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The
Effect of N-terminal Changes on Arginyl-tRNA ynthetase from Escherichia coli
LIU
Wen, LIU Mo-Fang, XIA Xian, WANG En-Duo*, WANG Ying-Lai
(
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology,
Shanghai
Institutes for Biological Sciences,
the Chinese Academy of Sciences,
Shanghai 200031, China )
Abstract An Asn2 deleted mutant of Escherichia
coli arginyl-tRNA synthetase deleted Asn2 and a chimera
mutant, in which the N-terminal 23 amino acid residues of yeast arginyl-tRNA
synthetase were appended to the N-terminus of Escherichia coli
synthetase, were synthesized and studied. The expression of the deletion and
chimera mutants in Escherichia coli formed inclusion bodies,
presumably due to improper folding of the proteins. Relative to the native enzyme, the deletion mutant showed
full amino acid activation activity and a 26% reduction in aminoacylation
activity, while the chimera mutant lost 93% and 96% activities in amino acid
activation and aminoacylation, respectively, and did not aminoacylate yeast
tRNAArg at all. The mutant deleted Asn2 and Ile3
was able to be expressed in Escherichia coli but not stable to be purified. The
emission maximum wavelength in the fluorescence spectra of the chimera mutants
shifted to longer one and the corresponding intensities decreased, when
compared with those of the native enzyme. The data show that the conformation
of the mutants are different and the tryptophan residues in the mutants are
more exposed than those in the native enzyme. An estimate of the secondary
structure of the mutant enzymes from their far ultraviolet CD spectra showed
that the chimera mutant contained less a-helix, more b-sheet
and slightly higher fraction of random coil, as compared with the native
enzyme. The results indicate that an intact N-terminal domain of E.coli
arginyl-tRNA synthetase is important to its activity and correct folding.
Key
words arginyl-tRNA
synthetase; N-terminal; activity; mutatation
Aminoacyl-tRNA synthetases
(aaRSs) catalyze, with a high degree of specificity, the attachment of amino
acids to their cognate tRNA molecules[1]. Biophysical, biochemical
and genetic studies have significantly deepened our understanding of the
structure and function of these enzymes[2, 3]. Generally, the overall reaction can be separated
into two steps. The first step is amino acid activation. In the second step,
the activated amino acid is transferred to the 3′end
of the cognate tRNA. Although the
20 aaRSs catalyze essentially the same reaction, they vary remarkably both in
primary and quaternary structure and show very limited sequence homology[4].
Based on conserved sequences and three-dimensional structures determined by
X-ray diffraction, the 20 aaRSs are classified into two groups of 10 enzymes
each[5, 6]. The class I
aaRSs, whose active sites contain a dinucleotide-binding fold (Rossmann fold),
display two signature amino acid sequences, HIGH and KMSKS. The class II aaRSs
are characterized by three homologous motifs and are built around an
antiparallel b-sheet
partly closed by helices that are the framework on which the active site is
constructed[5, 6].
Many
class I aaRSs from the cytoplasm and mitochondria of eukaryotes present a similar
picture, but have one major distinction from their bacterial counterparts. Some of the eukaryotic aaRSs, for example, glutaminyl-,
methionyl- and isoleucyl-tRNA synthetases (GlnRS, MetRS, IleRS) from Saccharomyces cerevisiae (S.cerevisiae), have an additional domain appended to
the N- or C-terminal end of the sequences that are homologous to their
respective prokaryote enzymes[7]. It is known that the appended domain of Neurospora
mitochondria tyrosyl-tRNA synthetase (TyrRS) is required for the novel RNA
splicing activity of this enzyme[8,
9]. However, the more general role of the appended
domain for enzyme activity is not understood.
Arginyl-tRNA
synthetase (ArgRS), glutamyl-tRNA
synthetase (GluRS) and glutaminyl-tRNA synthetase (GlnRS) are different from
the other 17 aaRSs, in that they
require their cognate tRNA in the amino acid activation[10, 11]. ArgRS from Escherichia coli (E.coli)
belongs to class I aaRSs, and is a
monomeric protein of 577 amino acid residues with a molecular mass of 64.7 kD[12]. By sequence alignment with all other
class I aaRSs, ArgRS has a longer
N-terminal region[13].
It is not known whether the longer N-terminal region of ArgRS is
necessary for its function. In
addition, yeast cytoplasmic ArgRS
is a monomeric protein of 607 residues with molecular mass 69.5 kD. ArgRS from yeast and E.coli
share a high degree of homogeneity,
except that yeast enzyme is longer than E.coli enzyme by
23 residues at the N-terminus[14]. Cross-recognition between ArgRSs and its cognate tRNAs from S. cerevisiae and E.coli
was studied in our laboratory. We
found that yeast ArgRS could arginylate E.coli tRNAArg, although at a lower efficiency than it
charged yeast tRNAArg,
while E.coli
ArgRS could acylate only E.coli
tRNAArg [15].
The tertiary structure of yeast ArgRS show that there is an additional
domain at its N-terminal, yet no
such domain is found in the other five class I aaRSs[14]. In order to investigate the effect of
the extra part at the N-terminus of yeast enzyme on the structure and function
of E.coli ArgRS, the
N-terminal peptide fragment consisting of the first 23 residues of yeast ArgRS
was transplanted to the N-terminus of E.coli ArgRS and the properties of
the chimera enzyme were studied.
Here, we report the effect of the changes at
the N-terminus of E.coli ArgRS on its structure and function.
1 Materials and Methods
1.1
Materials
Oligonucleotides
were synthesized on a Beckman DNA Synthesizer at Shanghai Institute of Plant
Physiology, the Chinese Academy of
Sciences. DEAE-Sepharose CL-6B and
Blue-Sepharose CL-6B were obtained from Pharmacia, Sweden. T4
polynucleotide kinase, T4 DNA
ligase, Taq DNA
polymerase, calf intestine
alkaline phosphatase and all restriction endonucleases were purchased from
Promega, USA. Radioactive L-[3H]arginine
(6.3×104
Ci/mol) was from Amersham,
England. E.coli tRNA2Arg
was isolated from an overproducing strain in our laboratory[16]. Yeast tRNAArg was purchased
from Shanghai Li-Zhu Dong-Feng Biochemical Factory. The genes, argS
and RRS1 encoding E.coli and yeast cytoplasm ArgRSs were gifts
from Dr. Gangloff and Eriani[12, 14]. The overproducing strain of E.coli ArgRS was
constructed in our laboratory[17,
18]. Plasmid pMFT7-5
was constructed by Che et al[19]. Purification of ArgRSs from E.coli and yeast was
performed by the published methods[14, 17].
1.2
Construction of plasmids containing the genes encoding the mutants of E.coli
ArgRS
Deletion
of Asn2 and double deletion of Asn2 and Ile3
were performed by PCR, using argS
as a template. Primers, CAT ATG
ATT CAG GCT CTT CTC TCA GAA AAA and CAT ATG CAG GCT CTT CTC TCA GAA AAA for
N-terminal of above two mutations,
and GCC AAG CTT CAC CAT AGG CTT for C-terminal, were used to synthesize DNA with NdeI
or HindIII restriction sites by PCR. The produced DNA was introduced into pMFT7-5. The recombined plasmids containing argS
deletion mutants were termed pMFT-argSD2
and pMFT-argSD2&3. The chimera ArgRS consists of E.coli
ArgRS and an N-terminal peptide fragment (1―23
residues) of yeast ArgRS. A DNA
fragment encoding 23 amino acid residues of yeast ArgRS flanked by NcoI
and HindIII restriction sites was generated by PCR and inserted into
corresponding sites of pTrc99B-argS[18] to yield the
recombinant plasmid, termed pTrc-argS–ad. The DNA sequences of the genes encoding
the both mutants were confirmed by dideoxy sequencing.
1.3
Expression and purification of ArgRS mutants from E.coli
transformants
E.coli
BL21(DE3) and TG1 were transformed with the above recombinant plasmids,
respectively. BL21(DE3)/pMFT-argSD2, BL21(DE3)/pMFT-argSD2&3
and TG1/pTrc-argS–ad were cultured at 37 ℃
in LB medium containing 100 mg/L ampicillin and induced by isopropylthio-b-D-galactoside
(IPTG) when A700 reach 0.7,
respectively. The cells
were harvested by centrifugation after 4 h continuous culture, and disrupted by sonication. After centrifugation of the crude
extract, the supernatants and
precipitates were analyzed by SDS-PAGE for ArgRS. Purification of mutant ArgRS from the supernatants was
performed through two-step chromatography on DEAE-Sepharose CL-6B and
HA-Ultrogel column as described previously[20].
1.4
Enzymatic assay
The
ATP～PPi exchange and aminoacylation
activities of ArgRS and its kinetic parameters were assayed as previously
described[21]. One unit was defined as the amount of enzyme which
charges 1 nmol arginine to tRNAArg in 1 min under the assay
conditions. The specific activity was defined as the units of enzyme per
milligram of protein. The concentration of purified ArgRS was determined by A280
of the enzyme solution. About 1.18 g/L protein was equal to 1 optical density
unit at 280 nm[22].
1.5
Fluorescence spectroscopy
The
fluorescence spectra were measured on a Hitachi F-4010 Fluorescence
Spectrophotometer. Excitation and emission wavelengths were 295 nm and 340 nm,
both with bandwidths of 5 nm. The scanning ranges of excitation and emission
fluorescence spectra were 220―300
nm and 300―400
nm, respectively. The measurements were made at 25 ℃
in l ml of 8 mmol/L MgCl2, 0.2 mmol/L DTT, and 200 mg/L enzyme[23].
1.6
Far-ultraviolet circular dichroism spectroscopy
Protein
samples at a concentration of 0.2 g/L in 20 mmol/L potassium phosphates were
analyzed on a Jasco J-715 spectropolarimeter purged with nitrogen at room
temperature. A 0.1 cm path-length cuvette was used and the spectra were
accumulated over five scans.
Estimation of the secondary structure was calculated according to the
method of Yang et al[24].
1.7
Determination of N-terminal amino acid sequence
The
N-terminal sequences of the mutated enzymes were determined on a Beckman Porton
LF3200 Protein/Peptide Sequencer.
2 Results
2.1
Expression and purification of the E.coli ArgRS mutants
Gene
constructs were confirmed by DNA sequencing. Over-expressed argSD2
and argSD2&3
could be detected by SDS-PAGE in the total proteins extracted after induction.
After disruption of the cells and centrifugation of the crude extract, most of the ArgRSΔ2
and ArgRSΔ2&3
were found in the centrifugation pellet,
only a small amount of the enzyme in the supernatant. This means that
the mutant enzyme, ArgRSΔ2
and ArgRSΔ2&3
formed inclusion bodies during over-expression (Fig.1). The mutant ArgRSΔ2
in the supernatant could be purified to a homogenity of 95% purity
(Fig.1). However ArgRSΔ2&3
could not be detected by both SDS-PAGE and assaying activity after
DEAE-Sepharose CL-6B chromatography because it was unstable and easy to be
degraded. In contrast, no inclusion body of ArgRS was found
during the over-expression of argS encoding the native enzyme[12,17, 18].
Fig.1 Analysis of ArgRSΔ2
by SDS-PAGE
Electrophoresis was carried out on a 12%
running gel and proteins were visualized by Coomassie brilliant blue R-250
staining. Lane 1 contains
molecular mass marker from Sigma,
with molecular mass of 97.4,
66.2, 55.0, 42.7, 40.0 and 31.0 kD.
Lane 2 contains proteins in the transformants containing argSΔ2.
Lane 3 contains proteins in the supernatant after disruption of cells and
centrifugation. Lane 4 contains
proteins in the pellet. Lane 5
contains renatured and purified ArgRSΔ2. The amount of protein in lane 3―5
was 5 mg.
The
chimera enzyme ArgS-AD was overproduced in the E.coli transformant
TG1/pTrc-argS–ad and formed inclusion bodies during its
expression as well (Fig.2). ArgRS-AD was purified to greater than 95% purity
from the supernatant by the same method as above[20] (Fig.2).
Fig.2 Analysis of ArgRS-AD by SDS-PAGE
Lane 1 contains molecular mass marker
from Sigma, with molecular mass of 97.4, 66.2, 55.0, 42.7, 40.0 and 31.0 kD.
Lane 2 contains proteins in the transformants containing argS–ad.
Lane 3 contains proteins in the supernatant after disruption of cells and
centrifugation. Lane 4 contains proteins in the pellet. Lane 5 contains
purified ArgRS-AD. Lane 6 contains purified ArgRS. The amount of protein in
each lane was 5 mg.
2.2 N-terminal amino acid sequences of the
two mutant enzymes
The
N-terminal sequences of the two purified mutants were determined by Edman
degradation and showed to be as expected from the designed sequences. The first
ten residues of ArgRSΔ2
were MIQALLSEKY, the same as those in the native enzyme with the second
residue, Asn, deleted. The N-terminal sequence of ArgRS-AD was MASTANMISQ
identical to the first ten amino acid residues in yeast ArgRS[14].
2.3
Enzyme activity of ArgRS mutants
Relative
to the native enzyme, purified
ArgRSΔ2 lost about 25% of its aminoacylation
activity, yet it still retained
almost full amino acid activation activity when compared with the native enzyme
(Table 1). In contrast, these two kinds of activities in
ArgRS-AD decreased dramatically to about 7% and 4% of those of the native
enzyme, respectively.
2.4 Kinetic constants of the two mutant
enzymes for aminoacylation
The
Km values of ArgRSΔ2
and ArgRS-AD for ATP were almost unchanged when compared with that of the
native enzyme, suggesting that the
N-terminus was not involved in ATP binding. The Km values of
ArgRSΔ2 for arginine and tRNAArg
were increased. In comparision, Km values of ArgRS-AD for
arginine and tRNAArg were higher than that of ArgRSΔ2
(Table 2).
2.5 Recognition of yeast tRNAArg by the
chimera mutant ArgRS-AD
Yeast
ArgRS purified from the E.coli transformant containing RRS1,
encoding yeast ArgRS, can catalyze the arginylation of yeast total tRNA under
standard conditions. However,arginylation was not detected with ArgRS-AD under
the same conditions (Fig.3), even with the incubation time extended to over 30
min. The data showed that yeast tRNAArg could not be recognized by
the chimera enzyme, although it has the same N-terminus as that of yeast ArgRS.
Fig.3 Arginylation of yeast tRNAArg
Arginylation
of 0.1 mmol/L yeast tRNAArg by 20 nmol/L mutant enzyme ArgRS-AD(○)
and by 20 nmol/L yeast ArgRS(●).
2.6 Fluorescence spectra of the mutants
A
relative decrease in the fluorescence intensity and a shifting of the emission
maxima from 337.4 nm to 340 nm were observed in the progression: ArgRS, ArgRSΔ2
and ArgRS-AD [Fig.4(A)]. This suggests that the conformation of the mutant
enzymes is more flexible and the tryptophan residues are more exposed. The
maximum excitation wavelength of ArgRS and its mutants did not shift. At the
maximum excitation wavelength the fluorescence intensity of ArgRS-AD decreased,
however the fluorescence spectra of ArgRS, ArgRSΔ2
were overlapping [Fig.4(B)].
Fig.4 Fluorescence spectra of ArgRS and its
mutants
(A) At 295 nm excitation wavelength
emission fluorescence spectra of ArgRS (―), ArgRSΔ2
(……), and ArgRS-AD (—-). (B) Excitation fluorescence
spectra of ArgRS and ArgRSΔ2
are overlapping (―), and that of ArgRS-AD is shown by a dash
line (—-), at 340 nm emission wavelength.
2.7 Far-UV CD spectra
The
a-helix,
b-sheet
and random coil contents of the proteins were estimated from their CD spectra
and are shown in Table 3. It indicated that the chimera mutation induced
significant conformation changes. In the mutant enzyme, the a-helix
content was lower, while the b-sheet
and random coil higher, than that in the native enzyme.
3
Discussion
It
has well been established that the class I aaRSs have an N-terminal
nucleotide-binding fold that contains the catalytic site, and in some class I
aaRSs, the C-terminal domain forms an all a-helix
and displays a module that could be participating in anticodon-binding. The
above result has been confirmed in the study of several class I aaRSs, such as E.coli
MetRS[25], B.stearothermophilus
TyrRS[26] and yeast ArgRS[14]. In a docking model of
binding of tRNA to yeast ArgRS, the N-terminal domain defines a module that
could be engaged in specific tRNA recognition and the tRNA variable pocket
interacts with the N-terminal domain of ArgRS[14]. Our current study
showed that N-terminal domain of ArgRS is crucial to its correct folding and is
important to maintain its activity catalyzing the aminoacylation reaction.
Firstly,
mutagenesis of ArgRS, such as in the cases of ArgRSΔ2,
ArgRSΔ2&3 and ArgRS-AD, resulted in the
formation of inclusion bodies during their gene expression. In contrast, even
when the native enzyme was overproduced 2 500 fold, the enzyme still appeared
in the supernatant after disruption of cells and centrifugation of crude
extract, no inclusion bodies were found[27]. These observations
showed that these mutations in the N-terminal domain could affect the correct
folding of this protein. Conformational change in chimera mutants was also
detected in the fluorescence spectra and far ultraviolet CD spectra. We have
previously obtained many single-site mutants of ArgRS, including five mutants
with substitution of Ala for Trp[28], four with substitution of Ala for Cys[29],
several with substitution of Ala for Lys[30, 31] and one with Asp2 for Asn2
(ArgRSN2D)[32], none of them formed inclusion bodies.
Particularly noteworthy is the fact that ArgRS-K306A lost almost all
of its activity, yet it did not formed inclusion bodies[31]. The
data in the present study suggests that an intact N-terminal domain might be a
key to the correct folding of the enzyme.
ArgRSΔ2&3
may be produced in the transformant. However, it can not be obtained by the
routine method of purification. It might be unstable and hydrolyzed during
purification.
Secondly,
the Km values of both ArgRSΔ2
and ArgRS-AD for ATP were almost the same as that of native E.coli
ArgRS. This indicated that the N-terminal domain was not involved in ATP
binding. On the other hand, although ArgRSΔ2
had full arginine activation activity, its aminoacylation activity was 74% of
the native enzyme. This, along with our previous results which demonstrated
that the substitution of the second amino acid residue at N-terminus, D, for N
did not affect the kinetic properties of this enzyme[32], showed
that the intact N-terminal domain might be involved in the interaction between
the enzyme and tRNAArg directly or indirectly. Because both
activities of ArgRS-AD were decreased dramatically, the additional N-terminal
of yeast ArgRS not only affected tRNAArg, but also affected the
interaction of the enzyme with arginine. The larger decreasement in the
aminoacylation activities of ArgRS-AD than those of ArgRSΔ2
might be the results of greater conformational changes of ArgRS-AD than that of
ArgRSΔ2. The exact N-terminal region of ArgRS
seems really necessary to its structure and function.
ArgRS
from S.cerevisiae, likes several other class I eukaryote aaRSs, such as
GluRS, MetRS and IleRS, has another domain appended to the N-terminus, in
addition to an active site containing a “body”
that is closely homologous to its E.coli relative[14]. The
role of the appended domain for enzyme activity is unclear. Attachment of the
extra appended domain of the yeast GlnRS to the E.coli enzyme enabled
the E.coli-yeast protein to function as a yeast enzyme, in vitro
and in vivo, suggesting
that the appended domain might be involved in species-specific recognition of
tRNA[33]. The present study demonstrated that the chimera enzyme,
ArgRS-AD, could recognize neither E.coli tRNAArg nor yeast tRNAArg
efficiently. The role of the
appended domain of yeast ArgRS remains unclear. In higher eukaryotes, it was
assumed that the extra domains of some aaRSs might be involved in the formation
of multi-enzyme complex[10,
34―36],
however the existence of a multi-synthetase complex in yeast is controversial[10, 37]. According to the present
results, it could be deduced that,
unlike GlnRS, the appended domain of yeast ArgRS did not participate in
specie-specific recognition of tRNA; ArgRS is different from GlnRS in the
aspect of structure and function.
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Received: September 18, 2001Accepted: November
2， 2001
This work was supported by grant (Number
39730120) of the National Natural Science Foundation of China and (Number
KSCX-2-2-04) of the Chinese Academy of Sciences
*Corresponding author: Tel,
86-21-64374430; Fax, 86-21-64338357; e-mail, [email protected]