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Crystal Structures of the CP1 Domain from Thermus thermophilus Isoleucyl-tRNA Synthetase and Its Complex with l-Valine

2004; Elsevier BV; Volume: 279; Issue: 9 Linguagem: Inglês

10.1074/jbc.m312830200

ISSN

1083-351X

Autores

Ryuya Fukunaga, Shuya Fukai, Ryuichiro Ishitani, Osamu Nureki, Shigeyuki Yokoyama,

Tópico(s)

Protein Structure and Dynamics

Resumo

Isoleucyl-tRNA synthetase (IleRS) links tRNAIle with not only its cognate isoleucine but also the nearly cognate valine. The CP1 domain of IleRS deacylates, or edits, the mischarged Val-tRNAIle. We determined the crystal structures of the Thermus thermophilus IleRS CP1 domain alone, and in its complex with valine at 1.8- and 2.0-Å resolutions, respectively. In the complex structure, the Asp328 residue, which was shown to be critical for the editing reaction against Val-tRNAIle by a previous mutational analysis, recognizes the valine NH3+ group. The valine side chain binding pocket is only large enough to accommodate valine, and the placement of an isoleucine model in this location revealed that the additional methylene group of isoleucine would clash with His319. The H319A mutant of Escherichia coli IleRS reportedly deacylates the cognate Ile-tRNAIle in addition to Val-tRNAIle, indicating that the valine-binding mode found in this study represents that in the Val-tRNAIle editing reaction. Analyses of the Val-tRNAIle editing activities of T. thermophilus IleRS mutants revealed the importance of Thr228, Thr229, Thr230, and Asp328, which are coordinated with water molecules in the present structure. The structural model for the Val-adenosine moiety of Val-tRNAIle bound in the IleRS editing site revealed some interesting differences in the substrate binding and recognizing mechanisms between IleRS and T. thermophilus leucyl-tRNA synthetase. For example, the carbonyl oxygens of the amino acids are located opposite to each other, relative to the adenosine ribose ring, and are differently recognized. Isoleucyl-tRNA synthetase (IleRS) links tRNAIle with not only its cognate isoleucine but also the nearly cognate valine. The CP1 domain of IleRS deacylates, or edits, the mischarged Val-tRNAIle. We determined the crystal structures of the Thermus thermophilus IleRS CP1 domain alone, and in its complex with valine at 1.8- and 2.0-Å resolutions, respectively. In the complex structure, the Asp328 residue, which was shown to be critical for the editing reaction against Val-tRNAIle by a previous mutational analysis, recognizes the valine NH3+ group. The valine side chain binding pocket is only large enough to accommodate valine, and the placement of an isoleucine model in this location revealed that the additional methylene group of isoleucine would clash with His319. The H319A mutant of Escherichia coli IleRS reportedly deacylates the cognate Ile-tRNAIle in addition to Val-tRNAIle, indicating that the valine-binding mode found in this study represents that in the Val-tRNAIle editing reaction. Analyses of the Val-tRNAIle editing activities of T. thermophilus IleRS mutants revealed the importance of Thr228, Thr229, Thr230, and Asp328, which are coordinated with water molecules in the present structure. The structural model for the Val-adenosine moiety of Val-tRNAIle bound in the IleRS editing site revealed some interesting differences in the substrate binding and recognizing mechanisms between IleRS and T. thermophilus leucyl-tRNA synthetase. For example, the carbonyl oxygens of the amino acids are located opposite to each other, relative to the adenosine ribose ring, and are differently recognized. Aminoacyl-tRNA synthetases (aaRSs) 1The abbreviations used are: aaRS, aminoacyl-tRNA synthetase; IleRS, isoleucyl-tRNA synthetase; ValRS, valyl-tRNA synthetase; LeuRS, leucyl-tRNA synthetase. catalyze the esterification of an amino acid to its cognate tRNA. This reaction proceeds in two steps: the synthesis of an aminoacyladenylate, as an activated intermediate, from the amino acid and ATP, and the transfer of the aminoacyl moiety to the 3′-terminal of the cognate tRNA to yield the aminoacyl-tRNA (1Fersht A.R. Kaethner M.M. Biochemistry. 1976; 15: 3342-3346Crossref PubMed Scopus (180) Google Scholar). To maintain accurate protein biosynthesis, each aaRS must discriminate between its cognate amino acid and other similar amino acids (2Pauling L. In Festschrift Arthur Stroll. Birkhäuser-Verlag, Basel1957: 597-602Google Scholar, 3Freist W. Sternbach H. Cramer F. Eur. J. Biochem. 1988; 173: 27-34Crossref PubMed Scopus (31) Google Scholar). Some aaRSs, including the isoleucyl-, leucyl-, and valyl-tRNA synthetases (IleRS, LeuRS, and ValRS, respectively), have a specific editing activity that hydrolyzes the misaminoacylated tRNAs ("post-transfer editing") (4Jakubowski H. Goldman E. Microbiol. Rev. 1992; 56: 412-429Crossref PubMed Google Scholar, 5Englisch S. Englisch U. von der Haar F. Cramer F. Nucleic Acids Res. 1986; 14: 7529-7539Crossref PubMed Scopus (78) Google Scholar, 6Baldwin A.N. Berg P. J. Biol. Chem. 1966; 241: 839-845Abstract Full Text PDF PubMed Google Scholar). For example, IleRS also recognizes valine, which is smaller than the cognate isoleucine by only one methylene group, and mischarges it with tRNAIle. Then, the mischarged Val-tRNAIle is hydrolyzed to valine and tRNAIle in the post-transfer editing pathway. As for IleRS, another editing pathway (pre-transfer editing) also exists, in which the misactivated Val-AMP is directly hydrolyzed to valine and AMP in the presence of tRNAIle (7Fersht A.R. Biochemistry. 1977; 16: 1025-1030Crossref PubMed Scopus (202) Google Scholar, 8Hale S.P. Auld D.S. Schmidt E. Schimmel P. Science. 1997; 276: 1250-1252Crossref PubMed Scopus (79) Google Scholar). Biochemical experiments linked the specific location of the editing site to the connective polypeptide 1 (CP1) domain, a large insertion in the aminoacylation catalytic Rossmann-fold domain (9Lin L. Hale S.P. Schimmel P. Nature. 1996; 384: 33-34Crossref PubMed Scopus (92) Google Scholar). Previously, we determined the crystal structures of the Thermus thermophilus full-length IleRS complexed with isoleucine and with valine and showed that the editing site is in the highly conserved threonine-rich region of the CP1 domain (10Nureki O. Vassylyev D.G. Tateno M. Shimada A. Nakama T. Fukai S. Konno M. Hendrickson T.L. Schimmel P. Yokoyama S. Science. 1998; 280: 578-582Crossref PubMed Scopus (316) Google Scholar). In addition, the crystal structure of Staphylococcus aureus IleRS complexed with tRNAIle and mupirocin (an analogue of isoleucyl-adenylate) revealed that the 3′-terminal of tRNAIle is located in the CP1 domain (although it was not completely resolved) (11Silvian L.F. Wang J. Steitz T.A. Science. 1999; 285: 1074-1077Crossref PubMed Scopus (360) Google Scholar). This suggested that when a nearly cognate amino acid is charged to a tRNA, the acceptor stem flips from the aminoacylation site to the editing site, while the rest of the tRNA remains bound. However, the B factors of many atoms in the CP1 domains of these structures were high and some residues were disordered, since the CP1 domain is quite mobile relative to the rest of the protein (10Nureki O. Vassylyev D.G. Tateno M. Shimada A. Nakama T. Fukai S. Konno M. Hendrickson T.L. Schimmel P. Yokoyama S. Science. 1998; 280: 578-582Crossref PubMed Scopus (316) Google Scholar, 11Silvian L.F. Wang J. Steitz T.A. Science. 1999; 285: 1074-1077Crossref PubMed Scopus (360) Google Scholar). Furthermore, in our previous study (10Nureki O. Vassylyev D.G. Tateno M. Shimada A. Nakama T. Fukai S. Konno M. Hendrickson T.L. Schimmel P. Yokoyama S. Science. 1998; 280: 578-582Crossref PubMed Scopus (316) Google Scholar), the omit map electron density for valine was not clear enough for us to determine its orientation precisely, and the coordinates were determined by analogy with the related ValRS system (12Fukai S. Nureki O. Sekine S. Shimada A. Tao J. Vassylyev D.G. Yokoyama S. Cell. 2000; 103: 793-803Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). The crystal structure of T. thermophilus ValRS complexed with tRNAval accurately revealed the location of the 3′ terminus of tRNAval in the CP1 domain with complete resolution, although that of threonine (which is edited by ValRS) remains to be elucidated (12Fukai S. Nureki O. Sekine S. Shimada A. Tao J. Vassylyev D.G. Yokoyama S. Cell. 2000; 103: 793-803Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Based on the location of valine in the IleRS CP1 domain and that of the tRNA 3′-terminal adenosine in the ValRS CP1 domain, we built the structural models for Val-tRNAIle in IleRS and Thr-tRNAval in ValRS, assuming that their binding modes are analogous (12Fukai S. Nureki O. Sekine S. Shimada A. Tao J. Vassylyev D.G. Yokoyama S. Cell. 2000; 103: 793-803Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). However, differences between their binding modes were subsequently reported (13Nordin B.E. Schimmel P. J. Biol. Chem. 2002; 277: 20510-20517Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). In addition, no water molecules could be assigned around the valine bound to the CP1 domain in the full-length T. thermophilus IleRS structure. Therefore, the precise valine-binding mode and the mechanisms by which the IleRS CP1 domain selectively recognizes valine and catalyzes the hydrolysis of Val-tRNAIle remained to be elucidated. Recently, the crystal structure of T. thermophilus LeuRS complexed with the post-transfer editing substrate analogue was determined (14Lincecum T.L. Tukalo M. Yaremchuk A. Mursinna R.S. Williams A.M. Sproat B.S. Van Den Eynde W. Link A. Van Calenbergh S. Grotli M. Martinis S.A. Cusack S. Mol. Cell. 2003; 11: 951-963Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). In comparison with IleRS, the CP1 domain is inserted at a different point in the enzyme, and its rotational orientation differs by nearly 180° from IleRS (14Lincecum T.L. Tukalo M. Yaremchuk A. Mursinna R.S. Williams A.M. Sproat B.S. Van Den Eynde W. Link A. Van Calenbergh S. Grotli M. Martinis S.A. Cusack S. Mol. Cell. 2003; 11: 951-963Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 15Cusack S. Yaremchuk A. Tukalo M. EMBO J. 2000; 19: 2351-2361Crossref PubMed Scopus (231) Google Scholar). The structure provided the precise substrate-binding mode, in which conserved Thr residues recognize the substrate (14Lincecum T.L. Tukalo M. Yaremchuk A. Mursinna R.S. Williams A.M. Sproat B.S. Van Den Eynde W. Link A. Van Calenbergh S. Grotli M. Martinis S.A. Cusack S. Mol. Cell. 2003; 11: 951-963Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). However, the LeuRS crystal structures revealed that both the pre- and post-transfer editing analogues are bound in one site in the CP1 domain, although a mutational analysis showed that distinct residues are needed between the pre- and post-transfer editing reactions in IleRS (16Hendrickson T.L. Nomanbhoy T.K. de Crecy-Lagard V. Fukai S. Nureki O. Yokoyama S. Schimmel P. Mol. Cell. 2002; 9: 353-362Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Therefore, the substrate-binding modes may differ between IleRS and LeuRS. In the present study, to study specifically the post-transfer editing mechanism of IleRS, we isolated the T. thermophilus IleRS CP1 domain (201-384 amino acids), which contains the post-transfer editing site, but lacks a part of the pre-transfer editing site, based on the previous model (12Fukai S. Nureki O. Sekine S. Shimada A. Tao J. Vassylyev D.G. Yokoyama S. Cell. 2000; 103: 793-803Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 16Hendrickson T.L. Nomanbhoy T.K. de Crecy-Lagard V. Fukai S. Nureki O. Yokoyama S. Schimmel P. Mol. Cell. 2002; 9: 353-362Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). We determined its crystal structure at 1.8-Å resolution. Then, we determined its cocrystal structure with valine at 2.0-Å resolution. In the complex structure, the valine is located in the same pocket as in the case of the modeled Val-tRNAIle (12Fukai S. Nureki O. Sekine S. Shimada A. Tao J. Vassylyev D.G. Yokoyama S. Cell. 2000; 103: 793-803Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 16Hendrickson T.L. Nomanbhoy T.K. de Crecy-Lagard V. Fukai S. Nureki O. Yokoyama S. Schimmel P. Mol. Cell. 2002; 9: 353-362Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar); however, its orientation within the pocket is quite different. This new valine-binding mode can explain the mutational data well, unlike the previous model. Furthermore, we examined the post-transfer editing activity of some mutant IleRSs. Based on the high resolution structural data and these and other mutational data, we created a new structural model of the Val-adenosine moiety of Val-tRNAIle in the editing state. In this model, the substrate recognition differs from that in the LeuRS system. Protein Preparation—The gene fragment encoding the T. thermophilus IleRS CP1 domain (201-384 amino acids with the initiating methionine) was subcloned into pET26b (Novagen). The construct was designed to have only the post-transfer editing site, but not the pre-transfer editing site, based on the previous model (12Fukai S. Nureki O. Sekine S. Shimada A. Tao J. Vassylyev D.G. Yokoyama S. Cell. 2000; 103: 793-803Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 16Hendrickson T.L. Nomanbhoy T.K. de Crecy-Lagard V. Fukai S. Nureki O. Yokoyama S. Schimmel P. Mol. Cell. 2002; 9: 353-362Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), to study specifically the post-transfer editing mechanism. The plasmid was transformed into E. coli strain JM109(DE3). For protein overexpression, the cells were grown to an OD600 of 0.8, and the expression was induced with 1 mm isopropyl-β-d-thiogalactopyranoside for 4 h. The cells were harvested and sonicated in 50 mm K2HPO4 buffer (pH 6.0) containing 5 mm MgCl2, 10 mm dithiothreitol, and 1 mm phenylmethylsulfonyl fluoride. The insoluble cell debris was removed by centrifugation at 15,000 × g for 30 min at 4 °C. The supernatant was heat-treated at 70 °C for 30 min to denature the E. coli proteins. The heat-treated mixture was centrifuged at 15,000 × g for 1 h at 4 °C. Then, 2.4 m (NH4)2SO4 was added to the supernatant to a final concentration of 0.8 m. The mixture was applied to a 50 ml column of butyl-Toyopearl (Tosoh) equilibrated with 50 mm K2HPO4 buffer (pH 6.0) containing 0.8 m (NH4)2SO4, 5 mm MgCl2, and 1 mm dithiothreitol. The CP1 domain was eluted with a linear gradient of 0.8-0 M (NH4)2SO4. The fractions containing the CP1 domain were pooled, dialyzed against 50 mm Tris-HCl buffer (pH 7.5) containing 5 mm MgCl2 and 1 mm dithiothreitol, and loaded onto a Mono Q column (Amersham Biosciences) using an Amersham fast protein liquid chromatography system. The enzyme was eluted with a linear gradient of 0-1 m NaCl. The CP1 domain protein was further purified on a UnoQ column (Bio-Rad) using an fast protein liquid chromatography system eluted with a linear gradient of 0-1 M NaCl and was dialyzed against 10 mm Tris-HCl buffer (pH 8.0) containing 5 mm MgCl2 and 5 mm β-mercaptoethanol. The final purity of the protein was monitored by SDS-PAGE. Crystallization and Data Collection—For crystallization, the hanging drop vapor diffusion method was used, by mixing 1 μl of the protein solution (10 mg·ml-1) with 1 μl of the reservoir solution (50 mm Hepes-NaOH buffer (pH 7.0) containing 2.0 m (NH4)2SO4 and 5% 2-propanol) and by equilibrating this mixture against 500 μl of the reservoir solutions at 20 °C. Crystals (space group P41212; unit-cell parameters a = b = 102.7 Å, c = 83.8 Å) were grown for 3 days to dimensions of ∼0.3 × 0.3 × 0.3 mm3. To obtain cocrystals of the CP1 domain complexed with valine, reservoir solutions containing 100 mm valine were used, and the crystals thus obtained were transferred to a solution containing 55 mm Hepes-NaOH buffer (pH 7.0), 2.2 m (NH4)2SO4, 5.5% 2-propanol, and 100 mm valine, 24 h before the data collection. The diffraction datasets of the ligand-free crystal were collected at beamline BL41XU at SPring-8 (Harima) to 1.8-Å resolution, and those of the complex crystal were collected at beamline BL44XU at SPring-8 to 2.0 Å resolution. A single crystal, flash-frozen at a temperature of -173 °C, was used for each experiment. Before flash-cooling, the crystals were transferred into a cryoprotective solution containing 20% (v/v) ethylene glycol. The ligand-free data sets were processed using the HKL2000 program (17Otwinowski Z. Minor W. Methods Enzymology. 1997; 276: 307-326Crossref Scopus (38617) Google Scholar), and the complex data sets were processed using MOSFLM/SCALA (18Leslie A.G. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1696-1702Crossref PubMed Scopus (485) Google Scholar). Data collection statistics are summarized in Table I.Table IData collection and model refinement statisticsData setCP1 domainCP1 domain + valineData collectionSpace groupP41212P41212Unit-cell parameters (Å)a = 102.73a = 102.27b = 102.73b = 102.27c = 83.83c = 84.03Resolution range (Å)50.0−1.850.0−2.0Unique reflections4148930184Redundancy15.88.9Completeness (%) (last shell)99.1 (98.2)98.8 (99.2)I/σ (last shell)45.3 (3.7)7.1 (2.6)Rsym (%) (last shell)8.1 (28.2)4.7 (28.5)Structure refinementNo. of reflections: working set/test set39682/208328646/1492No. of atoms28062843No. of water molecules251166R factor (%): working set/test set20.3/25.020.3/24.8Average B factor (Å2)40.248.4Root mean square bonds (Å)0.0200.011Root mean square angles (°)1.981.65 Open table in a new tab Structure Determination and Refinement—We carried out molecular replacement with MOLREP (19Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 39: 1022-1025Crossref Scopus (4175) Google Scholar), starting with the coordinates of the CP1 domain from the full-length T. thermophilus IleRS, which we previously determined at 2.5-Å resolution (Protein Data Bank ID, 1ILE) (10Nureki O. Vassylyev D.G. Tateno M. Shimada A. Nakama T. Fukai S. Konno M. Hendrickson T.L. Schimmel P. Yokoyama S. Science. 1998; 280: 578-582Crossref PubMed Scopus (316) Google Scholar). The rotated and translated model was first subjected to rigid-body refinement using 20-2.5 Å data sets collected from the ligand-free crystal. The model refinement was carried out using the program CNS (20Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar). After several rounds of Cartesian coordinate energy minimization, simulated annealing, B factor refinement, automatic water picking, and manual revision of the model, using the program O (21Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard Acta Crystallogr. Sect. D Biol. Crystallogr. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar), R and Rfree decreased to 20.3% and 25.0%, respectively. A random sample containing 5% of the total reflections in the data sets was excluded from the refinement, to calculate Rfree. Using the data sets of the complex crystals, molecular replacement was carried out with the coordinates of the ligand-free CP1 domain structure. The structure refinement was performed in the same way. After building the valine ligand, further rounds of refinement were performed. The model refinement of the complex structure finally converged to an R value of 20.3% and an Rfree of 24.8%, with good stereochemistry. Ramachandran plot analysis using the program PROCHECK (22Laskowski R.A. MacArthur M.W. L. M.A. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 28-291Crossref Google Scholar) showed that all of the residues in both the ligand-free and complex structures, except for one residue in each structure, were in the most favored or additionally allowed regions. Omit maps for the valine and surrounding water molecules were calculated at the end of the refinement. Post-transfer Editing Assay of Mutant IleRSs—The full-length T. thermophilus IleRS gene was cloned into pET28c (Novagen). This plasmid encodes IleRS with a His tag on its C-terminal end. The mutations, T228A, T229A, T230A, T233A, T228A/T230A, T229/T230A, T230A/T233A, and D328A, were introduced using a QuikChange mutagenesis kit (Stratagene). Plasmids were transformed into E. coli strain BL21(DE3). Cells were cultured and harvested by the same procedure used for the CP1 domain. Cells were sonicated in 30 mm Tris-HCl buffer (pH 7.5) containing 500 mm NaCl, 5 mm MgCl2, 10 mm imidazole, 5 mm β-mercaptoethanol, and 1 mm phenylmethylsulfonyl fluoride (buffer A). The insoluble cell debris and the E. coli proteins were removed by the same procedure used for the CP1 domain. The supernatant was applied to a 1-ml HiTrap chelating column (Amersham Biosciences) chelated with Ni2+ ions and equilibrated with buffer A. After the column was washed with buffer A, the mutant IleRSs were eluted with 30 mm Tris-HCl buffer (pH 7.5) containing 500 mm NaCl, 5 mm MgCl2, 500 mm imidazole, and 5 mm β-mercaptoethanol and were dialyzed against 150 mm Tris-HCl buffer (pH 7.5) containing 150 mm KCl and 10 mm MgCl2. The final purity of the proteins was monitored by SDS-PAGE. [14C]Val-tRNAIle was prepared as described previously (23Giege R. Kern D. Ebel J.P. Grosjean H. de Henau S. Chantrenne H. Eur. J. Biochem. 1974; 45: 351-362Crossref PubMed Scopus (67) Google Scholar, 24Schmidt E. Schimmel P. Science. 1994; 264: 265-267Crossref PubMed Scopus (138) Google Scholar). Deacylation assays were performed at 37 °C in 150 mm Tris-HCl buffer (pH 7.5) containing 10 mm MgCl2, 150 mm KCl, [14C]Val-tRNAIle (1.4 μm, 1000 cpm/pmol), and each mutant IleRS (20 nm). Aliquots were removed at specific time points and quenched on filter papers (Whatman, 3 mm) equilibrated with 10% trichloroacetic acid. The filters were washed 3 times with 5% trichloroacetic acid and once with 100% ethanol. The radioactivities of the precipitates were quantitated by scintillation counting. Overall Structure—We determined the high resolution crystal structure of the isolated T. thermophilus IleRS CP1 domain, containing the post-transfer editing site, at 1.8 Å resolution (Protein Data Bank ID, 1UDZ). The root-mean-square deviation between the isolated CP1 domain and the CP1 domain in the full-length IleRS over all Cα atoms is 0.91 Å, which is small enough to show that these two structures are quite similar to each other. Indeed, the secondary structures and the locations of the individual residues are almost the same, and therefore, the structure of the CP1 domain in this study reflects that of the full-length IleRS. Next, we determined the cocrystal structure of the CP1 domain complexed with valine at 2.0-Å resolution (Protein Data Bank ID, 1UE0) (Fig. 1A). Its overall structure is also quite similar to that of the CP1 domain in the full-length IleRS (Fig. 1B), and thus, no large conformational change occurs upon the valine binding. Valine-binding Mode—The editing active site is formed mainly by one β-strand (β8) and two almost parallel α-helices (α1 and α5). In the Fo - Fc omit map of the complex structure, there is a strong and clear electron density that could be attributed to the valine molecule in the editing active site (Fig. 2A), and there are some electron densities that could be attributed to water molecules around the valine. The high resolution complex structure revealed the precise valine recognition mechanism (Fig. 2, B and C, and Fig. 4A). The valine NH3+ hydrogen bonds to the Oδ of Asp328 and the α-CO of His319, and the valine COO- group hydrogen bonds to the Oγ atoms of Thr229 and Thr230, and the α-CO groups of Trp227 and Phe324, through water molecules. The valine side chain is surrounded by the side chains of Thr233, His319, Ala321, and Phe324, and the α-CO of Phe324. All of these residues are highly conserved in the IleRSs from other species (Fig. 6A). We previously proposed a valine-binding mode in the post-transfer editing state, which was modeled on the basis of the electron density corresponding to valine (although it was not clear enough to locate it unambiguously) in the full-length IleRS structure, and the 3′-terminal adenosine of tRNAVal in the T. thermophilus ValRS· tRNAVal cocrystal structure (12Fukai S. Nureki O. Sekine S. Shimada A. Tao J. Vassylyev D.G. Yokoyama S. Cell. 2000; 103: 793-803Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). In the present structure, the valine is located in the same pocket as in the case of the modeled Val-tRNAIle, but its orientation within the pocket is different from that in the model (Fig. 3).Fig. 4The valine-binding pocket is just as large as valine.A, the contact surface representation of the nearly cognate valine and the amino acid binding pocket of the IleRS CP1 domain (stereo view). Valine just fits in the pocket. B, the contact surface representation of the cognate isoleucine model and the amino acid binding pocket of the IleRS CP1 domain. An additional methylene group clashes with the side chains of His319.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6Comparison of the binding modes of the nearly cognate amino acid between IleRS and LeuRS and structural model of the Val-adenosine moiety of Val-tRNAIle in IleRS.A, sequence alignment of conserved segments of the CP1 domain. The residues that affected the post-transfer editing activity of IleRS, when substituted by Ala, are shown with thick arrows, and the residues that did not are shown with thin arrows. B, structural comparison between the IleRS CP1 domain complexed with valine, determined in this study (shown in gray), and LeuRS complexed with a post-transfer editing analogue (2′-(l-norvalyl)amino-2′-deoxyadenosine) (shown in red) (stereo view). The residue labeling conforms to that of T. thermophilus IleRS. C, structural model of the 2′-OH valylated adenosine moiety of Val-tRNAIle (stereo view). The CP1 domain is shown in gray. The structural model of the substrate was built without moving the valine in the determined structure (yellow). The two water molecules are located in the same places as in the determined structure (light blue). Ionic bonds and hydrogen bonds are shown by dashed yellow lines. The water molecule that hydrogen bonds to Asp328 is favorably located to act as a nucleophile in the ester bond hydrolysis reaction. In addition, the adenosine ribose 3′-OH group is favorably located to act as a nucleophile in the transacylation reaction.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3Comparison of the valine orientations in this and previous studies (stereo view). The valine and the CP1 domain of this study are shown in gray, and those in the full-length IleRS are shown in black. The valine locations and the conformations of the valine-binding sites of these two CP1 domain structures are almost the same. However, the valine molecules are differently oriented.View Large Image Figure ViewerDownload Hi-res image Download (PPT) This valine-binding mode can nicely explain the mutational data of the E. coli H319A mutant IleRS, which the previous model could not. The H319A mutant hydrolyzes not only the nearly cognate Val-tRNAIle but also the cognate Ile-tRNAIle (16Hendrickson T.L. Nomanbhoy T.K. de Crecy-Lagard V. Fukai S. Nureki O. Yokoyama S. Schimmel P. Mol. Cell. 2002; 9: 353-362Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). In the present structure, valine fits well in the editing active site (the distance between the Nϵ2 of His319 and the Cγ of valine is 3.5 Å) (Fig. 4A), but when the isoleucine model is placed in the same site, the additional methylene group clashes with the side chain of His319 (the distance between the Nϵ2 of His319 and the Cδ of isoleucine would be 2.1 Å) (Fig. 4B). If this His319 residue was mutated to Ala, then the distances between the Ala Cβ and the valine Cγ or the isoleucine Cδ would be 5.4 or 4.7 Å, respectively. These data support the idea that the location of the valine in the determined structure represents the post-transfer editing state, while the previous model for the valine-binding mode could not explain this mutational analysis. Therefore, we concluded that the valine-binding mode in the present structure precisely reflects that of the valyl moiety of Val-tRNAIle in the editing state. Post-transfer Editing Assay of Mutant IleRSs—Previous biochemical experiments using E. coli IleRS showed that the T242A (corresponding to Thr229 in T. thermophilus IleRS) mutant is defective in the total editing activity (whether it affects post- or pre-transfer editing or both is unknown), while the T241A, T243A, and T246A E. coli IleRS mutants (Thr228, Thr230, and Thr233 in T. thermophilus IleRS, respectively) can still perform the total editing activity (10Nureki O. Vassylyev D.G. Tateno M. Shimada A. Nakama T. Fukai S. Konno M. Hendrickson T.L. Schimmel P. Yokoyama S. Science. 1998; 280: 578-582Crossref PubMed Scopus (316) Google Scholar, 25Hendrickson T.L. Nomanbhoy T.K. Schimmel P. Biochemistry. 2000; 39: 8180-8186Crossref PubMed Scopus (45) Google Scholar). In addition, the D342A E. coli IleRS mutant (Asp328 in T. thermophilus IleRS) is defective in the post-transfer editing activity (26Bishop A.C. Nomanbhoy T.K. Schimmel P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 585-590Crossref PubMed Scopus (51) Google Scholar). Thus far, all of the mutant analyses have been carried out using E. coli IleRS, with most examining only the aspect of total editing and not distinguishing between pre- and post-transfer editing. For a more precise understanding of the post-transfer editing reaction, we analyzed the post-transfer editing activity of Ala-replaced mutants of T. thermophilus IleRS. As a result, the T228A, T229A, T228A/T230A, T229A/T230A, and D328A mutants had some defects, while T230A, T233A, and T230A/T233A retained the full activity (Fig. 5). These data show that Thr228, Thr229, Thr230 (detectable only when Thr228 is also mutated to Ala), and Asp328 (Thr241, Thr242, Thr243, and Asp342, in E. coli IleRS, respectively) play some role in the post-transfer editing reaction. Previous results using E. coli IleRS showed that Thr228 and Thr230 were not critical for the total editing (10Nureki O. Vassylyev D.G. Tateno M. Shimada A. Nakama T. Fukai S. Konno M. Hendrickson T.L. Schimmel P. Yokoyama S. Science. 1998; 280: 578-582Crossref PubMed Scopus (316) Google Scholar, 25Hendrickson T.L. Nomanbhoy T.K. Schimmel P. Biochemistry. 2000; 39: 8180-8186Crossref PubMed Scopus (45) Google Scholar). The Ala-replacing mutation of Thr230 leads to the defect only when Thr228 is also mutated to Ala, whereas the previous analysis examined only the single mutation of Thr230. The T228A defect is not as severe in the post-transfer editing, and in IleRS, the post-transfer editing was estimated to account for only 25% of the overall editing, while the remaining 75% was attributed to the pre-transfer editing (7Fersht A.R. Biochemistry. 1977; 16: 1025-1030Crossref PubMed Scopus (202) Google Scholar). By assuming that the T228A mutant is only defective in the post-transfer editing, but not in the pre-transfer editing, we can account for the previous mutational analysis by not being able to detect the slight defect of the T228A mutant in the total editing. The more severe defect of the T228A/T230A double mutant than that of the T228A single mutant also shows the importance of the Thr228 residue in the post-transfer editing (Fig. 5). A Model for the Editing Mechanism—We tried to build a structural model for the Val-adenosine moiety of Val-tRNAIle. First, we superposed T. thermophilus ValRS complexed with tRNAval onto the T. thermophilus IleRS CP1 domain. However, the 3′-terminal adenosine of tRNAval and valine could not be ester bonded, even with a slight adjustment. The distance between the OH group of the tRNAval adenosine ribose and the valine COO- group was too far (data not shown). Then, we superposed T. thermophilus LeuRS complexed with a post-transfer editing substrate analogue onto the T. thermophilus IleRS CP1 domain (Fig. 6B). This time, although there were some differences in the editing site architecture, especially in the adenine base recognition region and the conserved threonine-rich region, the distance between the OH group of the adenosine ribose and the valine COO- group was close enough for linkage with a slight adjustment. We translated the adenosine to allow the ribose 2′-OH group to form an ester bond with the valine COO- group, and rotated it around the 2′-C-O bond. We also rotated the χ angle of the adenine base to adjust for the differences between the adenine base recognition regions of IleRS and LeuRS (Fig. 6A). Finally, we could build a fine model for the Val-adenosine moiety of Val-tRNAIle, without moving any atom of the valine molecule in the determined structure (Fig. 6C). This modeled substrate is favorably hydrogen bonded by the editing active site residues and does not clash against any residue. We compared the substrate-binding mode of IleRS with that of LeuRS (Fig. 6B). The locations of the amino acid NH3+ groups and the Asp residues (Asp328 and Asp347 in T. thermophilus IleRS and LeuRS, respectively) are quite similar, and the amino acid NH3+ groups are recognized by the Asp residues in a similar way. These Asp residues are completely conserved among IleRSs, LeuRSs, and ValRSs (Fig. 6A) and are critical in the IleRS post-transfer editing reactions (Fig. 5 and Ref. 23Giege R. Kern D. Ebel J.P. Grosjean H. de Henau S. Chantrenne H. Eur. J. Biochem. 1974; 45: 351-362Crossref PubMed Scopus (67) Google Scholar) and in the LeuRS total editing reactions (25Hendrickson T.L. Nomanbhoy T.K. Schimmel P. Biochemistry. 2000; 39: 8180-8186Crossref PubMed Scopus (45) Google Scholar). Therefore, the interaction between the amino acid NH3+ group and the Asp residue must be vital for the editing reaction in both IleRSs and LeuRSs and maybe also in ValRSs. However, the orientations of the ester bond in the editing substrates are quite different between IleRS and LeuRS, and the threonine-rich region is closer to the substrate in LeuRS than in IleRS (Fig. 6B). In LeuRS, the amino acid carbonyl group is oriented toward the threonine-rich region and is recognized by Thr247 (Thr228 in IleRS) (Fig. 6B). In contrast, in IleRS, it is located opposite to the threonine-rich region and is recognized by the α-CO of His319 through the water molecule, not by the conserved threonine residue (Fig. 6C). In IleRS, only Thr229 hydrogen bonds to the substrate 5′-O, unlike the other conserved thereonine residues (Fig. 6C). These findings can account for the mutational data that Thr229 plays some role in the post-transfer editing, but Thr233 does not (Fig. 5). Furthermore, the water molecule, which hydrogen bonds to Asp328 and the NH3+ group of the substrate, is favorably located to act as the catalytic nucleophile for the ester bond hydrolysis (Fig. 6C). Conversely, in the determined valine-bound structure, Thr230 hydrogen bonds to the valine COO- group through the water molecule (Fig. 2, A and B), but the water molecule clashes with the ribose 3′-OH of the modeled substrate, and cannot exist in the same place in the model (data not shown). We will discuss the importance of Thr228 and Thr230 below. In the aminoacylation reaction by IleRS, valine is first mischarged to the 2′-OH of the tRNA 3′-terminal adenosine ribose (13Nordin B.E. Schimmel P. J. Biol. Chem. 2002; 277: 20510-20517Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Under normal conditions, valine is transacylated to the 3′-OH from the 2′-OH, and vice versa, and stays in equilibrium. Until now, we have discussed the 2′-OH valylated tRNAIle model. Biochemical analyses using aminoacyl-tRNAs with a deoxygenized 2′-OH or 3′-OH of the 3′-terminal adenosine ribose, which the amino acid cannot transacylate, suggested that IleRS specifically deacylates valine from the 3′-OH but not from the 2′-OH, whereas ValRS can deacylate the mischarged threonine from the 2′-OH (13Nordin B.E. Schimmel P. J. Biol. Chem. 2002; 277: 20510-20517Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Considering these data, we built the binding mode model of another editing substrate, the 3′-OH valylated adenosine moiety of Val-tRNAIle (Fig. 7). Unlike the case of the 2′-OH valylated model, to make the 3′-OH valylated model, we moved the valyl moiety from the observed location in the determined valine-bound structure. In the 3′-OH valylated model, the recognition modes of the NH3+ group and the adenine base are similar to those in the 2′-OH valylated model (Figs. 6C and 7). In the 3′-OH valylated model, Thr229 hydrogen bonds directly to the adenosine 5′-O, as in the 2′-OH valylated model, and Thr228 and Thr230 hydrogen bond to the adenosine 3′-O through the water molecule (Fig. 7), as observed in the determined valine-bound structure (Fig. 2, A and B). This water molecule, which hydrogen bonds to Thr228 and Thr230, is favorably located to act as a catalytic nucleophile for the ester bond hydrolysis (Fig. 7). In this model, Thr228 and Thr230 participate in the substrate recognition, in contrast to the 2′-OH valylated model, thus confirming the mutational analysis that revealed the importance of these residues in the editing reaction. Although Thr230 is highly conserved in IleRSs, it is replaced with the highly conserved Arg residue in LeuRSs and ValRSs (Fig. 6A). This is consistent with the data that the mischarged amino acid is deacylated from the 3′-OH only in IleRS (13Nordin B.E. Schimmel P. J. Biol. Chem. 2002; 277: 20510-20517Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) but is deacylated from the 2′-OH in ValRS (13Nordin B.E. Schimmel P. J. Biol. Chem. 2002; 277: 20510-20517Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) and maybe also in LeuRS (14Lincecum T.L. Tukalo M. Yaremchuk A. Mursinna R.S. Williams A.M. Sproat B.S. Van Den Eynde W. Link A. Van Calenbergh S. Grotli M. Martinis S.A. Cusack S. Mol. Cell. 2003; 11: 951-963Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Moreover, these two binding-mode models are not mutually exclusive. Instead, both binding mechanisms might be used. That is, first the 2′-OH valylated tRNAIle is bound to the editing site, and after the valyl moiety is transferred to the 3′-OH from the 2′-OH, it is deacylated from the 3′-OH. In the 2′-OH valylated model, the 3′-OH is favorably located to act as a catalytic nucleophile for the transacylation reaction (Fig. 6C). Concluding Remarks—The high resolution crystal structure of the T. thermophilus IleRS CP1 domain complexed with the nearly cognate valine provides a precise understanding of the selective valine recognition mechanism. In addition, we were able to build the binding mode models of the two substrates, the 2′-OH valylated and the 3′-OH valylated adenosine moieties of Val-tRNAIle. These structure models illustrate the mutational data well and suggest a specific editing mechanism for IleRS, which is distinct from those of LeuRS and ValRS. Actually, only in IleRS, the pre-transfer editing is predominant (75% of the total editing) (7Fersht A.R. Biochemistry. 1977; 16: 1025-1030Crossref PubMed Scopus (202) Google Scholar). However, we still need to determine whether the active site accommodates 2′-OH or 3′-OH valylated tRNAIle or both and to elucidate the precise binding mode. Moreover, the binding mode of the pre-transfer editing substrate is still unknown in IleRS, although in LeuRS, both substrates are recognized in the same binding site in similar manners (14Lincecum T.L. Tukalo M. Yaremchuk A. Mursinna R.S. Williams A.M. Sproat B.S. Van Den Eynde W. Link A. Van Calenbergh S. Grotli M. Martinis S.A. Cusack S. Mol. Cell. 2003; 11: 951-963Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). This structural study has provided significant clues toward elucidating the editing mechanism of IleRS. For a more precise understanding of both pre- and post-transfer editing, the determination of the IleRS structures complexed with nonhydrolyzable analogs of pre- and post-transfer editing substrates is required. We thank Dr. M. Kawamoto and Dr. H. Sakai (JASRI) and Dr. A. Nakagawa and Dr. E. Yamashita (Osaka University) for their help in data collection at the SPring-8 beamlines BL41XU and BL44XU, respectively. We are grateful to Dr. S. Cusack (EBI) for insightful discussions.

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