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

based on a structure of Arg-tRNA synthetase, tRNA, andan ATP analog ANP Michiko Konno1, Tomomi Sumida1,*, Emiko Uchikawa1, Yukie Mori1, Tatsuo Yanagisawa2,*, Shun-ichi Sekine2and Shigeuk

based on a structure of Arg-tRNA synthetase, tRNA, and

an ATP analog (ANP)

Michiko Konno1, Tomomi Sumida1,*, Emiko Uchikawa1, Yukie Mori1, Tatsuo Yanagisawa2,*,

Shun-ichi Sekine2and Shigeuki Yokoyama2

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

Mg2+ [amino acid + ATP fi aminoacyl-AMP +

pyrophosphate (PPi)] and the reverse reaction (amino-acyl-AMP + PPi 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

tRNAArgCCU )

(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-Gln-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, Arg-tRNAArgCCU and an ATP analog with Rfactor= 0.213 (Rfree= 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 tRNA synthetase-catalyzed deacylation of Arg-tRNA (Arg-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+(NH2)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; PPi, pyrophosphate.

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 cerevisiaeand porcine liver [7,8] and Glu-tRNA

syn-thetase (GluRS) from E coli K12 [9,10], the ATP–PPi

exchange reaction corresponding to the formation of

aminoacyl-AMP and its reverse reaction, amino

acid + ATP = aminoacyl-AMP + PPi, has never

been observed without tRNA In the presence of

cog-nate tRNA, the ATP–PPi 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 PPiand Mg2+[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, PPi, and Mg2+, whereas in the

absence of PPi, 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–PPi 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 tRNAArg inter-acting with ArgRS, we determined crystal structures of

a binary complex of Pyrococcus horikoshii ArgRS and tRNAArg

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 tRNAArg, 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 tRNAArgCCU Moreover, modeling of the relative posi-tions of Arg, A76 of tRNAArg 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–PPi 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, tRNAArgCCU, and the ATP analog ANP) and the binary complex (P horikoshii ArgRS and tRNAArgCCU) were obtained with Rfactor= 0.213 (Rfree= 0.253) at 2.0 A˚ resolution, and Rfactor= 0.201 (Rfree= 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, tRNAArgCCUand 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 tRNAArgICG [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),

gives a drastically decreased kcat value of 0.01 s)1 in

the aminoacylation reaction in comparison with the

kcat value of 8 s)1 for the wild-type ArgRS [14] In

S cerevisiae ArgRS bound to Arg and tRNA, the

a-NH2 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-aeArgRS, 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 horikoshiiArgRS 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 Oe1of 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 tRNACys (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 Mg2+ 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 tRNAArgCCU 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.

within 3.5 A˚ of protein residues The observed electron

density for the Mg2+ in this conformation is about

half of the electron density expected for an occupancy

of 1.0 for Mg2+ The presence of different orientations

for the PbNPc moiety of ANP attached and not

attached to Mg2+is manifested as low electron

densi-ties in the regions of Mg2+ and the PbNPc moiety

The salt bridge formed by Mg2+ between PbO and

PaO may retard the conformational inversion at Pa of

ATP in the reversible process of the ATP–PPi

exchange reaction, whereas the salt bridge formed by

Mg2+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–SO2–O–) or diaminosulfone (–NH–SO2–

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 amiami-noacyl-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 tRNAArg-assisted ATP–PPiexchange 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.

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–PPi exchange reaction

The 3¢-terminus of tRNA

In the 3¢-terminal G73–C74–C75–A76 sequence of tRNAArgCCU, 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 tRNAAla 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 tRNAArg

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 tRNAArgICGbound 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).

H135

H138

K132

S127

N129 H138

H135 K132 N129

S127

Q837 Q837

Fig 4 A final (2Fobs) 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+

observed in tRNAArgICG 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 tRNAVal (Protein Data Bank ID: 1GAX)

[17] and T thermophilus Leu-tRNA synthetase bound

to tRNALeu (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 tRNAMet

(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

tRNAMet does not progress On the other hand, in

E coli Cys-tRNA synthetase bound to tRNACys

(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 tRNAArg Among all tRNA species specific to each of the 20 amino acids, only tRNAArgisoacceptors have A at posi-tion 20 on the D-loop, with the excepposi-tion that four tRNAArgisoacceptors 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 tRNAArgplay an important role in tRNAArgbinding on ArgRS [13,28,29] Crystal structures of binary and ternary complexes of ArgRS and tRNAArgICG from

S cerevisiaeand Arg revealed that a base of D20 in the D-loop, which is specific to S cerevisiae tRNAArgICG, 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.

However, it was reported that, in the aminoacylation

reaction, the kcat and Km values for tRNAArgICG and

tRNAArgUCUon 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 tRNAArgare not important for the binding

of tRNAArgon ArgRS in the aminoacylation reaction

The crystal structure of free ArgRS from T

thermophi-lus has been determined, but that of the complex with

tRNAArghas not been determined yet [13] In T

ther-mophilusArgRS, 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

Kmfor tRNAArgand a large decrease in Vmaxin 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 Kmvalue for tRNAArgas that of the wild type

and does not affect the affinity of tRNAArg

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 C20a (extra

nucleo-tide inserted between nucleonucleo-tides 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 Nd2 and Od1

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, Od1 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 Ne2of Gln111 and Od1of 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 tRNAArgICG grow contains tRNA, l-Arg, ATP and

Mg2+ at sufficient concentrations for the aminoacyla-tion reacaminoacyla-tion, and (NH4)2SO4 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 kcatand Km for tRNAArgin 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 tRNAArgin 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 tRNAArg were not within 4.0 A˚ of those of D20 and A72 in S cerevisiae tRNAArg, respectively The main chains of the phospho-diester bridge of C5¢–O5–P–O3–C3¢ of G18–G19–A20– C20a in the D-loop in P horikoshii tRNAArg have no common conformation with those of G18–G19–D20– C20ain S cerevisiae tRNAArgICG

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 tRNAArgCCU, are within 1.3 A˚ of the corresponding

distances in S cerevisiae tRNAArgICG These facts

indi-cate that the framework of tRNAArgof 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 tRNAArgCCU 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)

N4H2 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 tRNAArgICG is also located in the

hydrophobic pocket formed by the aromatic ring of

the conserved Tyr565 On the other hand, the report

that the Km value for tRNAArgICG 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

tRNAMetCAU 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 tRNAMetCAU and tRNAArgCCG

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 tRNAArgCCU in

the complex of P horikoshii ArgRS and the

hydro-phobic pocket accepting C35 of tRNAArgICG in the

complex of S cerevisiae ArgRS The local structure

accepting G36 of tRNAArgICG in the complex of

S cerevisiae ArgRS is substantially equivalent to the

local structure accepting U36 of tRNAArgCCU in the

complex of P horikoshii ArgRS The reported

ture of T thermophilus ArgRS also has a local struc-ture that is quite similar to the local strucstruc-ture accepting U36 of tRNAArgCCU in the complex of

P horikoshii ArgRS

The relative orientation between A35 and U36 of tRNAMet

CAUbound 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 tRNAMetCCGin the formation of Arg-tRNAMetCCG on

E coliArgRS [29]

On the other hand, in tRNAMetCAU bound on

A aeolicusMet-tRNA synthetase, the conformation of the anticodon loop of C32–U33–C34–A35–U36–A37– A38 of tRNAMetCAU is largely different from that of C32–U33–C34–C35–U36–A37–A38 of tRNAArgCCU bound on P horikoshii ArgRS It is worth noting that the base of C32 is stacked on a C31ÆG39 base pair in tRNAMet

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 tRNAArgCCU 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 thermophilusArgRS 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 tRNAArgCCUon 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 tRNAArgCCU 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 tRNAArg, and measured the kinetic parameters of the

amino-acylation reaction for wild-type ArgRS and DN

Ar-gRS For wild-type ArgRS and DN ArgRS, the Km

values for tRNAArgCCU 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 tRNAArg

with the N-terminal domain makes a minor

contribu-tion to the aminoacylacontribu-tion reaccontribu-tion, but is not

essen-tial, and that DN ArgRS facilitates the aminoacylation

reaction of tRNAArgCCU well enough In particular,

the proper acceptance of C35 and U36 of tRNAArgCCU

on the plausible accepting structures may be

predomi-nantly contributory to the aminoacylation reaction of

tRNAArgCCUon P horikoshii ArgRS

Model building

Newly obtained structural information about the

posi-tion of ATP analog (ANP) in the ternary complexes of

P horikoshiiArgRS was successfully used for the

mod-eling of Arg, ATP and A76 of tRNA on P horikoshii

ArgRS for the tRNA-assisted ATP–PPiexchange

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 cerevisiaeArgRS, Arg and tRNAArgcorresponds 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-NH2 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-NH2 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 tRNAArg, 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 tRNAArg 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 Mg2+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 Mg2+and A76 of tRNAArg assisting the Arg-AMP formation reaction A model of NH2OH, enzymatically synthe-sized Arg-AMP and A76 of tRNAArg in the Arg-NHOH formation reaction in the presence of tRNAArg

is shown in Fig 7B

In the case of modeling the deacylation reaction of Arg-tRNAArg, coordinations of NH2–Ca–C and Cb of the Arg moiety of Arg-tRNAArg 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–PPi exchange reaction at

pH 6.0 in the absence of tRNAGluon the basis of the crystal structure of T thermophilus GluRS (Protein Data Bank IDs: 1N77, 1N78, and 2CV0) [32] Coordi-nations of NH2–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.

ATP–PPiexchange 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–PPiexchange reaction has been observed, but no

tRNA-independent ATP–PPi 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–PPi exchange reaction was

observed at much higher concentrations of Glu,

whereas the tRNA-assisted ATP–PPiexchange reaction

was observed at lower concentrations of Glu For

instance, the Kmvalue for Glu measured in the

tRNA-assisted ATP–PPi exchange reaction decreases

signifi-cantly by 102)103-fold in comparison with that in the

tRNA-independent ATP–PPi exchange reaction (the

Km values for Glu are 0.4 m in the absence of tRNA

and 6.6· 10)4m in the presence of tRNA at pH 7.7

for E coli W, 0.2 m and 7· 10)3m at pH 7.7 for

S cerevisiae, 0.4 m and 4· 10)3m 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–PPi exchange reaction on ArgRS [3], the ATP–PPi 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–PPi 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–PPi 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 PPiand Mg2+[6] Further-more, the pyrophosphorolysis of chemically synthesized Arg-AMP and the ATP–PPi exchange reaction cata-lyzed by ArgRS in the presence of tRNA have pH optima of 6.2 and 6.5, respectively The presence of PPi

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) HN2OH (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–Oc)–H–O2 form and ATP (orange) on T thermophilus GluRS in the Glu-AMP formation reaction in the absence of tRNA.