Structural and Mechanistic Basis of Pre- and Posttransfer Editing by Leucyl-tRNA Synthetase
2003; Elsevier BV; Volume: 11; Issue: 4 Linguagem: Inglês
10.1016/s1097-2765(03)00098-4
ISSN1097-4164
AutoresTommie L. Lincecum, M. A. Tukalo, Anna Yaremchuk, Richard S. Mursinna, Amy M. Williams, Brian S. Sproat, Wendy Van Den Eynde, Andreas Link, Serge Van Calenbergh, Morten Grøtli, Susan A. Martinis, S. Cusack,
Tópico(s)RNA Research and Splicing
ResumoThe 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 α-amino group of the noncognate amino acid and positions both substrates for hydrolysis. 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. 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 α-amino group of the noncognate amino acid and positions both substrates for hydrolysis. 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. 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. 1999Martinis S.A. Plateau P. Cavarelli J. Florentz C. Aminoacyl-tRNA synthetases: a new image for a classical family.Biochimie. 1999; 81: 683-700Crossref PubMed Scopus (75) Google Scholar). 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 1958Pauling L. The probability of errors in the process of synthesis of protein molecules.in: Prof. Dr. Artheur Stoll Fetschrift. Birkhauser Verlag, Basel1958Google Scholar). Experimentally, the rate of misincorporation of valine by IleRS was determined to be closer to 1 out of 3000 (Loftfield 1963Loftfield R.B. The frequency of errors in protein biosynthesis.Biochem. J. 1963; 89: 82-92Crossref PubMed Scopus (116) Google Scholar) due to the presence of an efficient editing mechanism which eliminates the initial selection errors (Baldwin and Berg 1966Baldwin A.N. Berg P. Transfer ribonucleic acid-induced hydrolysis of valyladenylate bound to isoleucyl ribonucleic acid synthetase.J. Biol. Chem. 1966; 241: 839-845Abstract Full Text PDF PubMed Google Scholar). 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 1992Jakubowski H. Goldman E. Editing of errors in selection of amino acids for protein synthesis.Microbiol. Rev. 1992; 56: 412-429Crossref PubMed Google Scholar) and class II enzymes ThrRS (Dock-Bregeon et al. 2000Dock-Bregeon A. Sankaranarayanan R. Romby P. Caillet J. Springer M. Rees B. Francklyn C.S. Ehresmann C. Moras D. Transfer RNA-mediated editing in threonyl-tRNA synthetase. The class II solution to the double discrimination problem.Cell. 2000; 103: 877-884Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) and ProRS (Beuning and Musier-Forsyth 2000Beuning P.J. Musier-Forsyth K. Hydrolytic editing by a class II aminoacyl-tRNA synthetase.Proc. Natl. Acad. Sci. USA. 2000; 97: 8916-8920Crossref PubMed Scopus (115) Google Scholar). The editing of misactivated homocysteine by several aaRSs, notably MetRS, is by a different mechanism than considered here (Jakubowski and Goldman 1992Jakubowski H. Goldman E. Editing of errors in selection of amino acids for protein synthesis.Microbiol. Rev. 1992; 56: 412-429Crossref PubMed Google Scholar). To enhance fidelity, the editing aaRSs rely on a "double sieve" mechanism for amino acid selection and discrimination as originally proposed by Fersht 1977Fersht A.R. Editing mechanisms in protein synthesis. Rejection of valine by the isoleucyl-tRNA synthetase.Biochemistry. 1977; 16: 1025-1030Crossref PubMed Scopus (197) Google Scholar, analyzed biochemically and genetically (e.g., Schmidt and Schimmel 1995Schmidt E. Schimmel P. Residues in a class I tRNA synthetase which determine selectivity of amino acid recognition in the context of tRNA.Biochemistry. 1995; 34: 11204-11210Crossref PubMed Scopus (78) Google Scholar) and first visualized in the crystal structures of IleRS (Nureki et al. 1998Nureki O. Vassylyev D.G. Tateno M. Shimada A. Nakama T. Fukai S. Konno M. Hendrickson T.L. Schimmel P. Yokoyama S. Enzyme structure with two catalytic sites for double-sieve selection of substrate.Science. 1998; 280: 578-582Crossref PubMed Scopus (305) Google Scholar, Silvian et al. 1999Silvian L.F. Wang J. Steitz T.A. Insights into editing from an Ile-tRNA synthetase structure with tRNAIle and mupirocin.Science. 1999; 285: 1074-1077Crossref PubMed Scopus (335) Google Scholar). 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. 2000Cusack S. Yaremchuk A. Tukalo M. The 2 Å crystal structure of leucyl-tRNA synthetase and its complex with a leucyl-adenylate analogue.EMBO J. 2000; 19: 2351-2361Crossref PubMed Scopus (221) Google Scholar, Fukai et al. 2000Fukai S. Nureki O. Sekine S. Shimada A. Tao J. Vassylyev D.G. Yokoyama S. Structural basis for double-sieve discrimination of L-valine from L- isoleucine and L-threonine by the complex of tRNA(Val) and valyl-tRNA synthetase.Cell. 2000; 103: 793-803Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, Nureki et al. 1998Nureki O. Vassylyev D.G. Tateno M. Shimada A. Nakama T. Fukai S. Konno M. Hendrickson T.L. Schimmel P. Yokoyama S. Enzyme structure with two catalytic sites for double-sieve selection of substrate.Science. 1998; 280: 578-582Crossref PubMed Scopus (305) Google Scholar, Silvian et al. 1999Silvian L.F. Wang J. Steitz T.A. Insights into editing from an Ile-tRNA synthetase structure with tRNAIle and mupirocin.Science. 1999; 285: 1074-1077Crossref PubMed Scopus (335) Google Scholar). The editing active site hydrolytically cleaves the misactivated aminoacyl-adenylate (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 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. 2000Fukai S. Nureki O. Sekine S. Shimada A. Tao J. Vassylyev D.G. Yokoyama S. Structural basis for double-sieve discrimination of L-valine from L- isoleucine and L-threonine by the complex of tRNA(Val) and valyl-tRNA synthetase.Cell. 2000; 103: 793-803Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, Nureki et al. 1998Nureki O. Vassylyev D.G. Tateno M. Shimada A. Nakama T. Fukai S. Konno M. Hendrickson T.L. Schimmel P. Yokoyama S. Enzyme structure with two catalytic sites for double-sieve selection of substrate.Science. 1998; 280: 578-582Crossref PubMed Scopus (305) Google Scholar). 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 ValRS-tRNAVal cocrystal structure and modeling of a charged threonine and threonyl-adenylate in the editing site (Fukai et al. 2000Fukai S. Nureki O. Sekine S. Shimada A. Tao J. Vassylyev D.G. Yokoyama S. Structural basis for double-sieve discrimination of L-valine from L- isoleucine and L-threonine by the complex of tRNA(Val) and valyl-tRNA synthetase.Cell. 2000; 103: 793-803Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). 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. 2002Hendrickson T.L. Nomanbhoy T.K. de Crecy-Lagard V. Fukai S. Nureki O. Yokoyama S. Schimmel P. Mutational separation of two pathways for editing by a class I tRNA synthetase.Mol. Cell. 2002; 9: 353-362Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) 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. 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. 1986Englisch S. Englisch U. von der Haar F. Cramer F. The proofreading of hydroxy analogues of leucine and isoleucine by leucyl-tRNA synthetases from E. coli and yeast.Nucleic Acids Res. 1986; 14: 7529-7539Crossref PubMed Scopus (78) Google Scholar, Apostol et al. 1997Apostol I. Levine J. Lippincott J. Leach J. Hess E. Glascock C.B. Weickert M.J. Blackmore R. Incorporation of norvaline at leucine positions in recombinant human hemoglobin expressed in Escherichia coli.J. Biol. Chem. 1997; 272: 28980-28988Crossref PubMed Scopus (68) Google Scholar, Lincecum and Martinis 2000Lincecum Jr., T.L. Martinis S.A. The tRNA synthetase proofreading and editing active sites: a novel antibiotic target.SAAS Bull. Biochem. Biotechnol. 2000; 13: 25-33Google Scholar). 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 sulfamoyl-analog 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. 1994Belrhali H. Yaremchuk A. Tukalo M. Larsen K. Berthet-Colominas C. Leberman R. Beijer B. Sproat B. Als-Nielsen J. Grübel G. et al.Crystal structures at 2.5 Å resolution of seryl-tRNA synthetase complexed with two analogs of seryl adenylate.Science. 1994; 263: 1432-1436Crossref PubMed Scopus (159) Google Scholar, Cusack et al. 2000Cusack S. Yaremchuk A. Tukalo M. The 2 Å crystal structure of leucyl-tRNA synthetase and its complex with a leucyl-adenylate analogue.EMBO J. 2000; 19: 2351-2361Crossref PubMed Scopus (221) Google Scholar, Dock-Bregeon et al. 2000Dock-Bregeon A. Sankaranarayanan R. Romby P. Caillet J. Springer M. Rees B. Francklyn C.S. Ehresmann C. Moras D. Transfer RNA-mediated editing in threonyl-tRNA synthetase. The class II solution to the double discrimination problem.Cell. 2000; 103: 877-884Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, Ueda et al. 1991Ueda H. Shoku Y. Hayashi N. Mitsunaga J. In Y. Doi M. Inoue M. Ishida T. X-ray crystallographic conformational study of 5′-O-[N-(L-alanyl)- sulfamoyl]adenosine, a substrate analogue for alanyl-tRNA synthetase.Biochim. Biophys. Acta. 1991; 1080: 126-134Crossref PubMed Scopus (75) Google Scholar). 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 Å for NvaAMS and 2.1 Å for Nva2AA) allows unambiguous positioning of the ligand, definition of the ribose pucker, and assignment of a network of water molecules and hydrogen bonds. 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 Å (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-Cα bond (psi torsional angle) resulting in an unusually bent conformation in which the α-amino group of the norvaline hydrogen bonds to the O2′ of the ribose (Figure 3A).Figure 3Interactions of the Pre- and Posttransfer Editing Substrate Analogs in the Editing Active Site of LeuRSTTShow full caption(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 Cα 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, Cα, C), adjacent to the point of hydrolysis, due to the conserved strong interaction of the α-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).View Large Image Figure ViewerDownload Hi-res image Download (PPT) (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 Cα 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, Cα, C), adjacent to the point of hydrolysis, due to the conserved strong interaction of the α-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). 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. 2000Cusack S. Yaremchuk A. Tukalo M. The 2 Å crystal structure of leucyl-tRNA synthetase and its complex with a leucyl-adenylate analogue.EMBO J. 2000; 19: 2351-2361Crossref PubMed Scopus (221) Google Scholar). 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 Å 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 α-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 hydrophobic 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. 2001Mursinna R.S. Lincecum Jr., T.L. Martinis S.A. A conserved threonine within Escherichia coli leucyl-tRNA synthetase prevents hydrolytic editing of leucyl-tRNALeu.Biochemistry. 2001; 40: 5376-5381Crossref PubMed Scopus (105) Google Scholar). 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. 2000Cusack S. Yaremchuk A. Tukalo M. The 2 Å crystal structure of leucyl-tRNA synthetase and its complex with a leucyl-adenylate analogue.EMBO J. 2000; 19: 2351-2361Crossref PubMed Scopus (221) Google Scholar) 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 γ-methyl group (Mursinna et al. 2001Mursinna R.S. Lincecum Jr., T.L. Martinis S.A. A conserved threonine within Escherichia coli leucyl-tRNA synthetase prevents hydrolytic editing of leucyl-tRNALeu.Biochemistry. 2001; 40: 5376-5381Crossref PubMed Scopus (105) Google Scholar). 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. 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 α-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. 2000Fukai S. Nureki O. Sekine S. Shimada A. Tao J. Vassylyev D.G. Yokoyama S. Structural basis for double-sieve discrimination of L-valine from L- isoleucine and L-threonine by the complex of tRNA(Val) and valyl-tRNA synthetase.Cell. 2000; 103: 793-803Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar), 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). 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 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 2002Nordin B.E. Schimmel P. Plasticity of recognition of the 3′-end of mischarged tRNA by class I aminoacyl-tRNA synthetases.J. Biol. Chem. 2002; 277: 20510-20517Crossref PubMed Scopus (51) Google Scholar). 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. 1986Englisch S. Englisch U. von der Haar F. Cramer F. The proofreading of hydroxy analogues of leucine and isoleucine by leucyl-tRNA synthetases from E. coli and yeast.Nucleic Acids Res. 1986; 14: 7529-7539Crossref PubMed Scopus (78) Google Scholar, Lincecum and Martinis 2000Lincecum Jr., T.L. Martinis S.A. The tRNA synthetase proofreading and editing active sites: a novel antibiotic target.SAAS Bull. Biochem. Biotechnol. 2000; 13: 25-33Google Scholar). 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 site-directed mutagenesis within the CP1 domain of E. coli LeuRS have also been reported to yield misaminoacylation of isoleucine to tRNALeu (Chen et al. 2000Chen J.F. Guo N.N. Li T. Wang E.D. Wang Y.L. CP1 domain in Escherichia coli leucyl-tRNA synthetase is crucial for its editing function.Biochemistry. 2000; 39: 6726-6731Crossref PubMed Scopus (112) Google Scholar, Chen et al. 2001Chen J.F. Li T. Wang E.D. Wang Y.L. Effect of alanine-293 replacement on the activity, ATP binding, and editing of Escherichia coli leucyl-tRNA synthetase.Biochemistry. 2001; 40: 1144-1149Crossref PubMed Scopus (21) Google Scholar, Mursinna and Martinis 2002Mursinna R.S. Martinis S.A. Rational design to block amino acid editing of a tRNA synthetase.J. Am. Chem. Soc. 2002; 124: 7286-7287Crossref PubMed Scopus (55) Google Scholar, Tang and Tirrell 2002Tang Y. Tirrell D.A. Attenuation of the editing activity of the Escherichia coli leucyl-tRNA synthetase allows incorporation of novel amino acids into proteins in vivo.Biochemistry. 2002; 41: 10635-10645Crossref PubMed Scopus (84) Google Scholar). We tested each of the mutant LeuRSs for isoleucylation activity to determine if the conserved aspar
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