Tài liệu Báo cáo khoa học: Modeling of tRNA-assisted mechanism of Arg activation based on a structure of Arg-tRNA synthetase, tRNA, and an ATP analog (ANP) ppt

Modeling of tRNA-assisted mechanism of Arg activation based on a structure of Arg-tRNA synthetase, tRNA, and an ATP analog (ANP) Michiko Konno 1 , Tomomi Sumida 1, *, Emiko Uchikawa 1 , Yukie Mori 1 , Tatsuo Yanagisawa 2, *, Shun-ichi Sekine 2 and Shigeuki Yokoyama 2 1 Department of Chemistry and Biochemistry, Graduate School of Humanities and Sciences, Ochanomizu University, Tokyo, Japan 2 Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Japan Introduction Most aminoacyl-tRNA synthetases (aaRSs) catalyze the formation of aminoacyl-AMP in the presence of Mg 2+ [amino acid + ATP fi aminoacyl-AMP + pyrophosphate (PP i )] and the reverse reaction (amino- acyl-AMP + PP i fi amino acid + ATP). Thus, the amino acid is converted into the reactive intermediate, Keywords aminoacyl-AMP formation; Arg-tRNA synthetase; deacylation reaction; pyrophosphorolysis; tRNA Correspondence M. Konno, Department of Chemistry and Biochemistry, Graduate School of Humanities and Sciences, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-Ku, Tokyo 112-8610, Japan Fax: +81 359785717 Tel: +81 359875718 E-mail: konno.michiko@ocha.ac.jp *Present address RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan Database The atomic coordinates and the structure factors have been deposited in the Protein Data Bank (ID 2ZUE for the ternary complex of ArgRS, tRNA Arg CCU and ANP, and ID 2ZUF for the binary complex of ArgRS and tRNA Arg CCU ) (Received 18 March 2009, revisied 11 June 2009, accepted 26 June 2009) doi:10.1111/j.1742-4658.2009.07178.x The ATP–pyrophosphate exchange reaction catalyzed by Arg-tRNA, Gln- tRNA and Glu-tRNA synthetases requires the assistance of the cognate tRNA. tRNA also assists Arg-tRNA synthetase in catalyzing the pyro- phosphorolysis of synthetic Arg-AMP at low pH. The mechanism by which the 3¢-end A76, and in particular its hydroxyl group, of the cognate tRNA is involved with the exchange reaction catalyzed by those enzymes has yet to be established. We determined a crystal structure of a complex of Arg- tRNA synthetase from Pyrococcus horikoshii, tRNA Arg CCU and an ATP analog with R factor = 0.213 ( R free = 0.253) at 2.0 A ˚ resolution. On the basis of newly obtained structural information about the position of ATP bound on the enzyme, we constructed a structural model for a mechanism in which the formation of a hydrogen bond between the 2¢-OH group of A76 of tRNA and the carboxyl group of Arg induces both formation of Arg-AMP (Arg + ATP fi Arg-AMP + pyrophosphate) and pyrophos- phorolysis of Arg-AMP (Arg-AMP + pyrophosphate fi Arg + ATP) at low pH. Furthermore, we obtained a structural model of the molecular mechanism for the Arg-tRNA synthetase-catalyzed deacylation of Arg- tRNA (Arg-tRNA + AMP fi Arg-AMP + tRNA at high pH), in which the deacylation of aminoacyl-tRNA bound on Arg-tRNA synthetase and Glu-tRNA synthetase is catalyzed by a quite similar mechanism, whereby the proton-donating group (–NH–C + (NH 2 ) 2 or –COOH) of Arg and Glu assists the aminoacyl transfer from the 2¢-OH group of tRNA to the phos- phate group of AMP at high pH. Abbreviations aaRS, aminoacyl-tRNA synthetase; ANP, adenosine-5¢-(b,c-imido)triphosphate; ArgRS, Arg-tRNA synthetase; D, dihydrouridine; GlnRS, Gln-tRNA synthetase; GluRS, Glu-tRNA synthetase; PP i , pyrophosphate. FEBS Journal 276 (2009) 4763–4779 ª 2009 The Authors Journal compilation ª 2009 FEBS 4763 i.e. aminoacyl-AMP, and then the aminoacyl-AMP bound on the aaRS may react with the hydroxyl group of the ribose at the 3¢-end CCA of tRNA to form ami- noacyl-tRNA. In the aminoacylation reaction of class I aaRSs with aminoacyl-AMP, the 2¢-OH group of the ribose of A76 tRNA attacks the carbonyl carbon atom of the –Ca–(CO)–O– moiety of aminoacyl-AMP. As lit- tle detailed structural information on the ATP-binding site of class Ia and class Ib aaRSs has been reported, clear molecular-scientific understanding of the activated complex formed between the amino acid and ATP in the reaction path of aminoacyl-AMP formation on the aaRSs has not been attained yet. In particular, the following detailed biochemical findings on the formation of aminoacyl-AMP and clo- sely related reactions have been reported for Arg-tRNA synthetase (ArgRS; EC 6.1.1.19) [1–6]. No success of consistent understanding has been achieved for the molecular reaction paths of aminoacyl-AMP formation and closely related reactions catalyzed by ArgRS. New models of the reaction paths involved in the formation of aminoacyl-AMP and closely related reactions observed for ArgRS will lead to improved understanding of the activated complex formed between the amino acid and ATP in the reaction path of aminoacyl-AMP formation on most aaRSs as well as ArgRS. For most aaRSs, the formation of aminoacyl-AMP does not require tRNA. On the other hand, for ArgRS from Escherichia coli, Bacillus stearothermophilus, Neu- rospora crassa, and Saccharomyces cerevisiae [1–5], Gln-tRNA synthetase (GlnRS) from E. coli W, S. cerevisiae and porcine liver [7,8] and Glu-tRNA syn- thetase (GluRS) from E. coli K12 [9,10], the ATP–PP i exchange reaction corresponding to the formation of aminoacyl-AMP and its reverse reaction, amino acid + ATP = aminoacyl-AMP + PP i , has never been observed without tRNA. In the presence of cog- nate tRNA, the ATP–PP i exchange reaction was observed for ArgRS, GlnRS, and GluRS. The cognate tRNA is also necessary for the ArgRS- catalyzed pyrophosphorolysis of chemically synthesized Arg-AMP in the presence of PP i and Mg 2+ [6]. It has also been reported that the in vitro ArgRS-catalyzed deacylation reaction of Arg-tRNA follows good first- order kinetics in solution at pH 6 containing excess amount of AMP, PP i , and Mg 2+ , whereas in the absence of PP i , the amount of Arg-tRNA decreases to 43% and then remains constant [11]. The detailed mechanism through which the 2¢-OH group of the ribose of the 3 ¢-end A76 of the cognate tRNA accelerates the ATP–PP i exchange reaction in the case of ArgRS remained unknown. In order to gain a clear molecular-scientific understanding of this mechanism and to clarify the orientation of the dihy- drouridine (D) loop containing A20 of tRNA Arg inter- acting with ArgRS, we determined crystal structures of a binary complex of Pyrococcus horikoshii ArgRS and tRNA Arg CCU and a ternary complex also containing the ATP analog adenosine-5¢-(b,c-imido)triphosphate (ANP); we found one reasonable mechanism, based on newly obtained structural information about the posi- tion of ATP bound on ArgRS. In order to understand the function of the N-terminal domain of ArgRS in relation to the binding mechanism of tRNA Arg ,we constructed an ArgRS mutant lacking the N-terminal domain (DN ArgRS). The experimental results showed that the DN ArgRS protein retains sufficient catalytic activity in the aminoacylation reaction for tRNA Arg CCU . Moreover, modeling of the relative posi- tions of Arg, A76 of tRNA Arg and ATP on ArgRS was undertaken to find the suitable position for the tRNA-assisted mechanism of Arg-AMP formation. We found that the formation of the hydrogen bond between the 2¢-OH group of A76 of tRNA and O2 of the carboxyl group induces the ATP–PP i exchange reaction and the pyrophosphorolysis reaction of syn- thetic Arg-AMP at low pH. Results Comparison of P. horikoshii ArgRS with those of Thermus thermophilus and S. cerevisiae The structures of the ternary complex (P. horikoshii ArgRS, tRNA Arg CCU , and the ATP analog ANP) and the binary complex (P. horikoshii ArgRS and tRNA Arg CCU ) were obtained with R factor = 0.213 (R free = 0.253) at 2.0 A ˚ resolution, and R factor = 0.201 (R free = 0.262) at 2.3 A ˚ , respectively. In the crystals grown in the presence of l-Arg, l-Arg was not visible in the electron density map. The overall structure of a ter- nary complex of P. horikoshii ArgRS, tRNA Arg CCU and the ATP analog is shown in Fig. 1, and sequence align- ments for ArgRSs from P. horikoshii , T. thermophilus and S. cerevisiae on the basis on three-dimensional structures are given in Fig. 2. Structures of S. cerevisiae ArgRS-bound arginine and tRNA Arg ICG [12] (Protein Data Bank ID: 1F7V) and ‘tRNA-free’ T. thermophilus ArgRS [13] (Protein Data Bank ID: 1IQ0), the Rossmann fold and the anticodon-binding domains of which were superim- posed onto those of P. horikoshii ArgRS, are shown in Fig. 3A,B. It has been reported that, in S. cerevisiae ArgRS, the Asn fi Ala mutation of Asn153, corre- sponding to Asn129 in P. horikoshii ArgRS (Fig. 2), Modeling of tRNA-assisted mechanism on ArgRS M. Konno et al. 4764 FEBS Journal 276 (2009) 4763–4779 ª 2009 The Authors Journal compilation ª 2009 FEBS gives a drastically decreased k cat value of 0.01 s )1 in the aminoacylation reaction in comparison with the k cat value of 8 s )1 for the wild-type ArgRS [14]. In S. cerevisiae ArgRS bound to Arg and tRNA, the a-NH 2 group of Arg is in close proximity to the car- bamoyl group of Asn153 in the loop between S5 and the signature sequence motif ‘HIGH’. In the three ArgRSs, an Asn with the same conformation was observed. The large difference found in the catalytic domain between P. horikoshii ArgRS and S. cerevisiae ArgRS concerns the relative orientations of the con- nective polypeptide domain and the inserted domain 1 to the Rossmann fold domain (Fig. 3A). In S. cerevisi- ae ArgRS, superimposition of ‘tRNA-free’ S. cerevisiae ArgRS on ‘tRNA-bound’ S. cerevisiae ArgRS reveals large movements of these domains [12]. Binding site of adenosine of ATP In Met-tRNA, Ile-tRNA, Val-tRNA and Leu-tRNA synthetases [15–18] belonging to class Ia, crystal struc- tures of complexes of aminoacyl-AMP analog bound mainly through the hydrophobic interaction of the aminoacyl moiety have been observed, whereas no ATP-bound protein of class Ia has been observed. In P. horikoshii ArgRS, the ATP analog (ANP) molecule was clearly found in the active site (Fig. 4). The obser- vation of the ANP-bound protein is due to the high hydrophobicity of ArgRS from the archaebacterium P. horikoshii living at very high temperature and the existence of the His417 residue. The adenine base of ANP with small values of average B-factor is stacked upon the aromatic ring of His417, which is specific to P. horikoshii ArgRS. The adenine base is in close prox- imity to the main chain of Val418 in the S16 strand, the N1–Val418 N and N6–Val418 O distances being 3.19 A ˚ and 3.47 A ˚ , respectively, and the 2¢-OH of the ribose is in close proximity to N of Gly384 and O e1 of Glu386 in the S14–H14 turn (Gly384–Ala385–Glu386– Gln387 turn), the distances being 2.71 A ˚ and 2.77 A ˚ , respectively. The distance between Ca of Glu386, the third residue in the S14–H14 turn, and Ca of Val418 in the S16 strand is 12.8 A ˚ , and the adenosine moiety is fitted into this hydrophobic groove. The Ala372– Ser373–Gln374–Gln375 turn in S. cerevisiae ArgRS and the Asp354–Val355–Arg356–Gln357 turn in T. thermophilus ArgRS have very similar backbone forms to that of the S14–H14 turn in P. horikoshii ArgRS. In aaRSs belonging to class I, the turn correspond- ing to the S14–H14 turn is almost conserved, as Gly ⁄ Ala-Xaa-Asp ⁄ Glu-Xaa (Xaa stands for any amino acid) and NH and C@O of the main chain of the resi- due corresponding to Val418 in the S16 strand are directed inside. In free E. coli Met-tRNA synthetase (Protein Data Bank ID: 1QQT) [19], the distance between Ca of the third residue, Asp296, in the S14– H14 turn and Ca of Val326 in the S16 strand is 12.7 A ˚ , in free T. thermophilus Ile-tRNA synthetase (Protein Data Bank ID: 1ILE) [20], the distance between the Ca atoms of the corresponding Asp553 and Ile584 is 13.4 A ˚ , in free P. horikoshii Leu-tRNA synthetase (Protein Data Bank ID: 1WKB) [21], the distance between Asp612 and Gly644 is 12.5 A ˚ , and in free T. thermophilus Val-tRNA synthetase (Protein Data Bank ID: 1IYW) [22], the distance between Asp490 and Val521 is 11.9 A ˚ . These distances within 0.9 A ˚ of the distance of 12.8 A ˚ in ArgRS indicate that, in these aaRSs, this space is the binding site of the adenosine moiety of ATP, and in E. coli Cys-tRNA synthetase bound to tRNA Cys (Protein Data Bank ID: 1U0B) [23], the distance between Asp229 and Val260 is 11.4 A ˚ . AMP weakly inhibits the binding of ATP in a competitive manner in the aminoacylation reaction [24]. The position of the Mg 2+ located between PbO (2.45 A ˚ and 3.01 A ˚ ) and PaO (2.97 A ˚ ) of ANP is not catalytic domain 'Stem contact fold' domain Anticodon-binding domain N-terminal domain ANP Fig. 1. Overview of the structure of P. horikoshii ArgRS complexed with tRNA Arg CCU and ATP analog (ANP). P. horikoshii ArgRS con- tains the N-terminal domain (residues 2–118; yellow), the catalytic domain [the Rossmann fold domain (residues 119–169, 238–269, 331–345, and 378–417; orange], the inserted domain 1 (residues 170–237; cyan), the connective polypeptide domain (residues 270– 330; blue), the inserted domain 2 (residues 346–377; green), the ‘stem contact fold’ domain (residues 418–503; red), and the antico- don-binding domain (residues 504–629; magenta). The tRNA back- bone is drawn with its phosphate chain traced as a thick green line, and ANP is shown in ball-and-stick representation. M. Konno et al. Modeling of tRNA-assisted mechanism on ArgRS FEBS Journal 276 (2009) 4763–4779 ª 2009 The Authors Journal compilation ª 2009 FEBS 4765 within 3.5 A ˚ of protein residues. The observed electron density for the Mg 2+ in this conformation is about half of the electron density expected for an occupancy of 1.0 for Mg 2+ . The presence of different orientations for the PbNPc moiety of ANP attached and not attached to Mg 2+ is manifested as low electron densi- ties in the regions of Mg 2+ and the PbNPc moiety. The salt bridge formed by Mg 2+ between PbO and PaO may retard the conformational inversion at Pa of ATP in the reversible process of the ATP–PP i exchange reaction, whereas the salt bridge formed by Mg 2+ between PbO and PcO does not, by any means, retard the conformational inversion at Pa of ATP. The reported aminoacyl-AMP analogs have sulfa- moyl (–NH–SO 2 –O–) or diaminosulfone (–NH–SO 2 – NH–) in place of Pa [–O–PO(OH)–O–] of AMP. Furthermore, the reported aminoacyl-AMP analogs are bound mainly through the interaction of the ami- noacyl moiety with the aminoacyl-tRNA synthetase. Therefore, the location of the adenosine moiety of the reported aminoacyl-AMP analogs may be somewhat perturbed by the strong binding of the aminoacyl moiety on the aminoacyl-tRNA synthetase. The con- formation of the sulfamoyl or diaminosulfone of the reported aminoacyl-AMP analogs may be also some- what perturbed by the strong binding of the aminoacyl moiety. For instance, the torsional angles of C3¢–C4¢– C5¢–O5¢⁄N¢ around the C4¢–C5¢ bond in ribose moie- ties of Ile-AMP analog [N-(isoleucinyl)-N¢-(adenosyl)- diaminosufone] (Protein Data Bank ID: 1JZQ) and Val-AMP analog [N-(valinyl)-N¢-(adenosyl)-diamino- sufone] (Protein Data Bank ID: 1GAX) are 52° and )169°, respectively. In contrast, the newly found location of the adeno- sine moiety of the ATP analog (ANP) is considered to be free from any perturbation. The conformation of Pa [–O–PO(OH)–O–] of ANP is also considered to be substantially free from any perturbation. Thus, the conformation of Pa [–O–PO(OH)–O–] of ANP is very suitable for use in constructing the model of the tRNA Arg -assisted ATP–PP i exchange reaction. The N of the side chain of Lys132, located three res- idues upstream from the signature sequence motif ‘HIGH’, is close to PaO, PbO and PcO of ANP, with Fig. 2. Sequence alignment of P. horikoshii ArgRS (PhRRS), T. thermophilus ArgRS (TtRRS) and S. cerevisiae ArgRS (ScRRS) on the basis of three-dimensional structures. The residues exposed on the surface of a-helices (colored in red) and b-strands (colored in blue) are aligned among the three ArgRSs. The Asn corresponding to Asn153 (ScRRS) is indicated by a green letter. Modeling of tRNA-assisted mechanism on ArgRS M. Konno et al. 4766 FEBS Journal 276 (2009) 4763–4779 ª 2009 The Authors Journal compilation ª 2009 FEBS distances of 2.67 A ˚ , 2.81 A ˚ , and 2.91 A ˚ , respectively. The fact that in P. horikoshii ArgRS, where the ‘KMSK’ motif is replaced by Lys424-Phe425-Ser426- Gly427, the first Lys424 of the current structure under- goes no interaction with the PbNPc moiety of ANP proves that, in P. horikoshii ArgRS, the ‘KFSG’ por- tion does not contribute to the ATP–PP i exchange reaction. The 3¢-terminus of tRNA In the 3¢-terminal G73–C74–C75–A76 sequence of tRNA Arg CCU , two transient forms were observed in the ternary complex (Fig. 5A) as well in the binary complex, depending on crystallization conditions; in the first stage, the base of G73 is stacked upon a G1ÆC72 base pair, and the conformation of the phos- phodiester bridge of C5¢–O–P–O–C3¢ between C72 and G73 is normal. This unusual structure was first observed by NMR analysis in the tRNA Ala acceptor end microhelix [25]. The C74–C75–A76 sequence is invisible in the electron density map, which indicates clearly that increased conformational flexibility around G73 is provided in the first stage. In another confor- mation of the 3¢-terminal end of tRNA Arg CCU in the second stage, the base of G73 is not stacked upon a G1ÆC72 base pair, and the conformation of the phos- phodiester bridge of C5¢–O–P–O–C3¢ between C72– G73 and G73–C74 is not of the normal helix type. This local conformation of C72–G73–C74 of the second stage is quite similar to the final stage confor- mation observed for tRNA Arg ICG bound to S. cerevisiae ArgRS in the tertiary complex [12]. The ribose of G73 and the bases of C75–A76 are invisible in the electron density map. Therefore, this newly observed transient form is the intermediate form, through which the con- formation of the 3¢-terminal end changes from the first stage to the final stage. The base of C74 is found near the surface of the connective polypeptide domain, which is a transient position, i.e. the hydrophobic cleft constructed by the side chains of Tyr300, Ala303, Val321, Arg324 and Ser325 in the connective polypep- tide domain. The relative orientation of G73 and C74 to the connective polypeptide domain is similar to that N-terminal domain Anticodon-binding domain 'Stem contact fold' domain Catalytic domain Inserted domain 1 Connective polypeptide domain Rossmann fold domain A B N-terminal domain Anticodon-binding domain 'Stem contact fold' domain Catalytic domain Inserted domain 1 Connective polypeptide domain Rossmann fold domain Fig. 3. (A) Comparison between two overall structures of P. horiko- shii ArgRS and S. cerevisiae ArgRS (white) bound to tRNA Arg ICG and arginine. The backbones of P. horikoshii tRNA and S. cerevisiae tRNA are drawn with the phosphate chain traced as a thick green line and a thick blue line, respectively. (B) Comparison between two overall structures of P. horikoshii ArgRS and ‘tRNA-free’ T. thermophilus ArgRS (white). V418 V418 H417 H417 Y415 Y415 E386 E386 G384 G384 H135 H138 K132 S127 N129 H138 H135 K132 N129 S127 Q837 Q837 Fig. 4. A final (2F obs ) F calc ) cross-validated r A -weighted omit map contoured at level 1.5r. The map was produced using the complex model without ANP and all the data from 40 A ˚ to 2.0 A ˚ resolution. A green sphere shows the Mg 2+ . M. Konno et al. Modeling of tRNA-assisted mechanism on ArgRS FEBS Journal 276 (2009) 4763–4779 ª 2009 The Authors Journal compilation ª 2009 FEBS 4767 observed in tRNA Arg ICG bound to S. cerevisiae ArgRS in the tertiary complex. It is predicted that the conformational change from the first stage to the final stage takes place in the absence of Arg, and the hydrophobic circumstance changes the hydration state around the phosphodiester bridges in C72–G73–C74– C75–A76. In T. thermophilus Val-tRNA synthetase bound to tRNA Val (Protein Data Bank ID: 1GAX) [17] and T. thermophilus Leu-tRNA synthetase bound to tRNA Leu (Protein Data Bank ID: 2BYT) [26], tRNA is left in the first stage, where the base of A73 is stacked on a G1ÆC72 base pair. Moreover, in Aqui- fex aeolicus Met-tRNA synthetase bound to tRNA Met (Protein Data Bank ID: 2CSX) [27], the base of A73 is still stacked on a G1ÆC72 base pair; that is, the change of the conformation of the 3¢-terminal end of tRNA Met does not progress. On the other hand, in E. coli Cys-tRNA synthetase bound to tRNA Cys (Protein Data Bank ID: 1U0B) [23], the structure such that the base of U73 is no longer stacked on a G1ÆC72 base pair allows the 3¢-terminal CCA end to enter into the active site. The D-loop of tRNA Arg Among all tRNA species specific to each of the 20 amino acids, only tRNA Arg isoacceptors have A at posi- tion 20 on the D-loop, with the exception that four tRNA Arg isoacceptors from S. cerevisiae have D or C. Detailed experiments with E. coli and T. thermophilus ArgRSs apparently suggested that the interaction with the middle base of the anticodon (C35) and A20 of tRNA Arg play an important role in tRNA Arg binding on ArgRS [13,28,29]. Crystal structures of binary and ternary complexes of ArgRS and tRNA Arg ICG from S. cerevisiae and Arg revealed that a base of D20 in the D-loop, which is specific to S. cerevisiae tRNA Arg ICG ,is positioned in close proximity to the side chains of Asn106, Phe109, and Gln111, which are included in the characteristic N-terminal domain of ArgRS [12]. A C B Fig. 5. The structure of tRNA Arg CCU on P. horikoshii ArgRS. (A) Two transient forms in the 3¢-terminal end of P. horikoshii tRNA Arg CCU . In the first stage (left side), the base of G73 is stacked upon a G1ÆC72 base pair, and C74–C75–A76 is invisible in the electron density map. In another conformation (right side), the base of G73 is not stacked, and the conformation of C72–G73–C74 is similar to that of the final stage of tRNA Arg ICU bound to S. cerevisiae ArgRS. (B) Packing arrangement of the bases of G19, A20 and C20 a of the D-loop of tRNA (green), and the side chains of Pro44, Phe47, Pro34, Leu38, Val82, Tyr85 and Asn87 in the N-terminal domain. (C) Packing arrangement of the bases of the anticodon loop (C32–U33–C34–C35–U36–A37–A38) of tRNA Arg CCU (green) and the anticodon-binding domain. Modeling of tRNA-assisted mechanism on ArgRS M. Konno et al. 4768 FEBS Journal 276 (2009) 4763–4779 ª 2009 The Authors Journal compilation ª 2009 FEBS However, it was reported that, in the aminoacylation reaction, the k cat and K m values for tRNA Arg ICG and tRNA Arg UCU on the Asn106 fi Ala, Phe109 fi Ala and Gln111 fi Ala mutant proteins of S. cerevisiae ArgRS are the same as those on the wild-type ArgRS [14]. This shows that the interaction between the N-ter- minal domain of S. cerevisiae ArgRS and D20 of the D-loop of tRNA Arg are not important for the binding of tRNA Arg on ArgRS in the aminoacylation reaction. The crystal structure of free ArgRS from T. thermophi- lus has been determined, but that of the complex with tRNA Arg has not been determined yet [13]. In T. ther- mophilus ArgRS, the Tyr77 fi Ala and Asn79 fi Ala mutants (Tyr77 and Asn79 correspond to Phe109 and Gln111 of S. cerevisiae ArgRS, on the basis of struc- tural comparison between S. cerevisiae ArgRS and T. thermophilus ArgRS) showed a notable increase in K m for tRNA Arg and a large decrease in V max in the am- inoacylation reaction at pH 7.5. On the other hand, it is very noticeable that the Asn79 fi Lys mutant, which is expected to be unable to form a hydrogen bond, has the same K m value for tRNA Arg as that of the wild type and does not affect the affinity of tRNA Arg . The additional N-terminal domain characteristic for ArgRS contains the core structure consisting of the b-sheet of four antiparallel b-strands and three helices on the N-terminal side and a long H4 helix and a loop continuing to the catalytic domain (Fig. 1). The core structure interacts weakly with the anticodon-binding domain. The hydrophobic interac- tions between the N-terminal domain and the bases of G19 and A20 in the complex of P. horikoshii ArgRS are shown in Fig. 5B. The bases of G18 and G19 of the D-loop form hydrogen bonds with the bases of U55 and C56 of the T-loop, respectively. The base of G19 interacts with the hydrophobic side chains of Pro44 and Phe47 in the N-terminal domain. The bases of A20 and C20 a (extra nucleo- tide inserted between nucleotides 20 and 21) of the D-loop are splayed out. The base of A20 is packed into the hydrophobic space surrounded by the side chains of Val82 and Tyr85 in the turn (Val82-Asn83- Gly84-Tyr85) between the S3 and S4 strands and the hydrophobic side chains of Pro34 and Leu38. N1 and N6 of the base of A20 lie close to N d2 and O d1 of the side chain of Asn87 in the S4 strand, with distances of 2.82 A ˚ and 2.97 A ˚ , respectively. The plane of the base of A20 and the end plane of the carbamoyl group of Asn87 are out of coplanar ori- entation, and the dihedral angle between these two planes was about 25°. In particular, O d1 of the car- bamoyl group of Asn87 is positioned far out of the base plane. Large values of average B-factor of resi- dues in the N-terminal domain (average B-factors of residues 2–118 in the N-terminal domain and resi- dues 119–629 in other domains are 49.9 A ˚ 2 and 29.5 A ˚ 2 , respectively) indicate that the D-loop does not have stable contact with the N-terminal domain. The relative orientation of the core structure of the N-terminal domain to the Rossmann fold domain and the anticodon-binding domain is substantially different among P. horikoshii ArgRS, S. cerevisiae ArgRS, and T. thermophilus ArgRS (Fig. 3A,B). In S. cerevisiae ArgRS, the base of D20 is surrounded by hydrophobic side chains of Phe109 in the turn (Asn106-Gly107- Pro108-Phe109) and Val70. O4 and N3 of D20 interact with N e2 of Gln111 and O d1 of Asn106, with distances of 2.75 A ˚ and 2.74 A ˚ , respectively [12]. The solution at pH 7.5 of crystallization drops from which crystals of the ternary complex of S. cerevisiae ArgRS, Arg and tRNA Arg ICG grow contains tRNA, l-Arg, ATP and Mg 2+ at sufficient concentrations for the aminoacyla- tion reaction, and (NH 4 ) 2 SO 4 and 1,6-hexanediol are used as precipitating agents [30]. This fact indicates that, even though all of the substrates required for the aminoacylation reaction are present at sufficient con- centration in the crystallization solution, the aminoacy- lation reaction does not occur during the long time needed for crystal growth, which suggests that tRNA- bound S. cerevisiae ArgRS observed in the ternary complex is by no means in a conformation that is fit to activate. The fact that k cat and K m for tRNA Arg in the aminoacylation reaction do not change in the Asn106 fi Ala, Gln111 fi Ala and Phe109 fi Ala mutants of S. cerevisiae ArgRS [14] indicates that those mutations have no influence on the orientation of tRNA Arg in wild-type ArgRS. When the Rossmann fold domain and the anticodon- binding domain in P. horikoshii ArgRS were superim- posed onto those of S. cerevisiae ArgRS, the C1s of A20 and C72 in P. horikoshii tRNA Arg were not within 4.0 A ˚ of those of D20 and A72 in S. cerevisiae tRNA Arg , respectively. The main chains of the phospho- diester bridge of C5¢–O5–P–O3–C3¢ of G18–G19–A20– C20 a in the D-loop in P. horikoshii tRNA Arg have no common conformation with those of G18–G19–D20– C20 a in S. cerevisiae tRNA Arg ICG . Distances between C1¢ of C72 of the 1Æ72 pair in the acceptor stem, C1¢ of G18 forming a hydrogen bond to U55 of the T-loop and C1¢ of C31 or G39 of 31Æ39 of the anticodon stem of tRNA were compared to con- firm the similarity of the three-dimensional structures of tRNAs. The C72 C1¢–G18 C1¢, C72 C1¢–C31 C1¢, C72 C1¢–G39 C1¢ and G18 C1¢–G39 C1¢ distances, except for G18 C1¢–C31 C1¢ in P. horikoshii tRNA Arg CCU , are within 1.3 A ˚ of the corresponding M. Konno et al. Modeling of tRNA-assisted mechanism on ArgRS FEBS Journal 276 (2009) 4763–4779 ª 2009 The Authors Journal compilation ª 2009 FEBS 4769 distances in S. cerevisiae tRNA Arg ICG . These facts indi- cate that the framework of tRNA Arg of the L-shape is conserved in these two cases. The anticodon loop of tRNA In the complex of P. horikoshii ArgRS, the base of C35 of tRNA Arg CCU is located in the hydrophobic pocket formed by the aromatic ring of Tyr587 at the C-terminal end of H23 and the hydrophobic side chains of Ile517 of H19 and Pro591, Val592 and Leu593 of the loop between H23 and H24 (Fig. 5C). N4H 2 and O2 of C35 are found within the distance of the hydrogen bonds with the main chain CO of Tyr587 (N4–O distance 2.69 A ˚ ) and with the main chain NH of Leu593 (O2–N distance 3.02 A ˚ ) of the turn of the loop, respectively. The base of C34 under- goes no interaction with the protein. The base of U36 is surrounded by the side chains of Tyr509, Ala512 and Ser516 on H19, and the C-terminal carboxyl group of Met629 in the C-terminal end; and O4 of U36 is in close proximity to NH of Met629 (O4–N dis- tance 3.02 A ˚ ). The base of A37 is stacked on a C31ÆG39 base pair, and the base of C32 is stacked on the base of A37. The base of A38 lies between the hydrophobic side chains of Leu451, Lys455 and Val471 in the ‘stem contact fold’ domain and the side chains of Pro505 and Met629. In S. cerevisiae ArgRS, the base of C35 of tRNA Arg ICG is also located in the hydrophobic pocket formed by the aromatic ring of the conserved Tyr565. On the other hand, the report that the K m value for tRNA Arg ICG on S. cerevisiae ArgRS with Tyr565 replaced by Ala is identical to that on the wild-type ArgRS [14] indicates that this conserved Tyr makes little contribution to the recog- nition of the base of C35. The report that a few tRNA Met CAU molecules are aminoacylated by Arg with E. coli ArgRS [29] suggests that when the back- bone of the anticodon stem is superimposed, the anticodon bases of tRNA Met CAU and tRNA Arg CCG should be oriented in the same direction and bind to almost the same region in the helix bundle structure of E. coli ArgRS. The reported structure of T. thermophilus ArgRS also has a quite similar hydrophobic pocket to the hydrophobic pocket accepting C35 of tRNA Arg CCU in the complex of P. horikoshii ArgRS and the hydro- phobic pocket accepting C35 of tRNA Arg ICG in the complex of S. cerevisiae ArgRS. The local structure accepting G36 of tRNA Arg ICG in the complex of S. cerevisiae ArgRS is substantially equivalent to the local structure accepting U36 of tRNA Arg CCU in the complex of P. horikoshii ArgRS. The reported struc- ture of T. thermophilus ArgRS also has a local struc- ture that is quite similar to the local structure accepting U36 of tRNA Arg CCU in the complex of P. horikoshii ArgRS. The relative orientation between A35 and U36 of tRNA Met CAU bound on A. aeolicus Met-tRNA synthe- tase (Protein Data Bank ID: 2CSX) [27] is appropriate to be accepted by the hydrophobic pocket and the local structure that are commonly found in the three ArgRSs. Thus, E. coli ArgRS is expected to have a similar hydrophobic pocket and local structure, which may successfully accept C35 and G36 of the mutated tRNA Met CCG in the formation of Arg-tRNA Met CCG on E. coli ArgRS [29]. On the other hand, in tRNA Met CAU bound on A. aeolicus Met-tRNA synthetase, the conformation of the anticodon loop of C32–U33–C34–A35–U36–A37– A38 of tRNA Met CAU is largely different from that of C32–U33–C34–C35–U36–A37–A38 of tRNA Arg CCU bound on P. horikoshii ArgRS. It is worth noting that the base of C32 is stacked on a C31ÆG39 base pair in tRNA Met CAU , but the base of A37 is stacked on a C31ÆG39 base pair in the case of the observed complex of P. horikoshii ArgRS and tRNA Arg CCU which caught its D-loop on the N-terminal domain of the ArgRS. The region from Asp456 to Glu466 in the superim- posed T. thermophilus ArgRS is not within 2.5 A ˚ of the corresponding region of S17, H18 and the X-loop from Ile490 to Glu500 in P. horikoshii ArgRS, whereas the C-terminal side from Gly467 in the X-loop in T. thermophilus ArgRS is within 1 A ˚ of that in P. hori- koshii ArgRS (Fig. 6). The structural difference of this region is due to difference of crystallization conditions. It was reported that structural difference in this region was observed in S. cerevisiae ArgRS proteins crystal- lized under different crystallization conditions [12,30,31]. The aminoacylation reaction for tRNA Arg CCU on wild-type ArgRS and ArgRS lacking the N-terminal domain (DN ArgRS) In order to clarify whether or not the binding of the D-loop of tRNA Arg CCU to the N-terminal domain contributes to the activation effect of tRNA on tRNA- assisted Arg-AMP formation reaction or the amino- acylation reaction, we constructed P. horikoshii ArgRS (residues 92–629; DN ArgRS) lacking the core region of the N-terminal domain from residue 1 to residue 91 in order to completely eliminate interactions between the N-terminal domain and the D-loop of tRNA Arg , and measured the kinetic parameters of the amino- Modeling of tRNA-assisted mechanism on ArgRS M. Konno et al. 4770 FEBS Journal 276 (2009) 4763–4779 ª 2009 The Authors Journal compilation ª 2009 FEBS acylation reaction for wild-type ArgRS and DN Ar- gRS. For wild-type ArgRS and DN ArgRS, the K m values for tRNA Arg CCU were 2.6 lm and 3.8 lm in 100 mm Hepes ⁄ NaOH buffer (pH 7.5), respectively, and the measured ratio of the V-value of DN ArgRS to that of wild-type ArgRS was [(8 ± 2) · 10 )2 ]. This indicates that the fixing of the D-loop of tRNA Arg with the N-terminal domain makes a minor contribu- tion to the aminoacylation reaction, but is not essen- tial, and that DN ArgRS facilitates the aminoacylation reaction of tRNA Arg CCU well enough. In particular, the proper acceptance of C35 and U36 of tRNA Arg CCU on the plausible accepting structures may be predomi- nantly contributory to the aminoacylation reaction of tRNA Arg CCU on P. horikoshii ArgRS. Model building Newly obtained structural information about the posi- tion of ATP analog (ANP) in the ternary complexes of P. horikoshii ArgRS was successfully used for the mod- eling of Arg, ATP and A76 of tRNA on P. horikoshii ArgRS for the tRNA-assisted ATP–PP i exchange reac- tion. In P. horikoshii ArgRS, the positions of the aden- osine and a-phosphate moieties of ATP bound thereon were assumed to be equivalent to those of ANP observed. The Arg-binding region in the complex of S. cerevisiae ArgRS, Arg and tRNA Arg corresponds to the region surrounded by the S5 strand, the HIGH loop, the H13 helix, the S14–H14 turn and the H14 helix in the Rossmann fold domain in P. horikoshii ArgRS. Referring to the distances between the a-NH 2 group of Arg and the main chain C@O of Ser151 and O of the side chain of Asn153, and the distances between the guanidinium moiety and the side chains of Glu148 and Asp351 in S. cerevisiae ArgRS, we pre- dicted the possible site for Arg in P. horikoshii ArgRS. In the predicted site, the distances between the a-NH 2 group of Arg and the main chain C@O of Ser127 and O of the side chain of Asn129 are set at 3.15 A ˚ and 3.04 A ˚ , and the distances between the guanidinium moiety and the side chain of Glu124 (S5) and Asp335 (H13) are set at 4.33 A ˚ and 3.66 A ˚ . Its carboxyl group is located at the proper position relative to Pa of ANP (ATP analog). In the ternary complex of S. cerevisiae ArgRS, the base of A76 is stacked on the side chain of Tyr347 on the helix corresponding to H13 in P. hori- koshii ArgRS, whereas in P. horikoshii ArgRS, the H13 helix deviates largely from that of S. cerevisiae ArgRS (Fig. 3A), and the conformation of the side chain of the corresponding Tyr331 is similar to that in the binary complex of S. cerevisiae ArgRS and tRNA Arg , the 3¢-terminal CCA of which is not visible in the electron density map. When A76 was moved within 2.5 A ˚ of the position of S. cerevisiae tRNA Arg bound to S. cerevisiae ArgRS superimposed on P. hor- ikoshii ArgRS, the 2¢-OH group of the ribose moiety was located in close proximity to the carboxyl group of the Arg. The Mg 2+ was coordinated to Pb@O and Pc@O of ATP, with a distance of 2.1 A ˚ . Figure 7A shows a model of Arg, ATP coordinated by Mg 2+ and A76 of tRNA Arg assisting the Arg-AMP formation reaction. A model of NH 2 OH, enzymatically synthe- sized Arg-AMP and A76 of tRNA Arg in the Arg- NHOH formation reaction in the presence of tRNA Arg is shown in Fig. 7B. In the case of modeling the deacylation reaction of Arg-tRNA Arg , coordinations of NH 2 –Ca–C and Cb of the Arg moiety of Arg-tRNA Arg were assumed to be essentially identical to those of the Arg predicted above, and the Arg moiety in the cyclic form was fitted in the space that is provided by the rearrangement of the side chain of Gln387 (Fig. 7C). We built a model for the Glu-dependent ATP–PP i exchange reaction at pH 6.0 in the absence of tRNA Glu on the basis of the crystal structure of T. thermophilus GluRS (Protein Data Bank IDs: 1N77, 1N78, and 2CV0) [32]. Coordi- nations of NH 2 –Ca–C and Cb of the Glu moiety of the intermediate of formed Glu-AMP were assumed to be identical to those of the observed Glu bound on GluRS (Fig. 7D). Ωloop Fig. 6. Comparison between regions of S17, H18 and the X-loop of P. horikoshii ArgRS and T. thermophilus ArgRS. Asn498, Phe499 and Glu500 in P. horikoshii ArgRS and Ser464, Phe465 and Glu466 in T. thermophilus ArgRS are shown in ball-and-stick repre- sentation. M. Konno et al. Modeling of tRNA-assisted mechanism on ArgRS FEBS Journal 276 (2009) 4763–4779 ª 2009 The Authors Journal compilation ª 2009 FEBS 4771 Discussion ATP–PP i exchange reaction and pyrophosphorolysis reaction at low pH In the cases of ArgRS from E. coli, B. stearothermophi- lus, N. crassa, and S. cerevisiae [1–5], GlnRS from E. coli W, S. cerevisiae, and porcine liver [7,8], and GluRS from E. coli K12 [9,10], the tRNA-assisted ATP–PP i exchange reaction has been observed, but no tRNA-independent ATP–PP i exchange reaction has ever been observed. In the cases of GluRS from E. coli W, S. cerevisiae, porcine liver, and rat liver, the tRNA-independent ATP–PP i exchange reaction was observed at much higher concentrations of Glu, whereas the tRNA-assisted ATP–PP i exchange reaction was observed at lower concentrations of Glu. For instance, the K m value for Glu measured in the tRNA- assisted ATP–PP i exchange reaction decreases signifi- cantly by 10 2 )10 3 -fold in comparison with that in the tRNA-independent ATP–PP i exchange reaction (the K m values for Glu are 0.4 m in the absence of tRNA and 6.6 · 10 )4 m in the presence of tRNA at pH 7.7 for E. coli W, 0.2 m and 7 · 10 )3 m at pH 7.7 for S. cerevisiae, 0.4 m and 4 · 10 )3 m at pH 7.7 for porcine liver, and 0.2 m and 6.7 · 10 )4 at pH 7.6 for rat liver, respectively [7,8,33]). In the absence of tRNA, any Arg-AMP and Gln-AMP are not detectable as intermediates formed by ArgRS and GlnRS, respec- tively [4,34–36]. As phosphodiesterase-treated tRNA and mutated tRNA containing C instead of A at the 3¢-end eliminate catalytic activity for the ATP–PP i exchange reaction on ArgRS [3], the ATP–PP i exchange reaction on ArgRS requires A at the 3¢-end of tRNA. In the presence of the tRNA treated with periodate, which oxidizes the 2¢-OH and 3¢-OH groups of the ribose of A at the 3¢-end to convert them into dialdehyde groups, the ArgRS, GlnRS and GluRS enzymes were incapable of catalyzing the ATP–PP i exchange reaction [1,4,5,7,33]. These facts indicate that the hydroxyl group of the ribose of the 3¢-terminal A76 of tRNA is essential for the ATP–PP i exchange reaction on ArgRS, GlnRS, and GluRS. The cognate tRNA is also necessary for the ArgRS- catalyzed pyrophosphorolysis of chemically synthesized Arg-AMP in the presence of PP i and Mg 2+ [6]. Further- more, the pyrophosphorolysis of chemically synthesized Arg-AMP and the ATP–PP i exchange reaction cata- lyzed by ArgRS in the presence of tRNA have pH optima of 6.2 and 6.5, respectively. The presence of PP i AB CD Fig. 7. Modeled intermediates on P. horikoshii ArgRS or T. thermophilus GluRS in the ATP–PP i exchange reaction, the Arg-NHOH formation reaction, and the deacylation reaction. (A) Arg (cyan), ATP (orange) coordinated by Mg 2+ and A76 (green) of tRNA assisting the Arg-AMP for- mation reaction on P. horikoshii ArgRS. The Mg 2+ is indicated by a green sphere. (B) HN 2 OH (cyan), enzymatically synthesized Arg-AMP (orange) and A76 (green) of tRNA in P. horikoshii ArgRS in the Arg-NHOH formation reaction in the presence of tRNA. (C) Arg-A76 (green) of Arg-tRNA with the Arg moiety with the cyclic configuration and AMP (orange) on P. horikoshii ArgRS in the deacylation reaction of Arg-tRNA. The torsional angles of the side chain of Gln387 (cyan) were changed from the original structure. (D) A Glu with the cyclic configuration (cyan) in the C–O c) –H–O2 form and ATP (orange) on T. thermophilus GluRS in the Glu-AMP formation reaction in the absence of tRNA. Modeling of tRNA-assisted mechanism on ArgRS M. Konno et al. 4772 FEBS Journal 276 (2009) 4763–4779 ª 2009 The Authors Journal compilation ª 2009 FEBS [...]... ArgRS and S cerevisiae ArgRS, a large conformational change of the anticodon loop, the D-loop, and the T-loop, and deviation of the inclination of base planes of the acceptor stem and the anticodon-binding stem of tRNAArgCCU from S cerevisiae tRNAArgICG The repeats of the model building and refinements using the o program [44] and the cns program [45] gave a better electron density map, and an ANP molecule... between O1 and Pa of ATP through formation of the trigonal bipyramid coordination around Pa At the same time, the H–O2 bond of H–O2–C of the a- carboxyl group of Arg is transferred to the O2–C bond to form the double bond Modeling of tRNA-assisted mechanism on ArgRS A7 6 A7 6 Arginine ' ' ' ' ' ' ' ' ' ' Arg- AMP ATP Fig 8 Reaction scheme of formation of Arg- AMP from Arg and ATP in the presence of tRNA at low... aminoacylation reaction and in the ATP PPi exchange reaction at pH 6.8 are 1.3 · 10)4 m and 6.7 · 10)4 m, respectively [33] The newly determined structure of the ATP analog (ANP) bound on P horikoshii ArgRS has been used to construct a model of the tRNA-assisted ATP PPi exchange reaction and a model of the tRNA-assisted pyrophosphorolysis reaction of Arg- AMP at low pH Modeling of the ArgRS-catalyzed deacylation... bond, and thereby the O–Pb bond is converted into O@Pb Finally, PPi is released in the form of Mg-PPi As O1 of the amino acid binds to Pa, and PPi is released from Pa on the reverse side, this reaction of Arg- AMP formation is an SN2 reaction The modeling of Arg, ATP and A7 6 of tRNA on P horikoshii ArgRS for the Arg- AMP formation reaction at low pH indicates that when the straight side chain of an Arg. .. Journal compilation ª 2009 FEBS 4775 Modeling of tRNA-assisted mechanism on ArgRS M Konno et al in the aminoacylation reaction at pH 7.5 and in the ATP PPi exchange reaction at pH 7.0 are 1.5 lm and 2.5 lm, respectively [39] For B stearothermophilus ArgRS, those for Arg at pH 8.0 are 5.7 lm and 7.4 lm, respectively [40], and for E coli K12 ArgRS, those in the aminoacylation reaction at pH 7.4 and in... consists of the following two steps: the first step is the formation of Arg- AMP from Arg- tRNA and AMP; the second step is the formation of Arg and ATP from Arg- AMP and PPi through a pyrophosphorolysis reaction Because the first step is an SN2 reaction, in the reaction intermediate, C of the Ca–(C@O)–O moiety of Arg- tRNA has an sp3 hybrid orbital The conversion from an sp2 to an sp3 hybrid orbital requires... form of the Arg moiety maintains a suitable conformation for the formation of an intramolecular hydrogen bond The intramolecular hydrogen bond, instead of tRNA, accelerates the pyrophosphorolysis step of this deacylation reaction The reported results of the deacylation reaction indicate that ArgRS has a binding affinity for Arg- tRNA with the cyclic form of the Arg moiety that is comparable to that for Arg- tRNA... and GlnRS The interaction between the 2¢-OH group and the O of the a- carboxyl group of Arg is thought to accelerate the pyrophosphorolysis reaction on ArgRS In a ternary complex of S cerevisiae ArgRS, tRNAArgICG, and Arg (Protein Data Bank ID: 1F7V) [12], the O of the 2¢-OH group in the 3¢-end A7 6 of ˚ ˚ ˚ tRNAArgICG is 3.18 A, 3.71 A and 3.57 A distant from the C, O1, and O2 (the O contacting Pa of. .. deacylation of ArgtRNA at high pH has also been performed on the basis of the common binding site of ATP and AMP on P horikoshii ArgRS The newly determined structure of the tRNAArgCCU bound on P horikoshii ArgRS provides further information on the plausible accepting structures for C35 and U36 of tRNAArgCCU In addition, the comparison of the reactivities of wildtype ArgRS and ArgRS lacking the N-terminal... pocket as in the Arg molecule bound to S cerevisiae ArgRS, its carboxyl group can locate in close proximity to Pa of ATP (Fig 7A) The a- carboxyl group of the Arg molecule can assume such a conformation that O2 forms a hydrogen bond with the 2¢-OH group of the ribose moiety of A7 6 of tRNAArg by rotation around Ca–C When Pa of ATP gains access to O1 of C@O1 of the a- carboxyl group of Arg, and two oxygen atoms . Modeling of tRNA-assisted mechanism of Arg activation based on a structure of Arg- tRNA synthetase, tRNA, and an ATP analog (ANP) Michiko Konno 1 ,. horikoshii ArgRS and tRNA Arg CCU and a ternary complex also containing the ATP analog adenosine-5¢-(b,c-imido)triphosphate (ANP); we found one reasonable mechanism,