Structural and Mechanistic Basis of Pre- and Posttransfer Editing by Leucyl-tRNA Synthetase

Molecular Cell, Vol. 11, 951–963, April, 2003, Copyright 2003 by Cell Press

Structural and Mechanistic Basis of Pre- and Posttransfer Editing by Leucyl-tRNA Synthetase Tommie L. Lincecum, Jr.,1,8 Michael Tukalo,2,8 Anna Yaremchuk,2,7,8 Richard S. Mursinna,1 Amy M. Williams,1 Brian S. Sproat,3 Wendy Van Den Eynde,3 Andreas Link,4 Serge Van Calenbergh,5 Morten Grøtli,6 Susan A. Martinis,1,* and Stephen Cusack2,* 1 Department of Biology and Biochemistry University of Houston Houston, Texas 77204 2 Grenoble Outstation of EMBL 6 rue Jules Horowitz, BP181 38042 Grenoble Cedex 9 France 3 RNA-TEC NV Minderbroedersstraat 17-19 B-3000 Leuven Belgium 4 Institute for Pharmacy University of Hamburg Bundesstrasse 45 D-20146 Hamburg Germany 5 Laboratory of Medicinal Chemistry Faculty of Pharmacy University of Gent Harelbekestraat 72 B-9000 Gent Belgium 6 Biotechnology Centre of Oslo University of Oslo Gaustadallee´n 21 N-0349 Oslo Norway 7 Institute of Molecular Biology and Genetics NAS of Ukraine 252627 Kiev, 3143 Ukraine

Summary The aminoacyl-tRNA synthetases link tRNAs with their cognate amino acid. In some cases, their fidelity relies on hydrolytic editing that destroys incorrectly activated amino acids or mischarged tRNAs. We present structures of leucyl-tRNA synthetase complexed with analogs of the distinct pre- and posttransfer editing substrates. The editing active site binds the two different substrates using a single amino acid discriminatory pocket while preserving the same mode of adenine recognition. This suggests a similar mechanism of hydrolysis for both editing substrates that depends on a key, completely conserved aspartic acid, which interacts with the ␣-amino group of the noncognate amino acid and positions both substrates for hydroly*Correspondence: [email protected] (S.C.), smartinis@uh. edu (S.A.M.) 8 These authors contributed equally to this work.

sis. Our results demonstrate the economy by which a single active site accommodates two distinct substrates in a proofreading process critical to the fidelity of protein synthesis. Introducton The aminoacyl-tRNA synthetases (aaRSs) are responsible for exclusively attaching a particular amino acid to its set of cognate tRNAs isoacceptors. The accuracy of this reaction is essential to the fidelity of protein synthesis and hence cell survival (reviewed in Martinis et al., 1999). However, distinguishing with sufficient accuracy aliphatic amino acids that are structurally similar, for instance only differing by a single methyl group, presents a fundamental challenge to the molecular recognition mechanism of the aminoacylation active site. For instance, the weakness of the additional van der Waals interactions of the extra methyl group of isoleucine compared to valine in the amino acid binding pocket of isoleucyl-tRNA synthetase (IleRS) was theoretically predicted to yield an error rate of as high as 1 out of 5 (Pauling, 1958). Experimentally, the rate of misincorporation of valine by IleRS was determined to be closer to 1 out of 3000 (Loftfield, 1963) due to the presence of an efficient editing mechanism which eliminates the initial selection errors (Baldwin and Berg, 1966). Subsequent biochemical analysis determined that a number of other aaRSs also have highly evolved proofreading mechanisms. To date, these include class I enzymes IleRS, ValRS, and LeuRS (reviewed in Jakubowski and Goldman, 1992) and class II enzymes ThrRS (Dock-Bregeon et al., 2000) and ProRS (Beuning and Musier-Forsyth, 2000). The editing of misactivated homocysteine by several aaRSs, notably MetRS, is by a different mechanism than considered here (Jakubowski and Goldman, 1992). To enhance fidelity, the editing aaRSs rely on a “double sieve” mechanism for amino acid selection and discrimination as originally proposed by Fersht (1977), analyzed biochemically and genetically (e.g., Schmidt and Schimmel, 1995) and first visualized in the crystal structures of IleRS (Nureki et al., 1998; Silvian et al., 1999). The first sieve encompasses the classical aminoacylation or “synthetic” active site which binds cognate amino acids but cannot adequately filter all closely related amino acids, notably those that are isosteric (e.g., valine/threonine) or slightly smaller (e.g., by only one methyl group, for example threonine/serine or isoleucine/valine). The second sieve is a distinct editing active site that hydrolyses noncognate amino acids that are misactivated or mischarged but, crucially, excludes on the basis of, for instance, size or hydrophilicity the correctly charged cognate amino acid. The editing site for IleRS, LeuRS, and ValRS lies within a discretely folded domain of about 200 residues, often called CP1, flexibly inserted into the Rossmann fold catalytic domain where aminoacylation occurs (Cusack et al., 2000; Fukai et al., 2000; Nureki et al., 1998; Silvian et al., 1999). The editing active site hydrolytically cleaves the misactivated aminoacyl-adenyl-

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the active sites for pre- and posttransfer editing are physically distinct or overlapping. Separate amino acid binding pockets for pre- and posttransfer editing substrates have been postulated based on modeling and comparative analysis of the homologous ValRS and IleRS cocrystal structures (Fukai et al., 2000; Nureki et al., 1998). The two sites are proposed to be distinct but proximal to each other and rely on a distinct set of amino acids to confer amino acid specificity. The prediction was based on the observed location of the terminal adenosine of the tRNAVal in the editing site in the ValRStRNAVal cocrystal structure and modeling of a charged threonine and threonyl-adenylate in the editing site (Fukai et al., 2000). Subsequently, two mutations in the closely related IleRS CP1 domain were described that were interpreted to predominantly affect either post- or pretransfer editing (Hendrickson et al., 2002) apparently supporting the notion of two distinct sites. Here we give an atomic resolution description of how the editing domain accommodates the two distinct substrates using high-resolution crystallographic structures of a class Ia synthetase, LeuRS, bound to nonhydrolyzable pre- and posttransfer editing substrate analogs. In contrast to previous hypotheses, our results show essentially overlapping sites and a common method of proofreading of the amino acid side chain for either the pre- or posttransfer editing substrate. We also combine structural and biochemical data to propose a mechanism for hydrolysis involving a water molecule attacking the adenylate or ester, without any direct catalytic effects of editing site residues. Results Figure 1. Editing Reactions, Editing Substrates, and Sequence Conservation in the Editing Domain (A) LeuRS aminoacylation and editing reactions. Editing reactions are indicated by the dashed arrows. Although tRNA has been shown to be a cofactor for pretransfer editing by IleRS (Baldwin and Berg, 1966), its role in LeuRS editing is unknown. (B) Diagrams of the analogs used in this work of LeuRS pre- and posttransfer editing substrates for the case of noncognate norvaline (Nva). Left: posttransfer substrate analog, 2⬘-(L-norvalyl)amino-2⬘deoxyadenosine (Nva2AA), mimicking the 3⬘ end of the aminoacyl2⬘-ester Nva-tRNALeu. Right: pretransfer substrate analog, 5⬘-O[N-(L-norvalyl)sulphamoyl]adenosine (NvaAMS), a sulfamoyl analog of norvalyl-adenylate. In each case, the labile ester linkages were replaced by a nonhydrolyzable amino linkage to permit structural studies. (C) Alignment of conserved regions within the editing (CP1) domain of selected LeuRS (L), ValRS (V), and IleRS (I) enzymes. The “threonine-rich region” contains two highly conserved threonines (arrowed) discussed in the text. In the second region, separated by a bracket, a conserved glycine-rich loop is followed by a completely conserved aspartic acid (arrowed) that was mutated to alanine. Abbreviations: Sc, S. cerevisiae; Ce, Caenorhabditis elegans; Hs, Homo sapiens; Nc, Neurospora crassa; Ec, E. coli; Tt, Thermus thermophilus; Bs, Bacillus subtilis; Gs, Geobacillus stearothermophilus; Sa, Staphylococcus aureus; cyt, cytoplasmic; mit, mitochondrial.

ate (called “pretransfer editing”) or the mischarged tRNA (called “posttransfer editing”), as shown in Figure 1A. Since the pre- and posttransfer substrates for editing are distinct, being respectively an aminoacyl-adenylate or an aminoacyl-ester (analogs of which are shown in Figure 1B), it is an intriguing question as to what extent

Structural Analysis of LeuRSTT Complexed with Editing Substrate Analogs LeuRS misactivates a diverse group of standard amino acids (e.g., isoleucine, methionine) and nonstandard metabolic amino acid intermediates (e.g., norvaline, norleucine, and homocysteine) (Englisch et al., 1986, Apostol et al., 1997, Lincecum and Martinis, 2000). We synthesized nonhydrolyzable analogs of the pre- and posttransfer editing substrates for the noncognate amino acid norvaline (Nva). These are respectively, (5⬘O-[N-(L-norvalyl)sulphamoyl]adenosine), a sulfamoylanalog of norvalyl-adenylate, designated NvaAMS and 2⬘-(L-norvalyl)amino-2⬘-deoxyadenosine, an amino analog of the terminal adenosine of charged tRNA, designated Nva2AA. In these two analogs, a hydrogen bond donating amide linkage with planar peptide geometry replaces the normal hydrogen bond accepting ester linkage to the amino acid, and in the case of NvaAMS, a sulfate replaces the normal phosphate (Figure 1B). Sulphamoyl analogs of aminoacyl-adenylates have been used in numerous structural studies of aminoacyl-tRNA synthetases (Belrhali et al., 1994; Cusack et al., 2000; Dock-Bregeon et al., 2000; Ueda et al., 1991). We also report synthesis and use for structural and biochemical studies of the nonhydrolyzable analog of the terminal adenosine of a charged tRNA. For both ligands, the high resolution of the crystallographic data (2.2 A˚ for NvaAMS and 2.1 A˚ for Nva2AA) allows unambiguous positioning of the ligand, definition

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of the ribose pucker, and assignment of a network of water molecules and hydrogen bonds. Pretransfer Editing Complex: The Structure of T. thermophilus LeuRS Complexed with 5ⴕ-O-[N-(L-Norvalyl)Sulphamoyl]Adenosine The structure obtained after soaking LeuRS crystals with the norvalyl-adenylate sulfamoyl analog (NvaAMS) shows very clear electron density for the compound in both the synthetic and editing active sites (Figure 2A), which are separated by 38 A˚ (Figure 2B). In the synthetic active site, NvaAMS has an extended conformation and specific interactions essentially identical to that previously described for the leucyl-adenylate analog (LeuAMS) except that the CD2 branched methyl group of leucine is lacking. The conformation of NvaAMS in the editing site is quite different from that in the synthetic site, with a significant rotation about the C-C␣ bond (psi torsional angle) resulting in an unusually bent conformation in which the ␣-amino group of the norvaline hydrogen bonds to the O2⬘ of the ribose (Figure 3A). The principal interactions of NvaAMS with residues in the editing active site are shown in Figure 3A. They involve the two universally conserved regions of the editing domain, the threonine-rich peptide (residues 247–252) and the region 327–347, which includes the highly conserved 333-GT/SG loop and the absolutely conserved aspartate 347 (Figure 1C). The adenine base stacks on Ile-337 (otherwise a conserved valine in most ILVRS [abbreviation for IleRS, LeuRS, and ValRS; Figure 1C]) and is specifically recognized by main chain interactions to the N1 and N6 positions, a frequently observed mode of adenine recognition (e.g., in the ATP binding site of class I synthetases) (Cusack et al., 2000). The highly conserved GT/SG motif (Figure 1C) is essential for maintaining the tight conformation of the adenine binding loop, both glycines having torsional phi/psi angles not allowed for other residues and the second one not being replaceable for steric reasons by a residue with a side chain. The ribose 3⬘OH is hydrogen bonded to Asp344, and both hydroxyl groups take part in an ordered network of water molecules linking them indirectly to Asp347, Tyr327, and the N3 of the base (data not shown). The sulfate of NvaAMS is tightly bound to the threonine-rich peptide, making a total of four hydrogen bonds to the side chain hydroxyls or main chain of Thr247 and Thr248. This is consistent with the fact that in all structures of LeuRSTT, a sulfate from the ammonium sulfate crystallization medium is found at this position in the vacant editing site. Furthermore, in a 2 A˚ structure of LeuRSTT with AMP bound in the editing site (data not shown), the conformation of the AMP is exactly the same as the AMS moiety of NvaAMS (data not shown), indicating that a phosphate (as in the true pretransfer substrate) can also bind in this position. The ␣-amino group of the norvaline is triply hydrogen bonded to the O2⬘ of the adenylate ribose, the carboxyl group of Asp347, and the main chain carbonyl oxygen of Met338. The carbonyl oxygen of the norvaline makes a hydrogen bond to an ordered water which itself is hydrogen bonded to the main chain amide of Met338 and carbonyl oxygen of Phe246. The norvaline side chain is bound in a largely hy-

drophobic pocket delimited by Met338, Val340, Thr252, and the aliphatic part of Arg249. Previous mutational analysis identified Thr 252 as a critical amino acid discriminant (Mursinna et al., 2001). Mutation to alanine (ecT252A) relaxes the specificity of the editing sieve allowing hydrolysis of Leu-tRNALeu, which of course is normally avoided. The crystal structure of T. thermophilus LeuRS (Cusack et al., 2000) showed that the conserved Thr252 lies at the bottom of a putative amino acid binding pocket and could block leucine binding by interfering with the side chains ␥-methyl group (Mursinna et al., 2001). We modeled a leucine side chain in place of that of norvaline in the NvaAMS complex and showed that indeed the additional methyl group would sterically clash with both Val340 and Thr252. Posttransfer Editing Complex: The Structure of T. thermophilus LeuRS Complexed with 2⬘-(L-Norvalyl)Amino-2⬘-Deoxyadenosine Clear electron density was observed for 2⬘-(L-norvalyl) amino-2⬘-deoxyadenosine (Nva2AA) bound in the editing site (Figure 2C). The principal interactions of this molecule with residues in the editing active site are shown in Figure 3B. It is immediately obvious that despite the different form of the two editing substrates, the mode of binding and recognition of the adenine base and the aminoacyl moiety of Nva2AA is very similar to that observed for NvaAMS (Figures 3A and 3C). Notably the ␣-amino group of the norvaline hydrogen bonds to the carboxyl group of Asp347 and the main chain carbonyl-oxygen of Met338, as for NvaAMS. However the ribose orientation is quite different, reflecting the different linkage to the amino acid in the two compounds. Interestingly, the threonine-rich peptide continues to play an important but different role in binding Nva2AA. The hydroxyl of Thr247 (a highly conserved threonine, exceptionally a serine, in all known ILVRS; Figure 1C) makes a hydrogen bond to the carbonyl oxygen of the norvaline, and both Thr247 and Thr248 (less conserved) make a total of three hydrogen bonds to the 3⬘OH of the ribose. Other notable differences are, first, that there is no direct role in binding the posttransfer substrate for the second but nonconserved aspartate, Asp344, whereas it hydrogen bonds to the 3⬘OH of the pretransfer substrate. Second, the side chain of Tyr332, disordered in the pretransfer complex, is fully ordered in the posttransfer complex and interacts at an angle with the purine base on the opposite face to Ile337 (Figure 3B). The hydroxyl group of Tyr332 hydrogen bonds to the O5⬘ of the Nva2AA and could presumably also interact with the phosphate of Ade76 in the full posttransfer editing complex including the tRNA. Importantly, we note that the position and conformation of the adenosine moiety of Nva2AA is very similar to that observed for the terminal Ade76 bound in the highly homologous editing site of T. thermophilus ValRS (Fukai et al., 2000), providing strong evidence that our structure indeed mimics a bona fide posttransfer complex. The ValRS complex structure also showed that the penultimate bases Cyt75 and Cyt74 do not make contacts with the editing domain, consistent with our observation that Nva2AA in itself has high affinity. Finally, we note that an extensive network of water molecules is associated with the Nva2AA substrate binding (Figure 3E).

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Figure 2. Electron Density of the Editing Substrates (A) Simulated omit map (Brunger et al., 1998) for the pretransfer substrate analog (NvaAMS) in the editing (top) and synthetic (bottom) active site. Resolution is 2.2 A˚. In both molecules, the ribose is in the C2⬘ endo conformation. (B) Location of the NvaAMS in the synthetic and editing active sites of LeuRSTT. (C) Unbiased difference map (2.0 A˚ resolution) for the posttransfer editing substrate analog (Nva2AA) in the editing site. The ribose is in the C3⬘ endo conformation. (D) Competitive inhibition of E. coli LeuRS editing of Ile-tRNALeu by Nva2AA. Editing of Ile-tRNALeu by wild-type E. coli LeuRS in the absence of inhibitor exhibited a KM of 0.2 ␮M.

Nva2AA, but Not Nva3AA, Competes with Mischarged Ile-tRNALeu for the Editing Site In contrast to LeuRSTT complexed to Nva2AA, no electron density was observed in the editing site of LeuRSTT upon soaking with the mischarged 3⬘ tRNA analog Nva3AA under the crystallization conditions tested and suggests that the LeuRS editing active site is specific for tRNA misaminoacylated at the 2⬘ hydroxyl. This is

supported by biochemical competition assays which showed that the posttransfer editing substrate analog Nva2AA inhibited hydrolysis of the mischarged tRNA product with a KI of 4.65 nM (Figure 3D). The implied tight binding of Nva2AA is consistent with the multiple interactions observed in the crystal structure, similar to that of the adenylate analog in both the editing and synthetic sites. High affinity of the mischarged 3⬘ end

Mechanisms of Editing by Leucyl-tRNA Synthetase 955

of the tRNA to the editing site is also consistent with the need to rapidly deacylate before the tRNA disassociates from the synthetase. Significantly, Nva3AA failed to effectively decrease LeuRS editing activity (data not shown). Thus, it appears that for LeuRS, the amino acid does not isomerise away from its initial point of attachment to the 2⬘OH of the ribose during posttransfer editing. In this respect, LeuRS behaves similarly to ValRS but differently from IleRS which specifically catalyses deacylation from the 3⬘OH and not from the 2⬘OH (Nordin and Schimmel, 2002). Mutation to Alanine of the Conserved Aspartic Acid in the Editing Site Primary sequence alignments of LeuRS, IleRS, and ValRS enzymes from prokaryotic and also eukaryotic cytoplasmic and mitochondrial origins revealed a completely conserved aspartic acid within the CP1 domain (Figure 1C). Based on this and also the conserved structural role found in both pre- and posttransfer editing complexes, we hypothesized that the aspartic acid may have a critical functional role in amino acid editing in all LeuRSs. We therefore substituted this aspartic acid in the S. cerevisiae, E. coli, and T. thermophilus enzymes to an alanine (ycD419A, ecD345A, and ttD347A, respectively). Each of the purified mutant enzymes aminoacylated tRNALeu with leucine at a significant, but slightly reduced, activity compared to the wild-type enzymes (data not shown). Isoleucine is misactivated by both the wild-type S. cerevisiae cytoplasmic and E. coli LeuRSs, but fails to be stably linked to tRNALeu, suggesting that it is removed by editing (Englisch et al., 1986; Lincecum and Martinis, 2000). Likewise, wild-type LeuRSTT shows a high rate of AMP formation in the presence of norvaline and tRNALeu, indicative of editing (Figure 4F). Insertion and sitedirected mutagenesis within the CP1 domain of E. coli LeuRS have also been reported to yield misaminoacylation of isoleucine to tRNALeu (Chen et al., 2000, 2001; Mursinna and Martinis, 2002; Tang and Tirrell, 2002). We tested each of the mutant LeuRSs for isoleucylation activity to determine if the conserved aspartate carried out a role in hydrolytic editing. While neither the wildtype S. cerevisiae cytoplasmic and E. coli LeuRSs yielded mischarged tRNALeu, both the ycD419A and ecD345A LeuRSs stably generated isoleucine misaminoacylated to tRNALeu (Ile-tRNALeu, Figure 4A). Similar results were obtained with the ttD347A mutant LeuRS (data not shown). To ensure that the TCA-precipitated radioactivity was directly related to charged tRNALeu, correctly charged and mischarged tRNAs were separated by acid gel electrophoresis (Martinis and Schimmel, 1992; Varshney et al., 1991). Figure 4B shows that ycWT and ycD419A yielded similar levels of correctly charged tRNALeu. Consistent with the isoleucine-mischarging activity based on TCA precipitation of tRNA (Figure 4A), the acid gel in Figure 4B shows that the mutant ycD419A enzyme stably produced tRNALeu linked to isoleucine. The wildtype enzyme failed to yield a detectable band representing the mischarged Ile-tRNALeu. The production levels of Ile-tRNALeu compared to Leu-tRNALeu are decreased, as would be expected since LeuRS misactivates isoleucine less efficiently than leucine (data not shown).

Methionine is also misactivated by LeuRS (Lincecum and Martinis, 2000; Martinis and Fox, 1997). Incorporation of methionine into the aminoacylation assay with the mutants ycD419A (Figures 4C and 4D) and ecD345A (Figure 4E) followed by either gel analysis or TCA precipitation recovery of the tRNA showed that both mutants formed Met-tRNALeu. As found with the isoleucine mischarging activity, the level of methionylation activity was significantly lower than that of Leu-tRNALeu production which would correspond to a relative decrease in activation (Lincecum and Martinis, 2000; Martinis and Fox, 1997). The combined mischarging phenotypes of methionine and isoleucine by both S. cerevisiae cytoplasmic and E. coli LeuRSs shows that the conserved aspartic acid plays a critical role in the amino acid editing active site. Previous work showed that mutation of a conserved threonine in E. coli LeuRS at position 252 to an alanine (ecT252A) altered specificity of the editing active site and hydrolyzed correctly charged Leu-tRNALeu (Mursinna et al., 2001). The ecT252 residue was hypothesized to be a fine discriminant for molecular recognition that blocks leucine from binding to the editing active site, which is fully consistent with the structural data presented above. Since the E. coli LeuRS T252A mutation recognizes an additional substrate (Leu-tRNALeu), it provided a unique opportunity to test the effects of the conserved aspartic acid in posttransfer editing of an alternate substrate. We combined the ecT252A with the ecD345A mutation to further analyze the latter’s effect on catalysis and the overall editing reaction. While the ecT252A mutation decreases apparent leucylation of tRNALeu, ecT252A/D345A restores activity to the level of the ecD345A LeuRS mutant that is also similar to wildtype enzyme (Figure 5A). The E. coli mutants as well as the wild-type enzymes were tested for their ability to hydrolyze correctly charged Leu-tRNALeu (Figure 5B). As expected, the ecD345A single mutation failed to edit Leu-tRNALeu. Previous results showed that ecT252A hydrolyzes leucine from tRNALeu (Mursinna et al., 2001). As Figure 5B illustrates, ecD345A restores leucylation activity of ecT252A mutant by preventing the hydrolysis of Leu-tRNALeu. The ecD345A mutation was also efficient in disrupting binding in the expanded editing pocket of the ecT252A modification to allow mischarging of isoleucine to tRNALeu (Figure 5C). These combined results strongly support the conclusion that the universally conserved aspartic acid plays a key role in the overall editing reaction of LeuRS whether by a pre- or posttransfer mechanism. Discussion The LeuRS Editing Domain Binds Preand Posttransfer Editing Substrates in Largely Overlapping Sites In this paper we have presented structural data on analogs of both the pre- and posttransfer editing substrates bound in the editing site of LeuRS. These results show how the editing site is capable of tightly binding two different, but related, compounds with the same set of interacting residues (Figures 3A–3C). The mode of binding is such that the common groups of the two substrates, the adenine and amino acid moieties, are

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Mechanisms of Editing by Leucyl-tRNA Synthetase 957

accommodated in the same specificity pockets and recognized in the same way. Moreover, the necessarily different conformations of the linking ribose (posttransfer) or ribo-phosphate (pretransfer) can be accommodated without need for any significant conformational adjustments to active site residues (Figure 3C). This remarkable economy of design obviates the need, at least in the case of LeuRS, to postulate distinct amino acid binding sites for the pre- and posttransfer editing substrates, each with full second sieve capability, as has been proposed for ValRS (Fukai et al., 2000) and IleRS (Fukai et al., 2000; Hendrickson et al., 2002). A corollary of our observations is that it would be impossible to bind the 3⬘ end of the tRNA to the editing domain simultaneously with the adenylate, as has been implied in a recent model for editing (Bishop et al., 2002) that was developed in part using the ValRS structural results and modeling predictions (Fukai et al., 2000). The Noncognate Amino Acid Binding Pocket In this and previous work we have identified two key conserved residues which when mutated individually to alanine have a critical effect on LeuRS editing activity. First, the Thr252 to alanine mutation results in an enzyme with no apparent activity in synthesis of Leu-tRNALeu since the product is now efficiently deacylated by the editing activity (Mursinna et al., 2001). The structural results confirm that this residue, together with ttVal340, form a constriction in the binding pocket sterically preventing binding of side chains branched at C␥ such as leucine. Modeling suggests that it is possible to bind isoleucine or methionine in the same pocket. Indeed this has recently been confirmed by structure determination at 2.7 A˚ resolution of the complex between LeuRSTT and the sulphamoyl analog of isoleucyl-adenylate (IleAMS) which clearly shows IleAMS bound in both the synthetic and editing sites (data not shown). Mutation of Thr252 to alanine relaxes the second sieve constraint allowing cognate leucine to be “mis-edited”. More recent results have shown that mutation of Thr252 to bulkier residues such as leucine, phenylalanine, or tyrosine restricts the size of the second sieve so that isoleucine and valine can be stably charged to tRNALeu without editing (Mursinna and Martinis, 2002; Tang and Tirrell, 2002). It has also been shown that for E. coli IleRS, an alanine mutation of ecHis333 (equivalent to Met338 in LeuRSTT, part of the amino acid binding pocket) leads to efficient edit-

ing of Ile-tRNAIle (Hendrickson et al., 2002), presumably by a similar mechanism. A Universally Conserved Aspartic Acid Critical for Overall Editing in LeuRS The second key residue to be identified in this work is residue ttAsp347 (ecAsp345, ycAsp419) which when mutated to alanine severely diminishes the editing activity in all three LeuRSs tested. The ability of the mutant enzyme to discriminate is thus limited to the capabilities of the aminoacylation active site, allowing accumulation of mischarged Ile-tRNALeu and Met-tRNALeu which are otherwise efficiently edited. Again the key role of this residue fully correlates with the structural results in that in both the pre- and posttransfer editing substrate complexes, ttAsp347 hydrogen bonds to the ␣-amino group of the amino acid and together with a second conserved hydrogen bond of the ␣-amino group to the main chain carbonyl oxygen of Met338 plays an essential role in binding and positioning the substrates for hydrolysis (see below for a discussion of the hydrolysis mechanism). The critical aspartic acid within the LeuRS editing domain is also universally conserved in the homologous CP1 editing domains of ValRS and IleRS. Substitution of the aspartic acid by alanine in ecIleRS (D342A) resulted in editing deficiencies in both post- and pretransfer hydrolysis of mischarged tRNAIle and misactivated aminoacyl-adenylate, respectively (Bishop et al., 2002). Substitution of the conserved aspartic acid with asparagine dramatically decreased editing activity similar to the alanine mutant. In contrast, replacement with glutamic acid retained significant levels of editing compared to wild-type IleRS, supporting the idea that the carboxylic acid moiety of the side chain of the conserved aspartic acid is important to editing. For ValRS and IleRS, this conserved aspartate was predicted to interact with the ␣-amino group of the amino acid of the mischarged tRNA (Fukai et al., 2000) but was presumed not to be involved in pretransfer substrate binding. The structural results presented here for LeuRS show that the aspartic acid has the same role in pre- and posttransfer editing. It is likely that this universally conserved aspartate also binds in the same way to the pre- and posttransfer editing substrates of IleRS and ValRS, and hence its substitution by alanine would be expected to greatly effect total editing in these systems as well (Bishop et al., 2002).

Figure 3. Interactions of the Pre- and Posttransfer Editing Substrate Analogs in the Editing Active Site of LeuRSTT (A) Diagram showing selected hydrogen bonds (green dotted lines) between editing site residues of LeuRSTT and the pretransfer editing substrate analog NvaAMS (orange). The two segments of the main chain shown (tt245-252 and tt327-347) correspond to the regions aligned in Figure 1C. The threonine-rich peptide (tt245-252) is in purple. (B) Diagram showing selected hydrogen bonds between editing site residues and the posttransfer editing substrate analog Nva2AA (orange). The orientation is the same as in (A). For clarity, some residues referred to in the text and which are in the same conformation in both structures are only included in either (A) (Asp251/Arg259 and Asp344) or (B) (Ile337). (C) Superposition of bound pre- (NvaAMS, orange) and post- (Nva2AA, black) transfer editing substrate analogs, obtained after superposing the C␣ positions of the editing domain of each complex. The two different substrates are accommodated with only minor changes to the active site conformation as indicated by the main chain traces. Substrates superposition is most precise at the norvaline backbone atoms (N, C␣, C), adjacent to the point of hydrolysis, due to the conserved strong interaction of the ␣-amino group with Asp347. (D) Stereo diagram showing network of water molecules (green spheres) and hydrogen bonds (dotted green lines) in the vicinity of the ester bond to be hydrolyzed for the case of the Nva2AA complex. The purple water molecule in the center, hydrogen bonds to Asp344 and the bridging amide of Nva2AA and is suitably placed to hydrolyze the ester bond. A similar situation is observed in the complex with NvaAMS (data not shown).

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Figure 4. Mischarging Activity of Wild-Type and Editing-Defective Mutants (A) Misaminoacylation of yeast tRNALeu with isoleucine. Symbols represent misaminoacylation of yeast crude tRNA by wild-type (ycWT, square) and ycD419A LeuRS (triangle). Inset shows isoleucylation of E. coli tRNA by wild-type (ecWT, square) and ecD345A LeuRS (triangle). (B) Acid gel analysis of yeast tRNALeu charged with 14C-labeled leucine or isoleucine. (C) Graphical analysis of the methionylation activity of tRNALeu by ycD419A (shown in [D]). Each phosphorimaged band for [35S]-Met-tRNALeu was integrated via MacBAS Image Gauge program. (D) Acid gel analysis of yeast tRNALeu mischarged with methionine by ycD419A LeuRS. (E) E. coli LeuRS misaminoacylation assay of tRNALeu with methionine using E. coli crude tRNA. Symbols represent the wild-type (ecWT, square) and ecD345A LeuRS (triangle). (F) The ttD347A mutant of LeuRSTT mutant shows very low activity in tRNALeu-dependent hydrolysis of NvaAMP by comparison with the wildtype protein.

Mechanism of Hydrolysis The pre- and posttransfer substrates contain respectively labile mixed anhydride or ester linkages and slowly degrade in aqueous solution due to hydrolysis by water attacking the carbonyl carbon. Catalytic enhancement of the hydrolysis rate would be expected to require the presence of either a base to generate the more nucleophilic OH⫺ and/or enhancement of the positive charge

on the carbonyl carbon, for example by strong hydrogen bonding to the carbonyl oxygen. Such a mechanism, often involving a conserved triad of residues and an oxyanion hole which stabilizes the tetrahedral intermediate, operates not only in serine proteases (which have to cleave the much more robust peptide bond) but also in several esterases (e.g., phospholipase, acetylcholinesterase, and cocaine esterase) (Turner et al., 2002).

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Figure 5. Suppression of the ecT252A Editing Specificity Defect (A) Leucylation assay of E. coli crude tRNALeu. Symbols represent aminoacylation by wild-type (ecWT, square), ecD345A (triangle), ecT252A LeuRS (circle), and ecT252A/D345A LeuRSs (diamond). (B) Deacylation of cognate Leu-tRNALeu. The symbols are described in (A). “X” indicates a control reaction that lacks enzyme. (C) Isoleucylation assay of E. coli crude tRNA. The symbols are described in (A).

However, neither extensive mutagenesis studies of conserved residues in IleRS (Hendrickson et al., 2002) or in LeuRS (Mursinna et al., 2001), notably in the threoninerich region, nor the structural results presented here have permitted identification of residues that can be considered as directly catalytic. There is no catalytic triad or obvious oxyanion hole. All the evidence points to the hypothesis that the largely rigid editing domain simply binds the substrates in a configuration favoring attack by a water molecule which itself is presumably appropriately positioned by the active site residues. The extent to which the editing site also contributes to stabilization of the transition state remains to be determined, for instance, by structural studies with transition state analogs as was recently done with the hairpin ribozyme (Rupert et al., 2002). In the high-resolution structures of both the pre- and posttransfer complexes, a potential attacking water is

found which hydrogen bonds to ttAsp344 and the bridging amide of either substrate analog (Figure 3E). However, mutation of ttAsp344 to alanine has little effect on editing (data not shown), and although highly conserved in bacterial LeuRS (occasionally substituted by an asparagine) and in ValRS, this position is occupied by an alanine or serine in archae or eukaryotic LeuRS (Figure 1C), suggesting that alternative residues could play the role of positioning the catalytic water molecule. For the posttransfer complex, the 3⬘OH of the ribose, which hydrogen bonds to Thr-248 and positions another water molecule close to the labile bond, could also have a role. A 3⬘OH-assisted mechanism for ester hydrolysis has frequently been invoked for posttransfer editing (Englisch et al., 1986; Nordin and Schimmel, 2002). The single most important factor in the correct positioning of either substrate is the fixing of the ␣-amino group of the amino acid by hydrogen bonding to universally conserved ttAsp347 and the main chain of ttMet338. This is evident from the fact that mutation of this aspartic acid to alanine has a major detrimental effect on overall editing by IleRS (Bishop et al., 2002) and various LeuRSs. On the other hand, ttThr247 and ttThr248 from the threonine-rich peptide (Figure 1C) are also clearly important in binding either the phosphate or ribose of the substrates (Figure 3), but individual mutations to alanine of these residues appear to have only small effects on editing (Mursinna et al., 2001). This is likely due to the fact that the pre- or posttransfer editing substrates form respectively four or three hydrogen bonds to the threonine-rich peptide (Figures 3A and 3B), only one of which would be removed by a threonine to alanine mutation. This is also consistent with the observations that for ecIleRS, double alanine mutants in the threonine-rich peptide (T242A/N250A) or a major disruption of the conformation (T242P) is required to have a dramatic effect upon editing (Hendrickson et al., 2000). (Thr242 in ecIleRS is equivalent to Thr248 in ttLeuRS). In ecIleRS, a T243R mutant has been proposed to have a specific deficiency in pretransfer editing (Hendrickson et al., 2002), apparently supporting the idea of distinct pre- and posttransfer editing sites. In LeuRS, the equivalent residue is already a conserved arginine/ lysine (ttArg249) that forms a highly conserved salt bridge with an aspartate/glutamate (ttAsp251) that is 2 residues away (249-RPD-251, Figure 3A). In ValRS, the same motif occurs (Figure 1C). This salt bridge is apparently absent in IleRS and replaced by a highly conserved Thr/Trp pair (243-TPW-245 in ecIleRS, Figure 1C). Introduction of an electrostatically uncompensated basic residue into the threonine-rich region of ecIleRS could specifically interfere with the binding of the negatively charged phosphate of the adenylate and perhaps explain a preferential effect on pretransfer editing of the T243R mutant. Concluding Remarks Four domains or proteins, with completely different structural frameworks, have to date been characterized which specifically deacylate charged tRNAs. This includes the editing domain of the three class Ia synthetases, IleRS, ValRS, and LeuRS, considered here, the editing domain of ThrRS (Dock-Bregeon et al., 2000) also

Molecular Cell 960

hypothesized to occur in AlaRS, (Sankaranarayanan et al., 1999), the putative editing domain of prokaryote-like ProRS (Beuning and Musier-Forsyth, 2000; Wong et al., 2002), and the D-Tyr-tRNATyr deacylase (Ferri-Fioni et al., 2001). However, for none of these proteins has structural information been obtained on editing substrate complexes, nor has the hydrolytic mechanism been elucidated. Here we have described crystal structures of complexes between a class Ia synthetase editing site and both the pre- and posttransfer editing substrates. These results show that LeuRS can accommodate both substrates in largely overlapping sites with the mode of binding and recognition for the adenine and noncognate amino acid moieties being essentially identical. A universally conserved aspartic acid forms a salt bridge with the ␣-amino group of the noncognate amino acid of both substrates. Mutation of this aspartic acid to alanine abolishes editing in LeuRS from three different species which have been shown to exhibit either preor posttransfer editing, or both. Similarly in IleRS, which is thought to edit in vitro predominantly by pretransfer editing (Fersht, 1977), the equivalent mutation eliminates overall editing (Bishop et al., 2002). These structural and biochemical results are consistent with a common and essential role of the aspartate in tightly binding and positioning the amino acid moiety of both pre- and posttransfer editing substrates: first, for proofreading the nature of the side-chain in the discrimination pocket and second for favoring hydrolysis by a water molecule attacking the carbonyl carbon. The high structural and sequence homology between the editing sites of the three class Ia synthetases, particularly LeuRS and ValRS, suggests that a similar unified mechanism operates in all three cases. This conflicts with the structural model previously proposed for ValRS and IleRS in which the amino acid pockets for noncognate amino acid would be significantly different for the pre- and posttransfer substrates (Fukai et al., 2000). However, this model was entirely based on the assumption that the adenosyl moiety (including the base and ribose) would be in the same conformation for both pre- and posttransfer substrates, which is clearly not the case for the ribose in the two corresponding structures presented in this work for LeuRS. The next major step toward understanding the functioning of LeuRS will be the determination of structures of the enzyme with cognate tRNA. These are required to shed light on the as yet unknown tRNA and editing domain configurations in the various steps of the aminoacylation and proofreading mechanism.

Talon metal affinity resin (BD Biosciences Clontech) (Mursinna et al., 2001). Aminoacylation Assays Leucylation and isoleucylation assays were carried out as described (Mursinna et al., 2001), except that either S. cerevisiae (5 mg/ml) or E. coli (4 mg/ml) crude tRNA was used rather than in vitro transcribed tRNALeu. Methionylation activities were analyzed similarly but included 20 ␮M [35S]-methionine (303 ␮Ci/ml; Amersham Biosciences). In addition, 10% trichloroacetic acid (TCA) containing 1 mM methionine was substituted to wash the mischarged tRNA. All reactions were repeated and averaged. Analysis of some wild-type and mutant tRNA synthetases has detected erroneously high levels of charged tRNA due to TCA coprecipitation of radiolabeled protein (Gillet et al., 1997; Martinis and Schimmel, 1992). Acid-gel analysis was performed to confirm the production of charged tRNA. Leucylation and isoleucylation assays were carried out as indicated above, except that 40 ␮M [14C]-leucine or [14C]-isoleucine (300 ␮Ci/ml) was substituted respectively for the 3 H-labeled amino acid. Methionylation reactions were conducted exactly as stated above. Each reaction was incubated at 37⬚C for 1 hr, quenched, and analyzed by acid gel analysis (Mursinna and Martinis, 2002). Editing and Inhibition Assays The overall editing of norvaline was assayed by measuring AMP formation in the absence or presence of 15 ␮M E. coli tRNALeu at 37⬚C (Englisch et al., 1986). Briefly, a 50 ␮l reaction contained 2–4 mM norvaline, 1 mM [14C]-ATP in 100 mM HEPES (pH 7.5), 25 mM KCl, 10 mM MgCl2, and 0.5 mM DTT. The reaction was started by addition of 1 ␮M enzyme and quenched by spotting 2 ␮l aliquots onto PEI cellulose plates (Sigma) that had been prewashed with water. AMP, ADP, and ATP were separated by TLC in 0.75 M KPi (pH 3.5). The nucleotide spots were identified with UV light, cut out, and the radioactivity counted. Reaction conditions for posttransfer editing have been described (Mursinna and Martinis, 2002). Kinetic constants were measured at 25⬚C in reactions that contained 60 mM Tris (pH 7.5), 10 mM MgCl2, and varied concentrations of 0.15–5 ␮M Ile-tRNALeu. Competition assays incorporated Nva2AA (0.5 nM–1 ␮M) or Nva3AA (0.05–300 ␮M). Each reaction was initiated with 8 nM wild-type E. coli LeuRS. Aliquots were quenched every 15 s by TCA precipitation. Synthesis of 5ⴕ-O-[N-(L-Norvalyl)Sulphamoyl]Adenosine 2⬘,3⬘-O-Isopropylidene-5⬘-O-sulfamoyladenosine was synthesized from 2⬘,3⬘-O-isopropylideneadenosine as described (Kristinsson et al., 1994) with slight modifications, and purified by silica gel column chromatography with an elution gradient of methanol in ethyl acetate (0%–4%). This material was then reacted with tert-butoxycarbonylL-norvaline N-hydroxysuccinimide ester and 1,8-diazabicyclo[5,4-0] undec-7-ene (Ueda et al., 1991). The product, 2⬘,3⬘-O-isopropylidene-5⬘-O-[N-(L-norvalyl)sulfamoyl]adenosine, was purified by Florisil gel column chromatography with an elution gradient of ethanol in dichloromethane (0%–15%). All protection groups were removed by incubation with trifluoroacetic acid/water (3:1 v/v) for 2 hr, followed by solvent evaporation. The crude NvaAMS product was purified on a preparative 25 mm C18 HPLC column with an elution gradient of water in acetonitrile and 0.1% trifluoroacetic acid (0%– 65%). NvaAMS was obtained in 38% overall yield.

Experimental Procedures Protein Mutagenesis and Purification LeuRSTT was expressed and purified as described (Yaremchuk et al., 2000). The S. cerevisiae CDC60 (Hohmann and Thevelein, 1992) gene was amplified by PCR using Pfu DNA polymerase and cloned into the plasmid pET-32 Ek/LIC (Novagen), to generate plasmid p32YL-2-3. Mutations were introduced via PCR (Mursinna et al., 2001) into either p32YL-2-3 yielding plasmid pHAPPY1-1-1-16, p15EC3-1 (Martinis and Fox, 1997) harboring the E. coli leuS gene yielding pHAPPY2-1-1-28, or pMURe10 (Mursinna et al., 2001) that encodes ecT252A yielding pHAPPY2-1-1-19. The yeast and E. coli LeuRSs were expressed respectively in E. coli strains Bl21-CodonPlus(DE3)-RIL and BL21(DE3)pLysS (Stratagene) and purified using

Structure Determination of T. thermophilus LeuRS Complexed with NvaAMS Crystals of LeuRSTT were grown in the presence of mercury chloride (Yaremchuk et al., 2000). These were soaked with 0.6 mM NvaAMS for 1 month. The soaked crystals were cryoprotected with 30% glycerol and diffraction data collected to 2.2 A˚ resolution on ESRF beamline ID14-EH1 and integrated with MOSFLM (Leslie, 1999) (Table 1). The structure was solved by molecular replacement (MOLREP) (CCP4, 1994), using the previously determined structure of LeuRSTT (Cusack et al., 2000) and refined to an R factor (Rfree) of 0.213 (0.234) using CNS (Brunger et al., 1998) with standard protocols (Table 1). A mercury atom is strongly bound to Cys-128 but otherwise does not influence the structure. As the NvaAMS was

Mechanisms of Editing by Leucyl-tRNA Synthetase 961

Table 1. X-Ray Crystallographic Data Collection and Refinement Statistics Crystal Contents Beamline/detector Wavelength Exposure/image Space group Cell dimensions (A˚) Resolution (A˚) Unique reflections Average redundancy Completeness (%) (highest bin) Rmerge (highest bin) Resolution (A˚) Work reflections Test reflections Rfree Rwork No. protein atoms No. substrate atoms No. solvent molecules No. metal atoms ⬍b⬎ protein ⬍b⬎ solvent ⬍b⬎ substrate Rms bonds (A˚) Rms angles (⬚) Ramachandran plot Favorable % Additional % Generous % Disallowed %

LeuRSTT ⫹ NvaAMS (Pretransfer Substrate Analog)

LeuRSTT ⫹ LeuAMS ⫹ Nva2AA (Posttransfer Substrate Analog)

ID14-EH1/ADSC Q4 0.934 A˚ 20 s/0.5⬚ C2221 a ⫽ 103.5 b ⫽ 152.9 c ⫽ 172.6 20.0–2.2 68288 4.4 (2.9) 98.1 (85.8) 0.053 (0.388) 20.0–2.2 64860 3388 (4.9%) 0.234 0.213 6241 60 (2 ⫻ NvaAMS) 197 water, 6 sulfate 1 Hg 46.8 43.2 42.2 (NvaAMS synthetic) 51.5 (NvaAMS editing) 0.0061 1.114

ID14-EH2/ADSC Q4 0.933 A˚ 25 s/1.0⬚ C2221 a ⫽ 101.9 b ⫽ 154.8 c ⫽ 175.1 25.0–2.1 77419 2.3 (1.8) 96.2 (84.2) 0.088 (0.350) 25.0–2.1 73546 3844 (4.8%) 0.225 0.193 6642 57 (LeuAMS, Nva2AA) 483 water 2 Zn 30.0 38.5 18.3 (LeuAMS) 27.6 (Nva2AA) 0.0058 1.243

90.8 8.7 0.3 0.1 (1, Ala 8)

94.2 5.8 0.0 0.0

soaked into pregrown apo-LeuRSTT crystals, the conformational changes described between the apo-state and that obtained by cocrystallization with LeuAMS do not fully occur, notably the ordering of the ZN-1 domain (Cusack et al., 2000). Measurements at 2.1 A˚ were subsequently made on LeuRSTT cocrystallized with NvaAMS (data not shown). This shows a fully ordered enzyme structure equivalent to that previously described for LeuRSTT-LeuAMS complex but with NvaAMS in the synthetic site and editing site. This confirms that binding of NvaAMS in the synthetic active site induces the same conformational changes as for LeuAMS. However, as the occupancy of NvaAMS in the editing site is only partial in this structure, the data on the soaked crystal is presented here. Synthesis of 2ⴕ-(L-Norvalyl)Amino-2ⴕ-Deoxyadenosine and 3ⴕ-(L-Norvalyl)Amino-3ⴕ-Deoxyadenosine In an overnight reaction under argon, N-␣-Fmoc-L-norvaline (120 ␮mol) was converted to its N-hydroxysuccinimide ester using di(Nsuccinimidyl)oxalate in dry acetonitrile containing one equivalent of pyridine (Takeda et al., 1983), followed by concentration in vacuo to about 1 ml. The Fmoc-protected version of Nva2AA or Nva3AA was then obtained by respectively adding dry 2⬘-amino-2⬘-deoxyadenosine or 3⬘-amino-3⬘-deoxyadenosine (100 ␮mol), dry DMSO (1 ml), and anhydrous N,N-diisopropylethylamine (100 ␮mol) to the crude N-hydroxysuccinimide ester. The clear solution was stirred for 24 hr under argon. The Fmoc group was removed by addition of 1 ml piperidine and stirred for 1 hr. The solvent was removed in vacuo. The crude Nva2AA or Nva3AA was dissolved in 0.5 ml N,N-dimethylformamide and 8 ml of 0.1 M aqueous ammonium bicarbonate added. After filtration, the supernatant containing Nva2AA or Nva3AA was purified by semipreparative reversed phase HPLC on a 19 ⫻ 300 mm ␮-Bondapak C18 column using a linear gradient from 0.1 M aqueous ammonium bicarbonate to 50% acetonitrile/50% aqueous ammonium bicarbonate. Fractions containing the desired product were evaporated to dryness in vacuo, and residual ammonium bicarbonate was removed by two lyophilisations from water. Nva2AA (or Nva3AA) was obtained as a white solid (21

mg) and the structure confirmed by NMR spectroscopy and fast atom bombardment mass spectroscopy run in the positive ion mode (observed [M ⫹ H]⫹ at m/z 366.2; The calculated molecular weight for C15H23N7O4 is 365.39). The purity of the final product was also confirmed by analytical reversed phase HPLC on a 4.6 ⫻ 250 mm XTerra RP8 column. Structure Determination of T. thermophilus LeuRS Complexed with Nva2AA Crystals of LeuRSTT were grown in the presence of the sulfamoyl analog of leucyl-adenylate (LeuAMS) (Yaremchuk et al., 2000), since the presence of the substrate leads to more ordered and robust crystals. These were soaked with 1 mM of either Nva2AA or Nva3AA for a few days. The soaked crystals were cryoprotected with 30% glycerol and diffraction data collected on ESRF beamline ID14-EH1 (data not shown). Preliminary data showed that the leucyl-adenylate analog bound exclusively in the synthetic active site (Cusack et al., 2000) and that no electron density was observed in the editing site when Nva3AA was soaked. However, when Nva2AA was soaked, additional density ascribable to it was observed in the editing site, but there appeared to be competition for binding with a sulfate ion from the crystallization medium which is always found to bind in the editing site adjacent to the threonine-rich peptide (Cusack et al., 2000). To overcome this problem, the crystals were transferred stepwise to a medium containing 1.0 M sodium citrate before soaking with Nva2AA. Data to 2 A˚ resolution were subsequently collected (Table 1), giving very clear and unambiguous electron density for the Nva2AA (Figure 2C). The structure was refined to an R factor (Rfree) of 0.193 (0.225) using CNS (Brunger et al., 1998) (Table 1). Acknowledgments This work was supported by The Human Frontiers Science Program (RGP0190/2001-M) (to S.A.M. and S.C.) as well as by grants to S.A.M. from The National Institutes of Health (GM63789) and The Robert A. Welch Foundation (E-1404). We would like to thank the

Molecular Cell 962

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Martinis, S.A., Plateau, P., Cavarelli, J., and Florentz, C. (1999). Aminoacyl-tRNA synthetases: a new image for a classical family. Biochimie 81, 683–700. Mursinna, R.S., and Martinis, S.A. (2002). Rational design to block amino acid editing of a tRNA synthetase. J. Am. Chem. Soc. 124, 7286–7287. Mursinna, R.S., Lincecum, T.L., Jr., and Martinis, S.A. (2001). A conserved threonine within Escherichia coli leucyl-tRNA synthetase prevents hydrolytic editing of leucyl-tRNALeu. Biochemistry 40, 5376– 5381. Nordin, B.E., and Schimmel, P. (2002). Plasticity of recognition of the 3⬘-end of mischarged tRNA by class I aminoacyl-tRNA synthetases. J. Biol. Chem. 277, 20510–20517. Nureki, O., Vassylyev, D.G., Tateno, M., Shimada, A., Nakama, T., Fukai, S., Konno, M., Hendrickson, T.L., Schimmel, P., and Yokoyama, S. (1998). Enzyme structure with two catalytic sites for doublesieve selection of substrate. Science 280, 578–582. Pauling, L. (1958). The probability of errors in the process of synthesis of protein molecules. In Fetschrift Prof. Dr. Artheur Stoll (Basel: Birkhauser Verlag), pp. 597–602 Rupert, P.B., Massey, A.P., Sigurdsson, S.T., and Ferre´-D’Amare´, A. (2002). Transition state stabilisation by a catalytic RNA. Science 298, 1421–1424.

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of the Escherichia coli leucyl-tRNA synthetase allows incorporation of novel amino acids into proteins in vivo. Biochemistry 41, 10635– 10645. Turner, J.M., Larsen, N.A., Basran, A., Barbas, C.F., 3rd, Bruce, N.C., Wilson, I.A., and Lerner, R.A. (2002). Biochemical characterization and structural analysis of a highly proficient cocaine esterase. Biochemistry 41, 12297–12307. Ueda, H., Shoku, Y., Hayashi, N., Mitsunaga, J., In, Y., Doi, M., Inoue, M., and Ishida, T. (1991). X-ray crystallographic conformational study of 5⬘-O-[N-(L-alanyl)- sulfamoyl]adenosine, a substrate analogue for alanyl-tRNA synthetase. Biochim. Biophys. Acta 1080, 126–134. Varshney, U., Lee, C.P., and RajBhandary, U.L. (1991). Direct analysis of aminoacylation levels of tRNAs in vivo. Application to studying recognition of Escherichia coli initiator tRNA mutants by glutaminyltRNA synthetase. J. Biol. Chem. 266, 24712–24718. Wong, F.C., Beuning, P.J., Nagan, M., Shiba, K., and Musier-Forsyth, K. (2002). Functional role of the prokaryotic proline-tRNA synthetase insertion domain in amino acid editing. Biochemistry 41, 7108–7115. Yaremchuk, A., Cusack, S., Gudzera, O., Grotli, M., and Tukalo, M. (2000). Crystallization and preliminary crystallographic analysis of Thermus thermophilus leucyl-tRNA synthetase and its complexes with leucine and a non-hydrolysable leucyl-adenylate analogue. Acta Crystallogr. D Biol. Crystallogr. 56, 667–669. Accession Numbers Coordinates and structure factors of the two structures have been deposited in the Protein Data Bank with access codes 1OBH (pretransfer analog complex) and 1OBC (posttransfer analog complex).