From the Institute of Molecular and Structural
Biology, Aarhus University, DK-8000 Aarhus C, Denmark and the
¶ Institute of Physical Biochemistry, University of
Witten/Herdecke, D-58448 Witten, Germany
Received for publication, March 12, 2001
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ABSTRACT |
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Elongation factor Tu (EF-Tu) undergoes a large
conformational transition when switching from the GTP to GDP forms.
Structural changes in the switch I and II regions in the G domain are
particularly important for this rearrangement. In the switch II region,
helix Elongation factor Tu
(EF-Tu)1 belongs to the
family of GTP-binding proteins, which generally cycle between an
active, GTP-bound form, in which they interact with the respective
downstream effectors, and an inactive, GDP-bound form. The proportion
between these two forms is regulated by other proteins such as guanine
nucleotide exchange factors and GTPase-activating proteins (GAPs) (1). The function of EF-Tu·GTP is to bind aminoacyl-tRNA (aa-tRNA) to form
a stable ternary complex that interacts with the A site of the
mRNA-programmed ribosome. Upon codon recognition, rapid GTP
hydrolysis takes place. As a consequence, EF-Tu switches into the GDP
conformation, releases the aa-tRNA into the A site, and dissociates
from the ribosome. Active EF-Tu·GTP then is regenerated by the
GDP-GTP exchange catalyzed by another elongation factor, EF-Ts.
The structures of GTPase family members reveal a common structural
GDP/GTP-binding core (the G domain) and several consensus sequence
elements defining the guanine nucleotide-binding site (1, 2). The
switch between the active and the inactive forms of the proteins
involves conformational changes in two common parts of the G domain:
the switch I region, which is usually positioned between helix The conformational transitions that take place in EF-Tu upon switching
between its GTP and GDP states have been elucidated from the x-ray
structures of EF-Tu·GDP from Escherichia coli (3-6) or
Thermus aquaticus (4) and EF-Tu·GDPNP from T. aquaticus (7) or Thermus thermophilus (8). It
was suggested that the hydrolysis of GTP and the release of the
The structure-function relations of the two glycines, Gly83
and Gly94, flanking helix Ribosomes, EF-Tu, and tRNAs--
Ribosomes from E. coli MRE600 and tRNAs were prepared as described (13, 14). Wild
type EF-Tu with a C-terminal Ser(His)6 tag and EF-Tu
mutants were purified as described (11, 12). Ribosome complexes with
blocked P sites were prepared by incubating the ribosomes with a
1.3-fold excess of AcPhe-tRNAPhe and 0.5 mg/ml of poly(U)
in buffer A (50 mM Tris-HCl, pH 7.5, 50 mM
NH4Cl, 10 mM MgCl2) for 30 min at
37 °C.
Binding of EF-Tu·GTP·Phe-tRNAPhe to the A
Site--
EF-Tu·GDP (20 pmol) were activated by incubation with 1 mM GTP, 3 mM P-enolpyruvate, 200 µg/ml
pyruvate kinase, and 1 mM dithiothreitol in 20 µl of
buffer B (50 mM Tris-HCl, pH 7.5; 50 mM
NH4Cl) supplemented with MgCl2 (0, 3, 5, 7, and
10 mM) for 15 min at 37 °C. Purified [14C]Phe-tRNAPhe (15 pmol; 1000 dpm/pmol)
were added in 5 µl of the appropriate buffer. Poly(U)-programmed
ribosomes (10 pmol) were added in 25 µl of buffer, and the reaction
was carried out for 3 min at 37 °C. The background was determined in
the absence of EF-Tu and was subtracted. Ribosome-bound
[14C]Phe-tRNAPhe was determined by filtering
the reaction through nitrocellulose filters that had been soaked in
buffer C (50 mM Tris-HCl, pH 7.5, 50 mM
NH4Cl, 7 mM MgCl4). The filters
were washed with 3 ml of buffer C and dissolved in scintillation
mixture QS361 (Zinsser Analytics) before determining the amount of
radioactivity retained on the filter in a scintillation counter.
Rapid Kinetic Methods--
Fluorescence stopped flow
measurements were carried out at 20 °C as described previously (13)
using an SX-18MV Spectrometer (Applied Photophysics). The fluorescence
of proflavin was exited at 436 nm and detected after passing a KV 500 filter (Schott), whereas the fluorescence of the mant-group was exited
at 349 nm and measured after passing a KV 408 filter (Schott). The
experiments were initiated by rapid mixing of equal volumes (60 µl
each) of the ternary complex EF-Tu·GTP·aa-tRNA and the ribosome
complex at the concentrations given in the figure legends.
Ternary complexes were formed from EF-Tu·GTP and
Phe-tRNAPhe charged in situ and were not
purified further, because the ternary complexes with EF-Tu mutants were
unstable and dissociated during gel filtration. The extent of
tRNAPhe(Prf16/17) charging was found to be ~75%. The
ratio between EF-Tu and aa-tRNA was optimized to ensure the best
possible signal-to-noise ratio, which was found when either EF-Tu or
aa-tRNA was present in 4-fold excess. The same amount of ternary
complex was formed independently of whether EF-Tu or aa-tRNA is present
in excess. The unpurified system gave results similar to those obtained
previously with ternary complex purified by gel filtration containing
wild type EF-Tu with a C-terminal Ser(His)6 tag (23) with
respect to both rates and fluorescence effects.
When the fluorescence signal originating from the proflavin label was
followed, the ternary complex was prepared by incubating 2.4 µM EF-Tu, 16 µM GTP, 3 mM ATP,
3 mM P-enolpyruvate, 0.6 µM tRNAPhe (Prf16/17), 1% phenylalanyl-tRNA synthetase, 1 mM phenylalanine, 10 µg/ml pyruvate kinase, and 0.05 µM EF-Ts in buffer A for 30 min at 37 °C. When
mant-GTP fluorescence was to be monitored, 2.4 µM EF-Tu
was incubated in buffer A with 1.2 µM EF-Ts and 2.4 µM mant-GTP for 15 min at 37 °C. Then 0.6 µM purified Phe-tRNAPhe was added, and the
incubation was continued for 10 min at room temperature. Where
indicated, the ratio between EF-Tu and Phe-tRNAPhe was
reverted without affecting the total amount of ternary complex formed.
mant-GTP was always present in the same molar amount as EF-Tu, whereas
EF-Ts was always present in half the molar amount as EF-Tu.
When EF-Tu·mant-GTP was present in excess over
Phe-tRNAPhe, that is under the conditions used to study the
conformational changes of Phe-tRNAPhe, the fluorescence
change obtained was small or absent because of the strong fluorescence
background of uncomplexed EF-Tu·mant-GTP. In the cases of wild type
EF-Tu and the mutant EF-Tu(G94A) where a fluorescence change could be
detected, the resulting data were used in numerical integration. The
data obtained with the excess of Phe-tRNAPhe over
EF-Tu·mant-GTP were only used for a qualitative evaluation of the
reaction steps. In the cases where apparent rates could be derived,
these appeared to be comparable irrespective of which component,
EF-Tu·mant-GTP or Phe-tRNAPhe, was in excess.
For quench-flow experiments, a KinTek quench-flow apparatus was used.
Equal amounts (16 µl each) of ribosome complex and ternary complex
were rapidly mixed at 20 °C, and the reaction was stopped by the
addition of the appropriate quenching solution (see below). To measure
the rates of GTP hydrolysis, the ternary complex was prepared by
incubating 0.6 µM EF-Tu, 4 µM
[
To measure the rates of dipeptide formation, the ternary complexes were
formed by the incubation of 2.4 µM EF-Tu, 16 µM GTP, 3 mM P-enolpyruvate, 10 µg/ml
pyruvate kinase, 0.6 µM purified [3H]Phe-tRNAPhe (4800-5300 dpm/pmol), and
0.05 µM EF-Ts in buffer A for 5 min at 37 °C. After
mixing with poly(U)-programmed ribosomes containing AcPhe-tRNAPhe in the P site, the reaction was terminated by
quenching with 1 M KOH, incubated at 37 °C for 1 h,
and then neutralized with acetic acid. The peptides formed were
analyzed by high pressure liquid chromatography as described
(13). Also in the absence of the factor, Phe-tRNAPhe was
able to slowly bind to the free A site (not shown). The background level of dipeptide formation caused in this way was measured in the
absence of EF-Tu, and the respective correction was made.
Determination of Rate Constants--
Apparent rate constants
were determined by exponential fitting, using one, two, or three
exponential terms (characterized by variable time constants,
kapp, and respective amplitudes) and a variable
for the final signal. Codon-independent initial binding of the ternary
complex to ribosomes was measured at a fixed concentration of
EF-Tu·GTP·Phe-tRNAPhe(Prf16/17) (0.3 µM
in the reaction) and different concentrations of nonprogrammed
ribosomes (0.3-1.0 µM). Apparent rate constants (kapp) were obtained by a single exponential
fitting, and rate constants k1 and
k During the binding of the ternary complex EF-Tu·GTP·aa-tRNA to
the ribosomal A site, eight steps can be distinguished by biochemical and pre-steady-state kinetic techniques (Fig.
1; for references, see Ref. 13). In the
first step, the ternary complex forms a labile initial binding complex
with the ribosome (rate constants of the forward and backward reactions
are defined as k1 and
k2 is flanked by two glycine residues:
Gly83 in the consensus element
DXXG at the N terminus and Gly94 at the C
terminus. The role of helix
2 was studied by pre-steady-state kinetic experiments using Escherichia coli EF-Tu mutants
where either Gly83, Gly94, or both were
replaced with alanine. The G83A mutation slows down the association of
the ternary complex EF-Tu·GTP·aminoacyl-tRNA with the ribosome and
abolishes the ribosome-induced GTPase activity of EF-Tu. The G94A
mutation strongly impairs the conformational change of EF-Tu from the
GTP- to the GDP-bound form and decelerates the dissociation of
EF-Tu·GDP from the ribosome. The behavior of the double mutant is
dominated by the G83A mutation. The results directly relate structural
transitions in the switch II region to specific functions of EF-Tu on
the ribosome.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and
strand
2 and varies in length, sequence, and structure among
different GTPases, and the more conserved switch II region,
which consists of helix
2 and the flanking loops. The N-terminal
flanking loop contains the consensus element DXXG, which is
involved in binding of the
-phosphate, the Mg2+ ion, and
a water molecule in the vicinity of the
-phosphate. Many of the
family members, e.g. EF-Tu, G
i1, and small
Ras-like GTPases, also contain one or more glycines located in the
C-terminal loop flanking the switch II region.
-phosphate induces the transition starting from Gly83
(E. coli numbering) at the N terminus of helix
2 by
breaking the hydrogen bond between the main chain amide nitrogen and
the
-phosphate. As a consequence, the peptide bond between
Pro82 and Gly83 flips by 150°, thereby
causing a rotation of helix
2 by 42°. Additionally, the position
of the helix is shifted from spanning residues 87-97 in the GTP form
to residues 83-93 in the GDP form. The switch II region is coupled to
the switch I region, which during the conformational switch undergoes a
remarkable structural change causing an
to
rearrangement in the
conserved part of the region (residues 53-59) (4, 5). The movements in
the two switch regions create a new surface of the G domain,
which, in turn, leads to a reorientation of domains 2 and 3, which
rotate by 90° and become separated from the G domain. Similar
structural transitions have been observed for helix
2 of other G
proteins, such as Ras (9) and G
i1 (10).
2 of EF-Tu, were previously
investigated by mutation to alanines either separately or in
combination, with the aim of restricting the flexibility of helix
2.
The mutant proteins were subjected to in vitro
characterization under steady-state conditions with respect to their
interactions with GTP, GDP, aa-tRNA, and nonprogrammed ribosomes.
Overall, the restriction of the flexibility at position 83 appeared to
cause a severe defect in the ability of the factor to interact
productively with the ribosome (11), although the substitution of
glycine with alanine at position 94 resulted in a conformation that
favored the binding of GTP (12). These findings prompted us to study
the interaction between the ternary complexes formed with these mutants
and the ribosome using pre-steady-state kinetic methods. The results
allow the identification of the partial reactions of EF-Tu on the
ribosome that are affected by the mutations.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
32P]GTP (1300-1900 dpm/pmol), 3 mM
P-enolpyruvate, 10 µg/ml pyruvate kinase, 2.4 µM
purified Phe-tRNAPhe, and 0.05 µM EF-Ts in
buffer A for 5 min at 37 °C. The GTPase reaction was terminated with
1 M HClO4, 3 mM potassium
phosphate, and the liberated inorganic phosphate was extracted as
described (13). The background caused by Pi contamination
in GTP and intrinsic GTPase activity of EF-Tu was measured both before
and after the quench-flow experiment and subtracted.
1 were determined from the linear
concentration dependence of kapp.
k
2 was assumed to be unaffected by the
mutations studied, and a value of 0.2 s
1 was taken from
previous experiments with wild type EF-Tu. The rate constants
k2-k6 were calculated by
global fitting of the combined sets of time courses measured by
monitoring proflavin fluorescence at five different ribosome
concentrations, mant-dGTP fluorescence, GTP hydrolysis, and dipeptide
formation. Fitting was performed by numerical integration using
Scientist for Windows software (MicroMath Scientific Software) as
described (13). The fit yielded a unique solution for the rate
constants k2-k6 as well
as for the fluorescence factors, provided the values for k1, k
1, and
k
2 and for the relative fluorescence of
aa-tRNA in the initial binding complex were fixed. For values that were
measured directly, standard deviations were calculated from the
variation of several experiments. For values calculated by global
fitting, the standard deviation for one parameter was determined for
the case when all other parameters, except the ones measured directly,
were allowed to change. That is, if a given parameter was set to a
value outside the range of standard deviation, no fit satisfying all
data sets could be obtained.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1, respectively). The subsequent codon
recognition (k2 and k
2) triggers the activation of the GTPase of EF-Tu
(k3), which is followed by instantaneous GTP
hydrolysis (kGTP). Subsequent release of
Pi induces the conformational transition of EF-Tu
from the GTP form to the GDP form (k4), whereby
the factor loses the affinity for aa-tRNA. As a result, the 3' end of
the aa-tRNA is free to accommodate in the 50 S A site
(k5) and takes part in rapid peptide bond
formation (kpep). In a parallel reaction,
EF-Tu·GDP dissociates from the ribosome
(k6).
View larger version (31K):
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Fig. 1.
Kinetic scheme of EF-Tu-dependent
aa-tRNA binding to the ribosomal A site. Kinetically resolved
steps are indicated by numbered rate constants, and steps that are
rate-limited by the preceding step are designated
kGTP and kpep. EF-Tu is
depicted differently in the GTP- and GDP-bound conformations, and
GTP* denotes the GTPase state.
A fluorescent tRNA derivative, tRNAPhe (Prf16/17), was used to monitor the steps of initial binding, codon recognition, and A site accommodation of aa-tRNA (13, 14), whereas a fluorescent GTP analog, mant-GTP, was used to monitor the rearrangement of EF-Tu to the GTPase-activated state and dissociation of EF-Tu·GDP from the ribosome (15); time courses were measured using the stopped flow technique. The rates of GTP hydrolysis and peptide bond formation were determined by the quench-flow technique. Ternary complexes were formed from EF-Tu·GTP and Phe-tRNAPhe charged in situ with either tRNA or the factor added in 4-fold excess (see "Experimental Procedures") and were used without subsequent purification.
Initial Binding of the Ternary Complex to the Ribosome--
The
labile binding of the ternary complex to the ribosomes in the absence
of mRNA or in the presence of a nonmatching codon in the A site
reflects the initial codon-independent step of A site binding that
precedes codon recognition (16). The rate constants of the reaction,
k1 and k1, were
determined by stopped flow fluorescence measurements in which the
ternary complex formed from Phe-tRNAPhe(Prf16/17) and EF-Tu
was rapidly mixed with nonprogrammed ribosomes, and the resulting
fluorescence change was recorded.
The initial binding of the ternary complex,
EF-Tu·GTP·Phe-tRNAPhe(Prf16/17), to the ribosome gives
rise to a rapid fluorescence increase (16). The apparent rate constant
of the reaction, kapp, increased linearly with
ribosome concentration for all three mutants as with wild type EF-Tu
(Fig. 2), yielding the values of
k1 and k1 summarized in
Table I. The G94A mutation had no
influence on initial complex formation, whereas the mutation at
position 83 lowered the association rate constant,
k1, about 20-fold. Additionally, k
1 was increased for the double mutant,
G83A/G94A, indicating a lower stability of this complex.
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Codon-dependent Binding of Ternary Complexes to the A Site-- The extent of stable, codon-dependent binding of EF-Tu·GTP·Phe-tRNAPhe was measured with poly(U)-programmed ribosomes using nitrocellulose filtration. EF-Tu mutations at positions 83 and 94 slightly destabilized the complex, such that the mutants required 7 mM Mg2+ for maximum binding of Phe-tRNAPhe compared with 5 mM for the wild type (data not shown). At 10 mM Mg2+, the conditions of kinetic experiments, stable ribosome binding was obtained with wild type and all EF-Tu mutants.
The structural transitions of aa-tRNA during ternary complex binding to
the A site were monitored by following the fluorescence changes of
tRNAPhe(Prf16/17). In the case of the wild type ternary
complex, the time course of A site binding comprised a fast increase of
fluorescence followed by a slower decrease (Fig.
3A), as observed previously with purified ternary complexes (14). Therefore, the assignment of
fluorescence changes to particular steps of A site binding was taken
from the previous data (Ref. 13 and references cited therein). The
fluorescence increase reflects both the initial binding of the ternary
complex to the ribosome and the codon recognition step (Fig. 1 and
Refs. 14 and 16). The subsequent steps of GTPase activation and GTP
hydrolysis do not change the fluorescence of proflavin. Rather, the
observed fluorescence decrease reflects the conformational change of
the aa-tRNA that is coupled to the transition of the factor from the
GTP to the GDP form and the release of aa-tRNA from EF-Tu·GDP. The
effect is partially masked by the fluorescence increase because of the
accommodation of aa-tRNA in the 50 S A site (14, 16). The fluorescence
changes observed with wild type EF-Tu and the mutant EF-Tu(G94A) are
qualitatively similar (Fig. 3A), although the consumption of
the high fluorescence state is extremely slow with the G94A mutant,
about 0.07/s, and is only observed upon prolonged incubation (Fig.
3B).
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The time courses obtained with the mutants with substitutions at
position 83 are different (Fig.
4A). A biphasic rapid increase of fluorescence was observed, whereas no decay could be detected. The
first step reflected in the fluorescence increase is comparable with
that of the wild type. The apparent rate of the first step increased
linearly with ribosome concentration (Fig. 4B), suggesting that the rate of binding of the ternary complexes was limited by the
second order initial binding step (Fig. 1). Neither EF-Tu(G83A) nor
EF-Tu(G83/94A) promote the formation of the high fluorescent intermediate observed for both the wild type and the mutant EF-Tu(G94A) (compare Figs. 3 and 4). Most likely, the high fluorescence
intermediate is not accumulated because the rate of initial complex
formation with the 83 mutants is slow compared with the wild type and
is rate-limiting for the subsequent steps.
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The second step observed in the time courses with the 83 mutants was very slow and independent of the ribosome concentration and was essentially identical to the rate of dipeptide formation (see below). In the presence of deacylated tRNA in the P site, where there is no peptide bond formation, similar time courses were observed (data not shown), indicating that the fluorescence change is caused by a conformational change of the aa-tRNA that limits the dipeptide bond formation rather than by the chemistry step. Thus, the slow step in the time courses of A site binding observed for the two 83 mutants is assigned to the rearrangement of aa-tRNA during the accommodation in the A site that precedes the peptidyl-transfer reaction.
GTPase Activation--
The formation of the GTPase-activated
conformation of EF-Tu was monitored by measuring the fluorescence
changes of a fluorescent GTP derivative, mant-GTP, upon ternary complex
binding to poly(U)-programmed ribosomes. In the case of the wild type
factor, the resulting time course showed a fast fluorescence increase
followed by a slower decrease (Fig. 5),
as observed earlier (15). The increase reflects the structural
rearrangement of EF-Tu, which brings the factor into the state
competent for GTP hydrolysis. Subsequent GTP cleavage initiates the
conformational change of EF-Tu from the GTP- to the GDP-bound state,
followed by release of aa-tRNA and dissociation of EF-Tu·GDP from the
ribosome. The latter step is reflected in the decrease of the
fluorescence signal, whereas GTP hydrolysis itself is not reported.
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Reliable measurements of the change in mant fluorescence required a reversion of the ratio of Phe-tRNAPhe and EF-Tu·mant-GTP in the experiment such that EF-Tu·mant-GTP was present in substoichiometric amounts relative to aa-tRNA (1:4; "Experimental Procedures"). With both wild type EF-Tu and mutant EF-Tu(G94A) a similar fluorescence increase reporting the GTPase activation was observed (Fig. 5A). The mutant EF-Tu(G94A) also showed the subsequent consumption of the high fluorescence state that is characteristic for the wild type factor, although the signal decrease was much slower for the mutant (Fig. 5B).
With mutant EF-Tu(G83A), the mant-GTP fluorescence increase was very small, indicating that either only part of the molecules were activated or that the environment of the mant group in the activated state is different in EF-Tu(G83A) and the wild type factor. The additional mutation of Gly94 partly restores the activation, so that the fluorescence increase of about 20% observed with EF-Tu(G83A/G94A) is comparable with that observed with wild type EF-Tu (Fig. 5C). With both EF-Tu(G83A) and EF-Tu(G83/94A), the rate of GTPase activation was slower than with the wild type EF-Tu, because of a lower rate of binding to the ribosome (Table I).
GTP Hydrolysis--
Kinetics of GTP hydrolysis were measured by
quench-flow ("Experimental Procedures") at two different
concentrations of poly(U)-programmed ribosomes, 0.5 and 1.0 µM. The data could be fitted with a single exponential
function (Fig. 6A). The rates
of GTP hydrolysis are similar for the wild type and the mutant
EF-Tu(G94A), although the extent of GTP hydrolysis was somewhat less
with the latter. In contrast, essentially no GTP hydrolysis was
observed with the two G83A mutants.
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Peptide Bond Formation-- Time courses of AcPhePhe dipeptide formation were measured by quench-flow. In the presence of wild type EF-Tu, the reaction was rapid and efficient (Fig. 6B). The extent of dipeptide formation was ~70% for the wild type and ~40% for the mutant EF-Tu(G94A), matching the efficiency of GTP hydrolysis. Unexpectedly, efficient dipeptide formation was also found with the mutants EF-Tu(G83A) and EF-Tu(G83A/G94A) (Fig. 6B), although both mutants were inactive in GTP hydrolysis.
Elemental Rate Constants of A Site Binding--
The results shown
in Figs. 3-6 provide qualitative information on the influence of the
mutations of Gly83 and Gly94 on the function of
EF-Tu on the ribosome. To quantify these effects, the elemental rate
constants of the A site binding were calculated by global fitting of
combined data sets comprising time courses obtained with all
observables, i.e. proflavin fluorescence at five
different ribosome concentrations, mant-GTP fluorescence, GTP
hydrolysis, dipeptide formation, as in Figs. 3-6. Rate constants k1 and k1 were
determined independently (Fig. 2 and Table I) and were used as fixed
values in the calculations. The dissociation rate constant of the codon
recognition complex, k
2, as obtained
previously with wild type EF-Tu, is small (0.2/s; Ref. 13) and is
determined by the interactions of aa-tRNA with the mRNA codon;
therefore, the value of k
2 was assumed to be small also with EF-Tu(G94A). For both the mutant EF-Tu(G94A) and wild
type, the rates of GTPase activation and GTP hydrolysis were indistinguishable under the experimental conditions applied. Therefore, the two steps are grouped for the global analysis. The values of
k2-k6 were obtained by
simultaneous fitting of all data sets to the scheme shown in Fig. 1 by
numerical integration as described (13). The resulting rate constants
are summarized in Table I. From the rate constants, time curves were
calculated for different concentrations of ribosomes; the results for
EF-Tu(G94A) are shown in Fig. 7 along
with the experimental data. The values of k1, k
1, k2,
k3, and k6 obtained for
the wild type EF-Tu are identical to those reported earlier (13). The
values of k4 and k5 are
somewhat lower than the published values, possibly because of the use
of unpurified ternary complexes and/or of EF-Tu containing a C-terminal
histidine tag in the present study. The G94A mutation decreases the
rate constants of codon recognition and GTPase activation; the effect
is moderate so that the resulting time courses of GTP hydrolysis are
similar to those obtained with wild type EF-Tu (Fig. 6A).
The major effect of the G94A mutation is a decrease of the rate
constant for the conformational change from the GTP form to the GDP
form by more than 300-fold. Additionally, the rate constant of
dissociation of EF-Tu·GDP from the ribosome is decreased 20-fold.
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The conclusion that the replacement of G94A strongly affects the conformational transition of EF-Tu toward the GDP form may be verified qualitatively on the basis of the time courses (Figs. 3, 5, and 6). Compared with the wild type, the rate of breakdown of the high fluorescent intermediates are strongly diminished with the G94A mutant; the rate of the fluorescence decrease is similar for both fluorescence labels and identical with the rate of peptide bond formation, 0.07/s, suggesting a common rate-limiting step for the reactions that follow GTP hydrolysis. According to the kinetic mechanism of A site binding (Fig. 1), this step is the conformational transition of EF-Tu from the GTP- to the GDP-bound form.
The analysis of the elemental rate constants of the A site binding of
the ternary complexes with EF-Tu(G83A) and EF-Tu(G83/94A) was not
possible, because the reactions of codon recognition and GTPase
activation (when observed) were limited by the rate of the bimolecular
initial binding step at all concentrations measured (Fig.
4B). The qualitative summary of the reactions that do take place with the two 83 mutants is given in Table I. Both mutants were
capable to promote productive binding of aa-tRNA to the A site, albeit
slowly, despite the lack of GTP hydrolysis. The EF-Tu(G83A) mutant had
a reduced GTPase activation and was deficient in GTP hydrolysis,
whereas the additional mutation G94A restored the rearrangement
attributed to GTPase activation, although GTP hydrolysis did not take
place. This suggests that the G83A mutation abolishes the GTP
hydrolysis, possibly by directly affecting the chemistry step.
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DISCUSSION |
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Role of Helix 2 in EF-Tu Binding to the Ribosome--
The
replacement of glycine 83 at the N terminus of helix
2 in the switch
II region of EF-Tu with alanine decreased the rate of initial,
codon-independent binding of the ternary complex to the ribosome
by a factor of 10. Although Gly83 is located on the side of
EF-Tu that contacts the ribosome (17), it is not likely that a single
substitution of glycine by alanine hinders the ribosome interaction
directly. Rather, the overall conformation of the ternary complex
appears to be changed, thereby affecting the association.
The switch I and II regions of EF-Tu are intimately involved in the
binding of aa-tRNA (18). Helix 2 points directly into the major
groove of the acceptor helix of the aa-tRNA. Tyr87,
Lys89, and Asn90 interact with residues at the
5' end of the tRNA, and Asp86 interacts with the phosphates
at positions 3 and 64 in a triangular arrangement over the major groove
of the acceptor stem (the residue numbers refer to E. coli
EF-Tu). The substitution of Gly83 with alanine and the
resulting displacement of helix
2 would probably bring a phosphate
group of the tRNA backbone closer to the carboxylate of
Asp86, which could impair the binding of aa-tRNA (18). In
fact, the Gly83 mutation resulted in a weakened complex
(11), although a functional ternary complex was formed. The unstable
nature of the ternary complexes formed by the mutants EF-Tu(G83A) and
EF-Tu(G83/94A), which may be further compromised on the ribosome,
explains why aa-tRNA was released into the A site part of the peptidyl
transferase center, even though GTP was not hydrolyzed by the 83 mutants.
Similar effects of the glycine in the N-terminal part of helix 2
have been reported for other GTP-binding proteins. In Ras, the mutation
of the homologous Gly60 to alanine inhibits the
conformational change induced by GTP binding and reduces the affinity
of Ras·GTP to its effector Raf (19). The analogous mutation in
G
s, G226A, abolishes the activation of the effector
adenylyl cyclase, and the mutant G
s is unable to undergo
the conformational transition required for the
GTP-dependent dissociation from the
subunits.
Furthermore, the mutation lowers the apparent affinity of
G
s for Mg2+ (20, 21). EF-Tu(G83A) behaves
similarly in that it requires higher concentrations of Mg2+
for full activity in A site binding compared with wild type and does
not sustain protein synthesis at low Mg2+ concentrations
(11). A molecular dynamics study of the conformational switch in Ras
(22) suggests that the formation of a transient complex between the
switch I and II regions established during conformational switching is
important for effector interaction. This may be hindered by mutation of
the pivotal glycine in the DXXG consensus motif.
Role of Glycine 83 in Ribosome-induced GTP Hydrolysis--
The
G83A mutation in EF-Tu abolishes GTP hydrolysis in the ternary complex
on the ribosome. Both the rapid GTP cleavage that is induced by correct
codon-anticodon interaction in the ribosome decoding center (this
paper) and the slow GTP hydrolysis brought about by binding to
nonprogrammed ribosomes (11) are suppressed. The catalytic water
molecule is held in position by hydrogen bonding with the main chain
amides of glycine 83 and histidine 84 as well as with the carboxyl
group of threonine 61 (7, 8). The introduction of an alanine side chain
at position 83 in the -phosphate-binding pocket creates a steric
clash with valine 20. Therefore, the replacement of Gly83
with an amino acid with a larger side chain may be expected to interfere with GTP binding. However, the binding of GTP or GDP was not
much affected by the mutation (11), indicating that Gly83,
by a movement of helix
2 as a whole, is shifted away from the GTP-binding site to accommodate an alanine at the N terminus of the
helix. It is possible that the presumed displacement of helix
2
changes the position of the catalytic water and thereby suppresses ribosome-stimulated GTP hydrolysis. Interestingly, the intrinsic GTPase
activity of EF-Tu was stimulated, rather than suppressed, by the G83A
mutation (11), indicating that intrinsic and ribosome-stimulated GTP
hydrolysis may have different mechanisms and suggesting that restricting the conformational flexibility at the N terminus of helix
2 impairs the proper positioning of EF-Tu for GTPase activation on
the ribosome.
The mutation in the subunit of the heterotrimeric G protein
G
i1 at the position Gly203, which is
analogous to Gly83 of EF-Tu, did not affect the intrinsic
GTPase activity (24). Although the rate of intrinsic GTPase is much
higher in G
than in EF-Tu, this may indicate that in both cases the
intrinsic reaction does not require a reorientation at the N-terminal
part of the switch II region. In fact, the comparison of the structures
of G
i1 in the GTP form, G
i1·GTP
S,
and in the transition state mimicked by the
Gi
1·GDP·AlF4- complex (25)
shows that the position of Gly203 does not change during
GTP hydrolysis, whereas the neighboring catalytically important
glutamine 204 rotates to stabilize the transition state. It is likely
that the formation of the ribosome-induced GTPase transition state of
EF-Tu involves an additional movement, which involves the glycine
residue at the N terminus of the switch II region and is precluded by
larger residues at that position.
A model of GTPase activation by GAP has been put forward for Ras and
two small Ras-like GTPases, Rho and Cdc42Hs (26-29). When the
structures of the Rho·RhoGAP·GDP·AlF4 complex
representing a transition state analog of GAP-stimulated GTP hydrolysis
and of the Cdc42Hs·RhoGAP·GDPNP complex representing the ground
state are compared, a rigid body rotation of RhoGAP of ~20° is
observed. The switch II region seems to act as a pivot point around
which the motion occurs on progression from the ground state to the
transition state (27). Assuming a similar rearrangement for EF-Tu, a
reduced rotational flexibility at the N-terminal glycine of helix
2
is likely to severely impair the transition to the activated state and
subsequent GTP hydrolysis. The proteins that inhibit the GTPase
activity of Cdc42, RhoGDI, or the effectors WASP and ACK have been
reported to bind to the switch II region of Cdc42 (30-32), further
emphasizing the importance of the switch II region for GTP hydrolysis.
The mechanism of GTPase activation in EF-Tu on the ribosome is not
known. Docking of the ternary complex onto the crystal structure of the
50 S ribosomal subunit indicates that parts of the switch I and II
regions are positioned between ribosomal protein L14 and the
sarcin-ricin loop (33). In this way, the ribosome may contribute
catalytically important residues and/or help in reorienting residues
within EF-Tu into the catalytically competent positions. The rate of
GTP hydrolysis in EF-Tu on the ribosome is controlled by the extent of
correct base pairing between the mRNA codon in the A site and the
anticodon of aa-tRNA in the ternary complex (34, 35). The signal for
GTPase activation has been shown to be transmitted (at least partially)
through the tRNA (36). It is conceivable that helix 2 of EF-Tu is in
intimate contact with both aa-tRNA and the ribosome and functions as a mediator of the communication between the components involved in the
elongation process. The G83A mutation weakens the binding of aa-tRNA to
EF-Tu in solution (11) and allows the dissociation of aa-tRNA from
EF-Tu·GTP on the ribosome (present data), suggesting that the
tRNA-dependent transmission of the signal for the
activation of GTP hydrolysis in EF-Tu is impaired by mutations of
glycine 83.
It is interesting to compare GTP-binding proteins with ATP-hydrolyzing
molecular motors such as myosin, kinesin, and others (37). In motor
ATPases, the glycine residue, which is structurally and functionally
equivalent to Gly83 in EF-Tu, is located in close vicinity
to the -phosphate of ATP in the loop that has a consensus sequence
DIXGFE. In myosin II from
Dictyostelium (38), the mutation of this glycine
(Gly457) to alanine abolished the ATPase activity, and the
rotation of the switch II loop at two pivotal residues,
Ile455 and Gly457, is believed to be important
for the transition from the ATP-bound to the ADP·Pi state
(39). Moreover, equivalent salt bridges are found between the switch I
and switch II regions in both EF-Tu and myosin. This indicates that the
NTPase activation in EF-Tu and myosin requires a similar structural
rearrangement, pivoting around the glycine in the N-terminal part of
the switch II region.
Transition of EF-Tu from the GTP to the GDP Conformation--
The
mutation of the C-terminal glycine in helix 2, Gly94, to
alanine decreases the rate of the rearrangement of EF-Tu from the GTP
to the GDP form by a factor of 300. Because the factor assumes the GDP
form very slowly, the release of aa-tRNA from the factor is also
delayed, resulting in the retardation of the subsequent reactions of
aa-tRNA, i.e. the accommodation in the A site and peptide
bond formation. In the GTP form, position 94 is found internally in
helix
2; in the structure of EF-Tu from T. aquaticus (7),
the corresponding Gly95 has
and
angles of
78°
and
34°, a backbone conformation that can be taken up by any amino
acid residue. Upon switching to the GDP conformation, helix
2 is
shifted with respect to its position in the primary structure and
terminates at position 94. In the GDP conformation, Gly94
takes up a new backbone conformation characterized by
and
angles of 85° and 20° (7). This set of angles is found in an area
of the Ramachandran plot where only the very flexible glycine residue
can enter. This explains why the conformational change to the GDP form
is inhibited by the mutation and indicates that the mutant protein will
assume an alternative, more GTP-like conformation, in keeping with the
observed slower dissociation of mutant EF-Tu·GDP from the ribosome.
The replacement of Gly94 with alanine dramatically
increases the affinity of EF-Tu for GTP and almost completely
eliminates the normal 200-fold preference for GDP over GTP (12).
Furthermore, EF-Tu(G94A)·GDP exhibited significant affinity for
aa-tRNA, in contrast to wild type EF-Tu·GDP. Thus, the reduced
flexibility at the C terminus of helix 2 seems to lock EF-Tu in a
GTP-like conformation.
The effect of the G83A mutation on the conformational changes of EF-Tu
following GTP hydrolysis could not be monitored, because no GTP
cleavage was observed within the time range of our experiments. The
structure of Gi1(G203A) was found to contain
GDP·Pi (24), suggesting that the analogous mutation G83A
in EF-Tu may affect the rearrangement of the factor from the GTP to the
GDP form. The stalling of the inorganic phosphate product indicates
that a conformational transition normally accompanies Pi
release and that this rearrangement is blocked by the mutation of the
glycine at the N terminus of helix
2.
The common structural features of GTPases and motor ATPases
suggest that they share similar strategies for undergoing the conformational change, and the mechanisms by which the presence of the
-phosphate is sensed appear to be similar (37). However, there is an
important difference between motor ATPases and regulatory GTPases. In
most GTP-binding proteins, GTP hydrolysis and subsequent release of
Pi is required to induce the dissociation from the downstream effectors. In contrast, dissociation of Pi from
motor proteins is slow, and the ADP·Pi form is crucially
important for the mechanochemical cycle. Note that whereas the
N-terminal part of the switch II region, including the pivotal glycine,
is conserved between GTPases and motor ATPases, the C-terminal part is
dissimilar, such that the motor proteins do not have a glycine residue
at the position homologous to Gly94 of EF-Tu. The
differences in the rate of Pi release are likely to be
related to the structure of the switch II region and may be influenced
by the flexibility at its C terminus. Further kinetic and structural
studies are necessary to fully understand the structural properties
that distinguish a molecular switch from a motor.
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ACKNOWLEDGEMENTS |
---|
We thank Yuri Semenkov and Vladimir Katunin for gifts of tRNA and aminoacyl-tRNA synthetase, Hartmut Nieman for the initial experiments, Gundula Talbot for editing of the manuscript, and Karen Margrethe Nielsen, Gitte Hartvigsen, Petra Striebeck, Simone Möbitz, and Astrid Böhm for expert technical assistance. We gratefully acknowledge Profs. Brian Clark and Wolfgang Wintermeyer for critically reading the manuscript and for valuable suggestions.
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FOOTNOTES |
---|
* This work was supported by the Program for Biotechnological Research of the Danish Natural Science Research Council, the Deutsche Forschungsgemeinschaft, and the Alfried Krupp von Bohlen und Halbach-Stiftung.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.
§ Supported by the Carlsberg Foundation.
To whom correspondence should be addressed: Inst. of
Physical Biochemistry, University of Witten/Herdecke, D-58448 Witten, Germany. Tel.: 49-2302-669205; Fax: 49-2302-669117; E-mail:
rodnina@uni-wh.de.
Published, JBC Papers in Press, April 13, 2001, DOI 10.1074/jbc.M102186200
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ABBREVIATIONS |
---|
The abbreviations used are: EF-Tu, elongation factor Tu; EF-Ts, elongation factor Ts; GAP, GTPase-activating protein; aa-tRNA, aminoacyl-tRNA; mant-GTP, 3'(2')-O-(N-methylanthraniloyl)-guanosine triphosphate.
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