Although strategies for the specific recognition of amino acid and tRNA are unique to each enzyme, two reaction steps in the esterification of amino acids to their cog-nate tRNAs are conse
Trang 1synthetase active site and its implication for specific
substrate recognition
Silvija Bilokapic1, Jasmina Rokov Plavec1, Nenad Ban2and Ivana Weygand-Durasevic1
1 Department of Chemistry, Faculty of Science, University of Zagreb, Croatia
2 Department of Biology, Institute of Molecular Biology and Biophysics, Swiss Federal Institute of Technology, Zu¨rich, Switzerland
The fidelity of protein synthesis depends on the correct
attachment of amino acids to the 3¢-ends of their
cog-nate tRNA species by aminoacyl-tRNA synthetases
(aaRS), a family of enzymes that bridges the
informa-tion gap between nucleic acids and proteins Although
strategies for the specific recognition of amino acid
and tRNA are unique to each enzyme, two reaction
steps in the esterification of amino acids to their
cog-nate tRNAs are conserved among all aaRS: (a) the
activation of amino acids with ATP by formation of
aminoacyl-adenylate, and (b) the transfer of the
aminoacyl-moiety from the aminoacyl-adenylate to the cognate tRNA substrate [1]
Sequence alignment and later structural data showed that aaRSs constitute a family of enzymes in which the same catalytic reaction is performed at two topologi-cally different structural domains Class I aaRSs are built around a canonical dinucleotide-binding fold (Rossmann fold) with the consensus motifs HIGH and KMSKS, which define two regions of sequence conser-vation for all class I aaRSs Class II synthetases share
an antiparallel b sheet partly enclosed by helices, thus
Keywords
conformational flexibility; motif 2 loop;
seryl-tRNA synthetase; specificity of substrate
recognition; synthetase:tRNA model
Correspondence
I Weygand-Durasevic, Department of
Chemistry, Faculty of Science, University of
Zagreb, Horvatovac 102a, 10000 Zagreb,
Croatia
Fax: +385 1 460 6401
Tel: +385 1 460 6230
E-mail: weygand@chem.pmf.hr
(Received 20 February 2008, revised 21
March 2008, accepted 26 March 2008)
doi:10.1111/j.1742-4658.2008.06423.x
Seryl-tRNA synthetase (SerRS) is a class II aminoacyl-tRNA synthetase that catalyzes serine activation and its transfer to cognate tRNASer Previ-ous biochemical and structural studies have revealed that bacterial- and methanogenic-type SerRSs employ different strategies of substrate recogni-tion In addition to other idiosyncratic features, such as the active site zinc ion and the unique fold of the N-terminal tRNA-binding domain, metha-nogenic-type SerRS is, in comparison with bacterial homologues, charac-terized by a notable shortening of the motif 2 loop Mutational analysis of Methanosarcina barkeriSerRS (mMbSerRS) was undertaken to identify the active site residues that ensure the specificity of amino acid and tRNA 3¢-end recognition Residues predicted to contribute to the amino acid spec-ificity were selected for mutation according to the crystal structure of mMbSerRS complexed with its cognate aminoacyl-adenylate, whereas those involved in binding of the tRNA 3¢-end were identified and mutagenized
on the basis of modeling the mMbSerRS:tRNA complex Although mMbSerRSs variants with an altered serine-binding pocket (W396A, N435A, S437A) were more sensitive to inhibition by threonine and cyste-ine, none of the mutants was able to activate noncognate amino acids to greater extent than the wild-type enzyme In vitro kinetics results also suggest that conformational changes in the motif 2 loop are required for efficient serylation
Abbreviations
aaRS, aminoacyl-tRNA synthetase; HTH, helix–turn–helix; mMbSerRS, archaeal (Methanosarcina barkeri) SerRS; Ni-NTA, nickel-nitrilotriacetic acid; SerRS, seryl-tRNA synthetase; SOL, serine ordering loop.
Trang 2creating a fold found only in the class II synthetases
and in their paralogues Three short conserved
sequence motifs (motifs 1, 2 and 3) are characteristic
of all class II synthetases [1]
The separation of aaRSs into two classes correlates
with the modes of tRNA binding: class I synthetases
approach the acceptor helix from the minor groove
side, whereas class II synthetases approach it from the
opposite, major groove side [2,3] The functional
differ-ences between the two classes can also be seen in the
mode of ATP binding: the ATP molecule adopts an
extended structure in the class I active site, whereas
bound ATP in the class II catalytic core has a bent
conformation Recent completion of representative
crystal structures for all 20 aaRSs has created a new
context to determine if synthetases of the same class
share kinetic features that parallel those based on
struc-ture [4,5] Again, a distinct mechanistic signastruc-ture has
been shown to divide the two classes of synthetase [6]
Seryl-tRNA synthetase (SerRS), which catalyzes
serylation of the corresponding set of isoaccepting
tRNAsSer, belongs to the class II aaRSs [7] The
crys-tal structure of Escherichia coli SerRS was the first
example of an aaRS that does not possess the
Ross-mann dinucleotide-binding fold [8] The enzyme is
homodimeric, with motif 1 involved in dimer interface
contacts As revealed by the crystal structures of two
prokaryotic seryl-tRNA synthetases from E coli and
Thermus thermophilus, each subunit possesses a
C-ter-minal active site domain typical for class II aaRSs,
whereas the first 100 N-terminal residues form an
anti-parallel a-helical coiled-coil crucial for the selection
and binding of tRNA [7] In the crystal structure of
T thermophilus SerRS complexed with tRNASer, the
N-terminal helical arm is buried between the TwC arm
and the long extra arm of tRNASer[9,10] The crystal
structure of mammalian mitochondrial SerRS from
Bos taurushas an N-terminal domain that also consists
of a long a-helical arm, and extensions in the N- and
C-termini of the enzyme ensure recognition of two
unusual mitochondrial tRNAsSer [11] In general, the
active sites of seryl-tRNA synthetases contain a very
long loop in motif 2 (the longest of all class II
synthe-tases), involved in binding ATP and the acceptor end
of tRNA [8,10,12]
With the first sequence of archaeal genome
(Methan-ococcus jannaschii) [13], it became apparent that it
encodes an atypical SerRS, later found in all
methano-genic archaea, and thus named methanomethano-genic-type
SerRS [14,15], to be distinguished from SerRSs found
in all other organisms (bacterial-type SerRSs) The
N-terminal region of this enzyme is significantly longer
than the corresponding domain of its bacterial-type
counterparts Accordingly, the crystal structure of Methanosarcina barkeri SerRS (mMbSerRS) [12] revealed two idiosyncratic features: a novel N-terminal tRNA-binding domain and a zinc ion in the active site Biochemical analysis confirmed and absolute require-ment of zinc for enzymatic activity In addition, meth-anogenic-type SerRS is, in comparison with bacterial homologues, characterized by a notable shortening of the motif 2 loop, questioning the mode of tRNA bind-ing Evidently, bacterial- and methanogenic-type SerRSs have diverse modes of substrate recognition [10,15–19] Although the widely distributed bacterial-type SerRS has been extensively characterized, the unusual methanogenic-type still represents an intrigu-ing puzzle in structure–functional and evolutionary terms Using mutational and kinetic analysis, we eva-luated the importance of mMbSerRS active site flexibility in the discriminating between substrates and the proper positioning of the tRNA CCA-end
Results
Structure-based design of mMbSerRS variants The positions of particular motifs along the mMbSerRS polypeptide are shown in Fig 1 Each mMbSerRS subunit consists of two domains linked by
a short flexible oligopeptide (L) The N-terminus (residues 1–165) is a mixed a⁄ b domain composed of a six-stranded antiparallel b sheet capped by a bundle of four helices (H1, H2, H3, H4) This fold is distinct from the coiled-coil of the tRNA-binding domain in bacterial-type SerRS, but presumably also interacts with the tRNA extra arm (see later) The catalytic module (residues 174–502), built upon eight anti-parallel strands surrounded by three a helices, repeats the general structure of the catalytic core in the bacte-rial enzyme The catalytic domain contains three class II signature motifs, designated M1, M2 and M3
in Fig 1A M1 motifs participate at the dimer inter-face, whereas M2 and M3 contain residues involved in serine, ATP and tRNA 3¢-end interactions The tetra-coordinated Zn2+ ion is bound to three conserved protein ligands (Cys306, Glu355 and Cys461; marked with asterisks in Fig 1A) and a water molecule, which dissociates from the zinc ion to allow coordination of the amino group of the serine substrate (Fig 1B) Accordingly, biochemical analysis revealed a loss of enzyme function as a consequence of alterations in zinc-binding residues [12] In this study, we aimed to characterize the other active site residues, which according to the crystal structure may influence the specificity of substrate recognition These are marked
Trang 3with a black lozenge in Fig 1A The mutations were
generated using site-specific in vitro mutagenesis of the
mMbSerRS gene, and N-terminally His-tagged
vari-ants with alanines (or valine in one case) at the
alter-ation sites were expressed and purified from E coli
All mutated enzymes possessed a characteristic class II
dimeric nature, as confirmed by gel-filtration
chroma-tography (not shown) CD spectra of the wild-type
and all mutants were similar, confirming that the
over-all fold is maintained (Fig 2) In addition, selected
mutants were crystallized and the structures were essentially identical to that of the wild-type enzyme (not shown)
Kinetic analysis of amino acid activation
by mMbSerRS variants with an altered serine-binding pocket
Serine binding in wild-type mMbSerRS is accompanied
by a significant localized conformational change in the
A
B
Fig 1 Structural and functional features of mMbSerRS (A) Idiosyncratic and general structural–functional motifs along the polypeptide chain
of one subunit H1–H4 represent four idiosyncratic helices in the N-terminal domain, L is a linker region, M1, M2 and M3 are class II signa-ture motifs, HTH and SOL denote idiosyncratic oligopeptide insertions into the mMbSerRS catalytic core Zinc-ion ligands are marked by asterisks (*) A black lozenge denotes other active site residues that are, according to a crystal or model structure, presumed to be impli-cated in the specificity of substrate recognition These are changed to alanine (or valine) in this study (B) View of the active site-bound seryl-adenylate analogue The characteristic extended conformation of seryl-adenylate in the active site of mMbSerRS can be observed Residues that interact with the analogue are indicated The active site zinc ion is in cyan.
200 210 220 230 240 250 260
–12 –10 –8 –6 –4 –2 0 2 4 6 8
3 (deg·cm
2 ·dmol
Wavelength (nm)
Fig 2 CD spectra of wild-type mMbSerRS
(h) and its mutants: N435A (s), S437A (n),
W396A ⁄ N435A S437A (,), R267A (X),
E338A (e), R347A (+).
Trang 4‘serine ordering loop’ (SOL, residues 394–410; Fig 1),
which brings the loop into the proximity of the zinc ion
and enables direct contact between Gln400 and the
carbonyl oxygen of the serine substrate [12] These
movements are required to position the carboxylate
oxygen for nucleophilic attack of the a-phosphate of
ATP Thus, according to the crystal structure, the
spec-ificity of serine recognition depends on: (a) the zinc ion,
(b) the size of the active site and (c) the hydrophilic
nature of the serine-binding pocket Despite precise
recognition of the cognate amino acid, functional
assays have shown slight, but notable, misactivation of
threonine by mMbSerRS, which does not seem to be
edited in vitro [12] To obtain further insight into the
discrimination strategy, we designed several
mMbS-erRS mutants and tested their ability to activate serine
and misactivate noncognate amino acids Because the
size of the active site depends predominantly on
Trp396, which is located at the bottom of the
serine-binding subsite and packs above the amino group of
the serine substrate (Fig 1B), by substituting alanine,
we expected to produce a variant with an enlarged
active site that was capable of accommodating a larger
noncognate amino acid However, formation of a
hydrophilic environment around Asn435 and Ser437
(which coordinate a structurally conserved water
mole-cule) may reduce the binding of amino acids with
hydrophobic side chains (e.g threonine) Accordingly,
the mMbSerRS triple-mutant (W396A⁄ N435A ⁄ S437A)
was expected to combine important misactivating
features, namely a larger binding site and increased
hydrophobicity
The kinetic parameters for serine activation were
determined using the pyrophosphate-exchange reaction
for the wild-type and five mMbSerRS variants with
alterations in the amino acid-binding pocket (W396A,
N435A, S437A, the double-mutant N435Al⁄ S437A
and the triple-mutant W396A⁄ N435A ⁄ S437A) All
mutants showed significant reductions in catalytic
effi-ciency (kcat⁄ Km) for serine compared with wild-type
mMbSerRS (Table 1) In accordance with structural
data and the sequence conservation of Trp396 (replacement by Phe in some methanogenic-type SerRSs is presumably functionally equivalent), muta-tion of this residue had deleterious effects on kcatand
Km for serine in seryl-adenylate synthesis (Table 1) Replacing Asn435 does not affect the affinity for serine
in the first step of the aminoacylation reaction as much
as mutation of Trp396 However, catalytic efficiency is seriously hampered The alteration S437A leads to the
Km value for serine being elevated by two orders of magnitude This residue is highly conserved in all SerRSs, although its direct interaction with serine has not been observed in bacterial- and methanogenic-type SerRS structures [12,16] CD spectrometry and X-ray analysis showed that the observed kinetic effect (Table 1) is not due protein misfolding (Fig 2) Having established the effect of alterations in both Asn435 and Ser437 on serine recognition, we investigated the catalytic properties of the enzyme using double substitutions The variant N435A⁄ S437A displayed kinetic parameters similar to those deter-mined for S437A (Table 1), suggesting that the large reduction in the affinity for serine was caused primar-ily by the mutation S437A Although mutations N435A⁄ S437A altered the chemical properties of the amino acid binding pocket significantly, the substrate specificity of the double mMbSerRS mutant was not relaxed Activation of noncognate amino acids in the ATP–PPiexchange reaction was not increased in com-parison with wild-type enzyme (not shown) This mutant showed decreased activation efficiency towards both cognate and noncognate substrates (serine and threonine, respectively) Although the triple-mutant W396A⁄ N435A ⁄ S437A showed a 103-fold decrease in serine affinity (Table 1) its kcat value was only three times lower than for the wild-type enzyme, suggesting
a compensatory effect for the mutations It seems that
in an enlarged amino acid-binding subsite orientation
of the carboxylate group is preserved and the attack
on ATP is presumably facilitated An electron-density map for the mutated enzyme in complex with serine
Table 1 Kinetic parameters for serine in ATP–PPiexchange reaction.
cat ⁄ K m (s)1Æ M )1) (k
cat ⁄ K m )rela
a (kcat⁄ K m )wt⁄ (k cat ⁄ K m )mut.
Trang 5(data not shown) revealed the ordering of 394–410
loop residues and interactions with Gln400, despite
replacing Trp396 with alanine
The influence of mutations in the serine-binding
pocket on magnesium binding
The characteristic conformation of ATP in the class II
active site is usually stabilized by hydrogen bonds with
motif 2 and motif 3 conserved arginine residues and
three Mg2+ ions In addition to phosphate groups,
acidic residues from the active site and water molecules
coordinate Mg2+ ions Interestingly, an exception has
been observed in the structure of HisRS: two
magne-sium cations are bound in the active site and a
HisRS-specific arginine residue occupies the same position as
the third Mg2+ and takes over its role in binding the
a-phosphate of the ATP molecule [20]
Magnesium ions play a crucial role in the
aminoa-cylation reaction because they decrease and delocalize
the negative charge of the triphosphate moiety of
ATP, which is in close proximity to the negatively
charged carboxylate group of the amino acid
sub-strate [21] The binding of ATP and the amino acid
substrate in the active site are linked, and incorrect
binding of ATP can affect serine binding and vice
versa [22] Asn435, together with Asp416 and Glu432,
contributes to binding of the Mg2+ ion between the
a- and b-phosphates of ATP Indeed, a 10-fold
increase in the Km value for serine has been observed
for the N435A mutant compared with the wild-type
enzyme Therefore, we assayed the activity
depen-dence of mMbSerRS variants comprising N435A
replacement on magnesium concentration in the
ATP–PPi exchange reaction at saturating levels of
serine and ATP substrates The double-mutant
N435A⁄ S437A and the triple-mutant N435A ⁄ S437A ⁄
W396A showed maximal reaction activity at
magne-sium concentrations that were higher than for the
wild-type enzyme or W396A mutant, supporting the
role of Asn435 in magnesium binding (Fig 3)
Kinetic analysis of aminoacylation by mMbSerRS
variants with an altered serine-binding pocket
The catalytic parametars of W396A, N435A, S437A,
the double-mutant N435Al⁄ S437A and the
triple-mutant W396A⁄ N435A ⁄ S437A were significantly
dif-ferent from those of the wild-type mMbSerRS in serine
activation The kinetic properties of the active site
mutants were further investigated in the
aminoacyla-tion reacaminoacyla-tion The producaminoacyla-tion and characterizaaminoacyla-tion of
different tRNAsSer substrates is described below The
data reveal that alteration in the SOL (W396A) has a more pronounced effect on adenylate synthesis than
on tRNA aminoacylation (Table 2) Also, mutations in Asn435 and Ser437 have a less deleterious influence on tRNA aminoacylation This suggests that tRNA bind-ing stabilizes the active site despite perturbations caused by the mutations
Although only four class I aaRSs, glutamyl-, glutaminyl-, arginyl- and lysyl-tRNA synthetases (GluRS, GlnRS, ArgRS and LysRS, respectively), are known to require their cognate tRNA for amino acid activation,
a number of other synthetases (including several class II representatives) [1], use tRNA to optimize the amino acid binding pocket Indeed, tRNA-mediated amino acid recognition has been documented for yeast SerRS [23,24] Our results show that tRNA also con-tributes to optimization of the active site in methano-genic-type SerRS, which can be detected when kinetic parameters for the mutated proteins are measured
Selectivity of the active site towards amino acid substrates
Based on previous biochemical and crystallographic studies [12], we assumed that of the 20 amino acids alanine, glycine, threonine and cysteine may potentially bind into the active site of methanogenic-type SerRS and cause mischarging problems The ability of these noncognate amino acids to impair the specific aminoa-cylation of tRNASer with serine was measured for wild-type and mutant mMbSerRS enzymes (Fig 4) Alanine and glycine were chosen because they are smaller than serine; yet they do not have similar chem-ical properties Valine was used as a positive control
0 0.2 0.4 0.6 0.8 1 1.2
Fig 3 Comparison of the activity dependence of wild-type and mMbSerRS mutants on magnesium concentration Activity was assayed in an ATP–PP i exchange reaction: wild-type (r), W396A ( ), N435A ⁄ S437A ( ), W396A ⁄ N435A ⁄ S437A (s).
Trang 6because it is bigger and cannot form the H-bonds that
are characteristic of serine in the active site Threonine
and cysteine were used because of their chemical
simi-larity with serine Binding of threonine may be
particu-larly likely because of the similarity between the
mMbSerRS and threonyl-tRNA synthetase (ThrRS)
catalytic domains, both of which contain a zinc ion
Serylation activity of the wild-type protein was 10%
lower in the presence of cysteine, and the inhibition of
N435A⁄ S437A and W396A ⁄ N435A ⁄ S437A mutants
was 55% and 70%, respectively (Fig 4) Interestingly,
the noncognate amino acid was not activated in the
ATP–PPiassay, indicating that the inhibition of
seryla-tion was not caused by cysteinyl-tRNASer formation
Cysteine may be a competitive inhibitor of
aminoacy-lation in the presence of tRNA or the –SH group
of cysteine may attack serylated-tRNA, resulting
in the formation of a serine–cysteine dipeptide and
deacylated tRNA, in accordance with the hypothesis
that aaRSs are capable of performing
thioester-depen-dent peptide synthesis [25,26] It remains to be
deter-mined which of two proposed scenarios is actually
taking place in methanogenic-type SerRS
In addition, inhibition of serylation with 6 mm threonine was 10–20% higher with mutated mMbSerRS variants than with wild-type enzyme Again, the mutants did not misactivate threonine (or other noncognate amino acids) more efficiently than the wild-type enzyme (data not shown) It seems that noncognate substrates are not bound in the proper orientation, which would allow their carboxyl group to attack the a-phosphate of bound ATP and lead to the completion
of the first step of the aminoacylation reaction
tRNA acceptor-stem binding by mMbSerRS motif 2 loop residues and the design of the mutants
Multiple sequence alignments of members of two distinct groups of SerRS showed that the motif 2 loop sequence, which according to crystal structures participates in acceptor-stem contact, is highly con-served within each type, but differs between them [12] The crystal structure of T thermophilus SerRS in com-plex with cognate tRNA revealed that Phe262 from this long loop interacts with the fifth base pair of the acceptor stem [10] In contrast to bacterial-type SerRS, the discriminator base (G73) and the first base pair
in the acceptor stem were shown to be important determinants of specific tRNASerrecognition in metha-nogenic-type enzyme [15] We assumed that the differ-ences in acceptor-stem recognition between the two SerRS types are due to differences in the motif 2 loop sequence and that each motif 2 loop is capable
of performing different but specific interactions with cognate tRNA
Because the acceptor end of tRNASerin the T ther-mophilus SerRS:tRNA co-crystal structure is disor-dered, we made a mMbSerRS:tRNA docking model using the crystal structures of ThrRS in complex with the cognate tRNA Because of the similar size of the motif 2 loop in methanogenic-type SerRS and ThrRS and the involvement of zinc ions in the recognition of amino acid substrates, the M barkeri loop was
homol-Table 2 Kinetic parameters for serine in aminoacylation reaction The tRNA used in these experiments was M barkeri tRNA Ser overpro-duced in E coli.
cat ⁄ K m (s)1Æ M )1) (k
cat ⁄ K m ) rela
a
(k cat ⁄ K m ) wt ⁄ (k cat ⁄ K m ) mut
0
20
40
60
80
100
120
Fig 4 Inhibition of mMbSerRS with noncognate amino acids.
Activity of wild-type mMbSerRS (black bar) and different mutants
(W396A, dark gray; N435A ⁄ S437A, gray; W396A ⁄ N435A ⁄ S437A,
white) in the presence of other amino acids The level of inhibition
was determined as a ratio of initial velocities of inhibited (6 m M
noncognate amino acid) and uninhibited reactions.
Trang 7ogy modeled based on the structure of the E coli
ThrRS loop We observed that the positioning of the
cognate tRNA in the active site of mMbSerRS would
be facilitated upon the conformational change of the
motif 2 loop, which was the only proximal protein
region in the proposed mMbSerRS:tRNA complex
able to mediate the interactions with the tRNA
accep-tor stem The model used to predict the amino acids
that might participate in the binding of the 3¢-end of
tRNA, which were therefore subjected to mutational
and kinetic analyses, is shown in Fig 5
Kinetic parameters for M barkeri tRNASer
amino-acylation by wild-type and mutant mMbSerRS enzymes
are given in Table 3 The results are consistent with
the involvement of several motif 2 residues (Glu338, Arg347, Gly340 and Gly341) in the serylation reaction Loss of the catalytic efficiency of mutant E338A is essentially the consequence of a decreased kcat value (Table 3), and is in agreement with previous studies on aspartyl-tRNA synthetase (AspRS) [27,28] and LysRS [29] Residue Glu338 in mMbSerRS is presumably functionally equivalent to Glu258 in T thermophilus SerRS The crystal structure of bacterial binary SerRS complexes reveals an interaction between Glu258 and N6 of ATP or Ser-AMS Upon tRNA binding, this residue forms a hydrogen bond with N2 of G73 How-ever, the side chain of Glu338 may adopt a different conformation, which would facilitate interactions with the O2¢ of C74 ribose, analogous to those observed in the structure of the E coli AspRS:tRNA complex Because G73 is a discriminator base in mMbSerRS, specific interactions, in addition to Glu338, must con-tribute to the importance of G73 The class-invariant Arg347 from the motif 2 loop in mMbSerRS is posi-tioned to interact with N1 of G73 It cannot be excluded, however, that upon tRNA binding the orien-tation of Arg347 side chain changes, allowing recogni-tion of C74 as in other class II synthetases The crystal structure of mMbSerRS shows that Arg347 and Glu338 also form contacts with ATP (Fig 1B) Our kinetic results revealed that replacement of these two residues with alanine affects tRNA aminoacylation (Table 3), which is consistent with the involvement of these two residues in both steps of the aminoacylation reaction in all class II synthetases [30] The R347A variant shows a greater reduction in kcat than the E338A variant (8-fold for E338A vs 175-fold for R347A) Moreover, Arg347 also contributes slightly to the binding of tRNA, as deduced from the twofold decrease in Km(Table 3)
Strikingly, two glycines in succession, Gly340 and Gly341, in the motif 2 loop are conserved among methanogenic-type SerRSs We analyzed the functional significance of this apparent flexibility for tRNA CCA-end binding and recognition of the G1:C72 iden-tity determinant In agreement with our model, the G340V⁄ G341A mutant showed a 410-fold diminished
Fig 5 Model of the acceptor end of tRNASerbound in the active
site of mMbSerRS The view is focused on the acceptor part of
tRNA in the active site of mMbSerRS The tRNA is shown as an
orange tube with the CCA-end and G73 shown as sticks The
cata-lytic domain of mMbSerRS in complex with Ser-AMS is shown in
blue The structure of mMbSerRS with the proposed motif 2 loop
conformation upon tRNA binding is depicted in gray Residues that
participate in the interaction with tRNA and that were tested are
shown as sticks It can be seen that the side chain of Ile342 points
away from tRNA molecule All molecular depictions were produced
using PYMOL (http://pymol.sourceforge.net).
Table 3 Kinetic parameters for M barkeri tRNA Ser aminoacylation with wild-type and mutant mMbSerRS enzymes The tRNA used in these experiments was M barkeri tRNA Ser overproduced in E coli.
mMbSerRS K m (l M) k cat (s)1) k cat ⁄ K m (s)1ÆlM)1) k cat ⁄ K m (s)1Æ M )1) k
cat ⁄ K m rel
Trang 8catalytic efficiency and a twofold higher Km(Table 3).
Thus, the flexibility of this region might be important
in avoiding steric clashes with tRNA and allowing
hydrogen bonding of the first base pair
The crystal structure of mMbSerRS revealed that
the unique 30-residue insertion between motifs 1
and 2 in methanogenic-type SerRSs adopts a helix–
turn–helix (HTH) fold The HTH fold of one
mono-mer is positioned above the catalytic core of the other
Interestingly, Arg267 from the beginning of helix 9 in
the HTH fold of one monomer is positioned towards
the active site of the other monomer and can
contrib-ute to interactions with the tRNA acceptor end In
the proposed model, Arg267 coordinates the ribose
O2¢ of C74 Substitution of Arg267 with alanine
significantly decreased the kcat value for tRNA
aminoacylation (Table 3) Amino acids subjected to
alterations in G340V⁄ G341A and R267A mutants
presumably interact with tRNA in the transition state
and contribute to structural rearrangements that result
in new contacts between the acceptor end of tRNA
and the active site of synthetase Furthermore, Gly340
and Gly341 from the motif 2 loop also contribute to
the affinity for tRNA Importantly, our kinetic data
indicate that Arg267 from the HTH-motif, belonging
to one monomer of the dimeric mMbSerRS, is
essen-tial for tRNA aminoacylation in the active site of the
other monomer The crystal structures of mMbSerRS
in complex with ATP or the seryl-adenylate analogue
do not reveal any interactions between this arginine
and small substrates Therefore, detected changes in
the kinetic parameters for this mutant can only be due
to tRNA CCA-end binding (Table 3) Thus, Arg267
from the HTH-motif seems to be essential for the
second, but not the first, step in the aminoacylation
reaction
Wild-type enzyme and mutants were tested for their
ability to bind tRNA using a gel-shift assay All
pro-teins were able to shift the cognate tRNA, although
some mutants form weaker complexes, especially
R347A, as shown in Fig 6
Variability of tRNA substrates for mMbSerRS
We encountered many difficulties in preparing
suffi-cient quantities of tRNA substrates to determine the
kinetic parameters with mMbSerRS variants Because
of the very inefficient in vitro transcription of M
bark-eri synthetic tRNASer genes we decided to attempt to
overproduce M barkeri tRNAsSer in E coli Both
tRNACGA and tRNAGGA isoacceptors were found to
be well expressed in vivo Kmvalues for serylation were
estimated for the expressed tRNAs and for in vitro
transcribed tRNACGA as a reference The Km and kcat values for both isoacceptors were similar and compara-ble with tRNASer obtained by in vitro transcription (Table 4) It was thus evident that the M barkeri tRNAsSer were processed correctly in the bacterial host
tRNAGGA acceptor made up 50–60% of the total tRNA and was used for subsequent experiments because
it was better expressed than tRNACGA, which contrib-uted only 25–30% Endogenous E coli tRNASer, 3.5%
in the same preparation, was negligible in comparison with the overexpressed M barkeri tRNAGGA Because
of the 25-fold higher kcat value (Table 4), E coli tRNASerwas not expected to influence the kinetic results obtained with in vivo produced M barkeri tRNASer To confirm this, in vivo expressed M barkeri tRNAGGA was purified on urea PAGE The kinetic parameters obtained with pure M barkeri tRNAGGAand mMbS-erRS were the same as for the ‘crude’ sample (data not shown)
Methanococcus maripaludis tRNAGCU gene tran-script was not efficiently recognized by wild-type mMbSerRS (Table 4), which is consistent with struc-tural differences between homologous and heter-ologous serine-specific tRNAs in the D-loops
Although M barkeri, in contrast to Me jannaschii and Me maripaludis, does not possess tRNASec [31], our experiments revealed charging of Me jannaschii tRNASecby mMbSerRS, comparable with the recogni-tion of tRNASec in the human and bacterial systems (Table 4)
Fig 6 Native PAGE analysis of complex formation between over-expressed tRNA Ser and mMbSerRS mutants (Upper) Wild-type mMbSerRS forms a more stable complex with overexpressed tRNASer in comparison with R347A mutant: lane 1, wild-type mMbSerRS (1.6 l M ); lanes 2–6, wild-type and tRNA (lane 2, 0.12 l M ; lane 3, 0.3 l M ; lane 4, 0.6 l M ; lane 5, 1.2 l M , lane 6, 2.4 l M ); lanes 7–10, R347A and tRNA (lane 7, 0.12 l M; lane 8, 0.3 l M; lane 9, 0.6 l M; lane 10, 1.2 l M ) (Lower) Complex formation between tRNA Ser (1.2 l M ) and different mMbSerRS mutants (1.6 l M ): lane 1, wild-type mMbSerRS; lane 2, N435A S437A; lane
3, W394A; lane 4, catalytic domain (does not make complex with tRNA; unpublished results); lane 5, catalytic domain mutant; lane 6, R347A; lane 7, G340V ⁄ G341A The gel was stained with Coomas-sie Brilliant Blue Arrow denotes the position of the complex.
Trang 9Switching the specificity for amino acid substrate
Although alteration of the three residues (W396A,
N435A and S437A) in the amino acid binding pocket
of mMbSerRS was expected to increase the level of
threonine misrecognition, no improvement in threonine
activation was observed with mutated enzymes Thus,
it appears to be far easier to abolish cognate amino
acid acivation than to replace it with noncognate
activation, despite redesigning the active site in terms
of size, polarity and hydrogen-bonding capacity
Specific amino acid recognition is therefore
substan-tially more resistant to mutation than structure-based
predictions would suggest, as observed previously for
other systems [32] Our results suggest that engineering
methanogenic-type SerRS in an attempt to change its
substrate specificity is not straightforward and may
possibly be achieved only by multiple alterations of the
active site residues Furthemore, contemporary
metha-nogenic-type SerRS and ThrRS may have diverged too
far to readily allow the switching of substrate
specifici-ties between the two, as seen for tryptophanyl-tRNA
synthetase (TrpRS) and tyrosyl-tRNA synthetase
(TyrRS) [32] By contrast, substrate-specificity
switching was possible for GlnRS⁄ GluRS, a much
more closely related pair of aaRSs, which have only
relatively recently diverged from a common ancestor
[33,34]
Flexibility of the motif 2 loop is required for tRNA
binding
The studies described here were undertaken to test the
role of the motif 2 loop in functional binding of
cog-nate tRNA Sequence variability in this loop which exists in different class II aaRSs clearly points to its role in the selectivity of tRNA recognition In order to study binding of the tRNA acceptor end into the active site of mMbSerRS, we superimposed the ThrRS
in complex with cognate tRNA onto the catalytic core
of methanogenic-type SerRS The most striking feature
of the obtained model is the severe steric clash between the acceptor end of tRNA and the motif 2 loop of mMbSerRS, especially with residue Ile342 (Fig 7) In the co-crystal structures of AspRS:tRNA and ThrRS:tRNA complexes, the conformation of motif 2 loop is closed, in comparison with the mMbSerRS motif 2 loop The clash seen in the mMbSerRS:tRNA model, combined with the results of our mutational and kinetic analyses, suggest that a conformational change in this loop is required upon tRNA binding Recognition by methanogenic SerRS relies, in addi-tion to the long extra arm, on G1:C72 in the cognate tRNA, which is achieved by the motif 2 residues (Figs 5 and 7) Two successive glycines in the motif 2 loop are conserved among all the methanogenic-type SerRSs Our biochemical experiments point to the importance of these residues for the flexibility of the tRNA 3¢-end binding region The ability of the loop to change its conformation upon tRNA binding is crucial for correct positioning of the tRNA acceptor end in the active site However, whether these residues also contribute to specific interactions with the tRNA G1:C72 pair remains to be investigated
Role of motif 2 and the SOL in the two steps of the aminoacylation reaction
Strucutural [12] and kinetic data (Table 3) showed that the SOL has a crucial role in cognate amino acid
Table 4 Kinetic parameters for wild-type mMbSerRS enzyme with various tRNA substrates in aminoacylation reaction Mb, Mm and Mj denote M barkeri, M maripaludis and Me jannaschii, respectively.
cat ⁄ K m (s)1Æ M )1) k
cat ⁄ K m rela
In vivo
Mb tRNA Ser
GGA
In vivo
Mb tRNA Ser
CGA
In vitro transcript
Mb tRNASer
In vitro transcript
Mm tRNA Ser
tRNASerfrom
total E coli tRNA
In vitro transcript
Mj tRNASec
a
k cat ⁄ K m (in vivo Mb tRNASerGGA ) ⁄ k cat ⁄ K m (tRNA).
Trang 10binding to mMbSerRS In addition, the motif 2 loop
participates in ATP binding and recognition of the
tRNA 3¢-end in all class II aaRSs The motif 2 loop of
T thermophilus SerRS is disordered in the absence of
substrate It adopts two different conformations upon
substrate binding: an ‘A-conformation’ in the presence
of ATP or adenylate or a ‘T-conformation’ when
tRNASer is bound In mMbSerRS, the motif 2 loop is
fully ordered in an apo-enzyme structure However, in
the presence of ATP or seryl-adenylate the motif 2
loop shifts (Fig 8) In addition, the side chains of the
conserved arginine residues in the motif 2 loop show a
concerted movement following substrate binding in the
active site In the apo-enzyme, the motif 2 Arg336
occupies the adenine-binding site and upon binding of
ATP or Ser-AMS shifts to form interactions with the
a-phosphate group Arg336 also interacts with the carbonyl oxygen of Ser-AMS, but not with serine Therefore, this residue can sense the presence of both substrates needed for the first step of the amino-acylation reaction
Fig 7 Proposed model of the mMbSerRS:tRNA complex Overall
view with ThrRS:tRNA Thr complex (1qf6) shown as a ribbon
repre-sentation and colored in orange The apo-structure mMbSerRS
(2cim) is shown as a blue transparent surface For clarity, only the
tRNA CCA-end and the first two base pairs are shown as sticks.
Two synthetases are superposed by means of their catalytic
domains The view reveals steric clashes between the mMbSerRS
motif 2 loop and the acceptor end of tRNA (circled) The gray arrow
indicates the likely movement of the mMbSerRS N-terminal domain
upon tRNA binding (Inset) Comparison of the motif 2 loops in
AspRS:tRNA (1c0a; red) ThrRS:tRNA (1qf6; orange) and
mMbS-erRS (2cim; blue) structures, resulting from the superpositions of
their entire catalytic domains The backbone of the tRNA Thr
accep-tor arm is shown (tRNAAsphas been omitted for clarity) The open
conformation of motif 2 loop in apo-structure of mMbSerRS causes
clashes with tRNA, especially Ile342.
Serine
ATP
serine loop
Beginning of motif 3 Motif 2
loop Helix 9
Fig 8 Conformational changes in the active site upon binding of the substrates The r.m.s.d of C a atoms after superposition of the catalytic domains in complex with different substrates and the cata-lytic domain of the apo-enzyme The binding of the small substrates
in the active site leads to conformational changes in the motif 2 loop and SOL The serine loop residues were excluded from calcu-lation of the r.m.s.d because they are not visible in the apo-struc-ture or the strucapo-struc-ture of the enzyme in complex with ATP However, it can be seen that the residues that are enclosing this loop are flexible and adopt a range of conformations.