site and trum of the binding free energies from the component frag- throughout the D2–D3 domains. The inherent structural ments in a way that is facilitated by the extended confor- flexibility of Trm5 may promote rapid release of the frag- mation of the bound AdoMet. ment, facilitating the return to the native structure and The different binding determinants of AdoMet versus explaining the escape from targeting. fragments to TrmD and to Trm5 provide a basis to under- stand the fragment selectivity. Even if a fragment binds with the same binding energy to both enzymes, the overall ener- Insight into selective targeting TrmD getic contribution of the fragment relative to AdoMet bind- Fragments of enzyme substrates, due to their smaller ing is different for the two enzymes. For example, while chemical space and easier accessibility to exploration, have adenosine binds with the same energy to both TrmD and been suggested as an attractive starting point to build frag- Trm5 (6.3 kcal/mol), it accounts for 84% of the total ment libraries for drug screening that may have the poten- binding energy of AdoMet to TrmD (7.5 kcal/mol), but tial to supersede traditional high throughput screening of only 66% of the total binding energy of AdoMet to Trm5 larger molecules. Fragment hits usually achieve only low (9.6 kcal/mol). The greater contribution of adenosine mM to high mM affinities with the possibility for further binding to the TrmD–AdoMet binding interaction than to elaboration into more potent inhibitors. The elaboration the Trm5–AdoMet interaction suggests a more stable can take place in one of four directions: evolution of initial binding of the fragment to TrmD than to Trm5. Analysis fragments by building larger and more complex molecules of the thermodynamic Kd as determined by kinetic mea- surements under rapid binding equilibrium (Fig. 4) con- firms that the adenosine affinity to TrmD is 2.4-fold higher than the affinity to Trm5 (Kd = 372 6 37 and 910 6 58 mM, respectively). This difference, after normalization by the inherent 12-fold difference of AdoMet affinity to the two enzymes (Kd = 5.2 6 0.4 and 0.44 6 0.09 mM, respectively), is enlarged to 29-fold, meaning that the fragment is 29-fold more stable in the AdoMet-binding site on TrmD than on Trm5 each time it outcompetes against AdoMet for the binding site. Due to the rapid binding equilibria under which the binding constants were measured, the Kd (= koff/kon) values can be used to calculate FIGURE 6. A model of adenosine targeting TrmD and Trm5. In the koffs assuming that kon is diffusion controlled. This calcu- active state, TrmD and Trm5 each possess an AdoMet-binding site, lation suggests that the adenosine fragment on TrmD is consisting of an adenosine and a methionine pocket. The AdoMet site more stable than on Trm5 with a 29-fold longer duration is located in a dimer interface of TrmD comprised of the N-terminal time. Direct measurements of fragment binding kinetics domain of monomer 1 (N1) and the C-terminal domain of monomer 2(C2), whereas it is located between the D2 and D3 domains of the to TrmD and Trm5 will more precisely assess the difference monomeric Trm5. In the adenosine-bound inactive state, TrmD in binding stability. Nonetheless, part of the stability of the maintains the same structure and binds adenosine in the already- fragment on TrmD may be provided by the inherent established adenosine pocket, whereas Trm5 shifts its adenosine structural rigidity of the trefoil-knot fold, as suggested by pocket and binds adenosine in a misfit conformation, leading to structural alterations of the enzyme. In TrmD, the stable binding of a biophysical analysis of a model knotted protein (King adenosine causes delayed release of the ligand, whereas in Trm5 the et al. 2010). Conversely, part of the instability of the unstable binding of adenosine can lead to rapid release of the fragment on Trm5 may derive from the inherent structural fragment, allowing refolding of the enzyme to its native structure. fluidity of the enzyme, which is indicated by a recent study The chemical structure of AdoMet is depicted below with a blue box showing the adenosine fragment and a red box showing the of Trm5 during catalysis (Christian et al. 2010a) and is methionine fragment. The AdoMet-binding site is shown in white, supported by the higher B factors associated with Trm5 whereas the adenosine ligand is shown in dark blue.

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Lahoud et al. that form additional interactions with the target protein; of adenosine. Details of structural analysis of the adenosine-bound chemically linking two or more fragments that bind to sep- TrmD and Trm5 structures are described in Supplemental Table arate but close enough sites on the target protein to gen- S1. erate a larger and higher-affinity molecule; using the target protein as a template to assemble fragments with SUPPLEMENTAL MATERIAL complementary functional groups so as to react together to generate a larger molecule with a higher potency; and Supplemental material is available for this article. optimization of initial fragments based on properties other than just the binding potency. Recent work has ACKNOWLEDGMENTS seen successful examples in all of these approaches each with the potential to develop from initial fragments into This work was supported by NIH grant GM081601 to Y.M.H. We high-affinity leads (Rees et al. 2004). thank Dr. Saulius Klimasauskas for AdoPropen and AdoButyn, In the pursuit of selective targeting of TrmD, we show Dr.ElmarWeinholdforAETA,Dr.SeWonSuhfortheH. influenzae here that the enzyme has the ability to fully accommodate TrmD expression plasmid, and Drs. Charles P. Scott, Saulius adenosine to the adenosine pocket in a conformation in- Klimasauskas, and Howard Gamper for productive discussion. Authors’ contributions: G.L., S.Y., and Y.M.H. designed the distinguishable from that in AdoMet or AdoHcy, suggest- research; G.L., S.G.I., K.Y., and T.I. performed the research; all ing that the energetic penalty upon binding AdoMet is due authors analyzed data and wrote the paper. to the inability to completely capture the free energy of binding the methionine portion of the molecule. Indeed, Received March 4, 2011; accepted April 15, 2011. the methionine portion of AdoMet when bound to TrmD adopts an unusual conformation that places the sulfonium ion in close proximity to the bridging 4-oxo group of the REFERENCES ribose, resulting in the bent conformation. This raises the Ahn HJ, Kim HW, Yoon HJ, Lee BI, Suh SW, Yang JK. 2003. Crystal possibility that a fragment-linking approach that links structure of tRNA(m1G37)methyltransferase: Insights into tRNA the adenosine fragment with derivatives of the methionine recognition. EMBO J 22: 2593–2603. fragment in structural or topological constraints that Alexandrov A, Chernyakov I, Gu W, Hiley SL, Hughes TR, Grayhack EJ, Phizicky EM. 2006. Rapid tRNA decay can result from lack of stabilize the bent conformation of AdoMet may offer an nonessential modifications. Mol Cell 21: 87–96. attractive route to develop selective targeting. 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Differentiating analogous tRNA methyltransferases by fragments of the methyl donor

Georges Lahoud, Sakurako Goto-Ito, Ken-ichi Yoshida, et al.

RNA published online May 20, 2011

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