From the Cellular Signaling Laboratory, RIKEN
Harima Institute, 1-1-1 Kouto, Mikazuki-cho, Sayo, Hyogo 679-5148, Japan, the § Department of Biophysics and Biochemistry,
Graduate School of Science, University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-0033, Japan, and the ¶ Institut de
Génétique et de Biologie Moléculaire et Cellulaire, 1 Rue Laurent Fries, 67404 Illkirch Cedex, France
Received for publication, October 24, 2000, and in revised form, December 4, 2000
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ABSTRACT |
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The presence of two short signature sequence
motifs (His-Ile-Gly-His (HIGH) and Lys-Met-Ser-Lys (KMSK)) is a
characteristic of the class I aminoacyl-tRNA synthetases. These motifs
constitute a portion of the catalytic site in three dimensions and play
an important role in catalysis. In particular, the second lysine of the
KMSK motif (K2) is the crucial catalytic residue for stabilization of
the transition state of the amino acid activation reaction (aminoacyl-adenylate formation). Arginyl-tRNA synthetase (ArgRS) is
unique among all of the class I enyzmes, as the majority of ArgRS
species lack canonical KMSK sequences. Thus, the mechanism by which
this group of ArgRSs achieves the catalytic reaction is not well
understood. Using three-dimensional modeling in combination with
sequence analysis and site-directed mutagenesis, we found a conserved
lysine in the KMSK-lacking ArgRSs upstream of the HIGH sequence motif,
which is likely to be a functional counterpart of the canonical class I
K2 lysine. The results suggest a plausible partition of the ArgRSs into
two major groups, on the basis of the conservation of the HIGH lysine.
Aminoacyl-tRNA synthetases (amino acid-tRNA ligases,
aaRSs)1 catalyze the
aminoacylation of cognate tRNAs in a two-step reaction. At the first
step, the aaRSs specifically bind the amino acid and ATP to activate
the amino acid through the formation of an aminoacyl-adenylate. At the
second step, the aminoacyl moiety is transferred to the 3'-terminal
adenosine (A76) of the tRNA. The 20 aaRSs are divided into two classes
(10 members each) on the basis of their evolutionarily distinct
ATP-binding catalytic domains (1, 2). The class I aaRSs are
characterized by the Rossman-fold architecture of the catalytic domain
and by the two short conserved sequence motifs, His-Ile-Gly-His (HIGH)
and Lys-Met-Ser-Lys (KMSK). These motifs, which are far apart in the
primary sequence, come together in the three-dimensional structure to
constitute a portion of the ATP-binding site. The second lysine in the
KMSK motif (K2) plays a key role in the catalysis, as its mutation causes the most severe defect in the enzymatic activity (3-6). In the crystal structure of the glutaminyl-tRNA synthetase
(GlnRS)·tRNAGln·ATP complex, this lysine interacts with
the General--
[32P]Sodium pyrophosphate and
L-[14C]arginine were purchased from
PerkinElmer Life Sciences. The Pyrobest DNA
polymerase for PCR and the restriction endonuclease NdeI
were obtained from Takara Shuzo Co., Ltd. (Shiga, Japan). The
restriction endonuclease BbrPI was from Roche Molecular
Biochemicals (Tokyo, Japan). T7 RNA polymerase was purified from
an overproducing strain, kindly provided by Dr. W. Studier (Stonybrook,
New York), according to Ref. 12.
Preparations of Proteins and RNA--
The wild-type T. thermophilus ArgRS was expressed in Escherichia coli
BL21(DE3) cells by using a T7-promotor-controlled expression vector.
The protein was purified by a combination of heat treatment (70 °C
for 30 min) and sequential chromatographies on DEAE-Sephacel ( Measurements of Enzyme Activities--
The arginine (and
tRNA)-dependent PPi-ATP isotopic exchange
reaction was carried out at 65 °C in 50 mM Hepes-NaOH
buffer (pH 7.2) containing 20 mM MgCl2, 40 mM KCl, 2 mM arginine, 20 µM
tRNAArg transcript, 4 mM ATP, 4 mM
[32P]sodium pyrophosphate (1.8 TBq
mol We have recently determined the structure of another class I
synthetase, T. thermophilus glutamyl-tRNA synthetase
(GluRS), bound to ATP, which is the first tRNA-free, ATP-bound
structure of a class I
synthetase.3 In contrast to
the ArgRSs, the overwhelming majority of GluRSs possess the highly
conserved KMSK signature motif (243KISK246 for
T. thermophilus GluRS). In the GluRS·ATP complex, the side chain of Lys246 (the class I K2) forms a salt bridge with
the
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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-phosphate of the ATP molecule (7). In the structures of the
tryptophanyl-tRNA synthetase·Trp-AMP and GlnRS·
tRNAGln·Gln-AMP analog complexes (8, 9), the K2 side
chain forms a salt bridge with the phosphate group of the adenylate.
Thus, it is now believed that K2 stabilizes the negatively charged
transition state of the first reaction step in the class I synthetases
(10). In agreement with the proposed function, K2 is strictly conserved in the class I aaRSs, with only a few exceptions. Arginyl-tRNA synthetase (ArgRS) is the only class I enzyme for which many species lack the apparent KMSK motif and, subsequently, the K2 counterpart. Recently, the atomic structures of the yeast Saccharomyces
cerevisiae (11) and Thermus
thermophilus2 ArgRSs
have been determined. In both enzymes, the KMSK motif is degenerate,
but 407GMST410 (yeast ArgRS) and
394QMSG397 (T. thermophilus ArgRS)
were shown to be its structural variants, in which the class I
principle lysine (K2) residues are absent. As the ATP-bound structures
are not available for both the yeast and T. thermophilus
enzymes, the mechanism by which these ArgRSs compensate for the lack of
K2 remains obscure.
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INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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2.5 × 20 cm) and FPLC PhenylSuperose (Amersham
Pharmacia Biotech, United Kingdom). The expression plasmid for
the mutant T. thermophilus ArgRS (K116G) was generated from
the vector for the wild-type enzyme. A 357-base pair-long DNA fragment
(NdeI-BbrPI sites) in the enzyme gene was
replaced by a PCR-amplified fragment that contains the AAG to GGG
mutation in the 116th codon (Lys
Gly). The target
mutation and integrity of the gene outside of the mutation site were
confirmed by DNA sequencing. The mutant ArgRS was expressed in the
E. coli cells and was purified according to the procedure
used for the wild-type enzyme. The purified mutant ArgRS shows the same
mobility on the SDS-polyacrylamide gel electrophoresis as the wild-type
enzyme. The wild-type and mutant ArgRSs exhibit the same circular
dichroism (CD) spectra with the calculated
-helical content of 45%,
which is consistent with the structural data. The two proteins are
thermostable: the temperature profiles of the CD (222 nm) are linear
and reversible in a wide temperature range (20-95 °C). T. thermophilus tRNAArg was synthesized by in
vitro transcription with T7 RNA polymerase and was purified as
described previously (13, 14).
1), and 50 nM (wild-type or
mutant) ArgRS. After various incubation times, a 10-µl aliquot of the
reaction was withdrawn, and the amount of the synthesized
[32P]ATP was measured as described previously (15). The
aminoacylation reaction was carried out at 65 °C in 100 mM Hepes-NaOH buffer (pH 7.5) containing 5 mM
MgCl2, 40 mM KCl, 4 mM ATP, 100 µM L-[14C]arginine, 20 µM tRNAArg transcript, and 100 nM
(wild-type or mutant) ArgRS. At various incubation times, an 8-µl
aliquot was removed, and the reaction was quenched by addition of
ice-cold trichloroacetic acid. It was washed with 5%
trichloroacetic acid on a filter paper (Whatman 3MM,
24 mm),
and the radioactivity of the acid-insoluble fraction (the synthesized
[14C]Arg-tRNAArg) was measured by a liquid
scintillation counter.
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ABSTRACT
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-phosphate of ATP (Fig.
1A), which is consistent with
its catalytic function. The structures of the catalytic domains are
well conserved between the two ArgRSs (yeast and T. thermophilus) and T. thermophilus GluRS (the root mean
square displacements are 1.92 and 1.88 Å for 75 and 74 C
atoms,
respectively), which suggests the similarity in the ATP binding mode.
To identify the functional counterpart of K2 in the ArgRSs,
the ATP-binding sites of the ArgRSs were compared with that of the
ATP-bound GluRS (Fig. 1). To our surprise, the superpositions showed
that the
ammonium groups of the lysine residues, Lys156
in the yeast and Lys116 in the T. thermophilus
ArgRSs, respectively, are located in close vicinity of the GluRS ATP
-phosphate (distances are 4.3 and 3.2 Å, respectively), without any
modification of the synthetase structures (Fig. 1). These lysine
residues protrude from the HIGH loops (the N-terminal loop of the HIGH
motif) to form a putative contact with the ATP
-phosphate oxygen
from the opposite direction of the GluRS KMSK lysine (K2). To
investigate the functional significance of the HIGH-loop lysine
residue, we prepared a T. thermophilus ArgRS with the
mutation of Lys116 to Gly (K116G), and its catalytic
activities were compared with those of the wild-type enzyme. The mutant
ArgRS (K116G) exhibits considerably lower activity for the arginine
(and tRNAArg)-dependent PPi-ATP
isotopic exchange, which is the reverse reaction of Arg-AMP formation
(first step of aminoacylation) (Fig.
2A). The initial rate
catalyzed by the mutant ArgRS is reduced by a factor of >1000, as
compared with that of the wild-type enzyme. Consistently, the arginine
charging on tRNAArg catalyzed by the mutant ArgRS (K116G)
is not detectable (Fig. 2B). Thus, we conclude that this
lysine residue plays a crucial role during the aminoacylation reaction,
in particular at the first step. It probably compensates for the lack
of the second lysine (K2) in the KMSK motif.
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Fig. 1.
Position of the HIGH-loop Lys in the ArgRS
structures. A, a stereo view showing the yeast
ArgRS catalytic site superposed on that of the ATP-bound T. thermophilus GluRS. In the GluRS active site (colored in
light blue), the second Lys (Lys246,
cyan) of the KMSK motif (green) interacts with
one of the -phosphate oxygens of the ATP molecule
(yellow). The ArgRS catalytic site (dark orange)
is superposed on that of GluRS without any modification of the
synthetase structure. Lys156 near the "HIGH" motif
(purple) is displayed in orange. B, the catalytic
site of T. thermophilus ArgRS (dark orange) is
superposed on that of the ATP-bound GluRS, and the HIGH-loop lysine
(Lys116) is shown in orange. The figures were
produced with the MOLSCRIPT (20) and RASTER3D (21) programs.
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Fig. 2.
The effects of the mutation of HIGH-loop
lysine on the ArgRS activities. A, arginine and tRNA
dependent PPi-ATP isotopic exchange reactions catalyzed by
the wild-type ( ) and mutant (K116G,
) T. thermophilus
ArgRSs. B, profiles of tRNAArg arginylation by
wild-type (
) and mutant (K116G,
) T. thermophilus
ArgRSs.
A sequence analysis shows that the ArgRSs can be divided into two major
groups, on the basis of the conservation of the HIGH-loop lysine (Fig.
3). The distribution of ArgRSs with the
HIGH-loop lysine extends to Eukarya, Archaea, and several taxons of
Bacteria (Proteobacteria /
subdivision,
Thermus-Deinococcus group, Cyanobacteria, and so on)
("HIGH group"). Remarkably, these ArgRSs do not possess a canonical
KMSK motif (11), which suggests that in these enzymes, the HIGH-loop
lysine is likely to play the role of the class I K2. In contrast, in
several bacterial classes (Proteobacteria
/
subdivision, most
Gram-positive bacteria, and Aquificales), a glycine residue substitutes
for the HIGH-loop lysine in the ArgRSs (Fig. 3). Consistently, these
bacteria preserve the KMSK motif, suggesting that the canonical class I
K2 lysine mediates the adenylate formation ("KMSK group").
Exceptionally, a few archaea lack both the HIGH-loop and KMSK lysines,
which suggests a somewhat different architecture of the active center
and/or mechanism of the catalysis (tentative group). As far as we see
from the sequences of the class I synthetases, the HIGH-loop lysine is
a unique characteristic of the ArgRSs, and the purpose of the drastic
migration of the catalytic residue in the primary sequences is not
clear. ArgRS is one of the three class I synthetases that possess a
tRNA-dependent mechanism of amino acid activation (16-18).
Thereby, one possibility is that the appearance of the HIGH-loop lysine
might be the result of the coevolution of the ArgRSs and
tRNAArg molecules from the subsequent organisms. It is
worth mentioning that some ArgRSs from the HIGH group contain KMSK-like
sites (E. coli etc.) (Fig. 3). Although the lysines of these
sites are shifted in sequence from their positions within the canonical
KMSK motifs (Fig. 3) (11), we cannot exclude here the possibility of
their catalytic role.
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The aaRSs are known to be potential targets for antibiotics. Mupirocin
is a natural catalytic inhibitor that is specific to the isoleucyl-tRNA
synthetases (IleRSs) from several Gram-positive and -negative bacterial
pathogens. Several artificial Ile-AMP analogs have been developed to
block the catalytic sites of the IleRSs of pathogens, but not to bind
the human IleRS (reviewed in Ref. 19). Similarly, it might be possible
to design Arg-AMP analogs that are sensitive to either the KMSK or
HIGH-loop lysine in the ArgRSs. This would be especially useful, as the
KMSK group includes the ArgRSs from many pathogens, such as
Rickettsia prowazekii, Helicobacter pylori,
Mycobacterium, and Mycoplasma species, etc., whereas all of the eukaryotic enzymes (including human) belong to the
HIGH group. Thus, the diversity of the catalytic mechanisms in the
ArgRS family could be utilized for the development of new selective
anti-infectives.
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FOOTNOTES |
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* This work was supported in part by grants-in-aid for Science Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan (to S. Y.) and a grant from the RIKEN Special Postdoctoral Researchers Program (to S. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence may be addressed. Tel.: 81-791-58-2838;
Fax: 81-791-58-2835; E-mail: dmitry@yumiyoshi.harima.riken.go.jp (
for D. G. V.) or Tel.: 81-3-5841-4392; Fax: 81-3-5841-8057; E-mail: yokoyama@biochem.s.u-tokyo.ac.jp (for S. Y.).
Published, JBC Papers in Press, December 5, 2000, DOI 10.1074/jbc.C000756200
2 A. Shimada, O. Nureki, and S. Yokoyama, unpublished result.
3 S. Sekine, O. Nureki, D. G. Vassylyev, and S. Yokoyama, unpublished result.
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ABBREVIATIONS |
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The abbreviations used are: aaRS, aminoacyl-tRNA synthetase; GlnRS, glutaminyl-tRNA synthetase; ArgRS, arginyl-tRNA synthetase; PPi, pyrophosphate; GluRS, glutamyl-tRNA synthetase; IleRS, isoleucyl-tRNA synthetase; PCR, polymerase chain reaction.
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REFERENCES |
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