From the Groupe de Biophysique-Equipe 2, Ecole Polytechnique, F-91128 Palaiseau Cedex, France
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ABSTRACT |
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Elongation factor (EF) Tu Thr-25 is a key residue
binding the essential magnesium complexed to nucleotide. We have
characterized mutations at this position to the related Ser and to Ala,
which abolishes the bond to Mg2+, and a double
mutation, H22Y/T25S. Nucleotide interaction was moderately destabilized
in EF-Tu(T25S) but strongly in EF-Tu(T25A) and EF-Tu(H22Y/T25S).
Binding Phe-tRNAPhe to poly(U)·ribosome needed a higher
magnesium concentration for the latter two mutants but was comparable
at 10 mM MgCl2. Whereas EF-Tu(T25S) synthesized
poly(Phe), as effectively as wild type, the rate was reduced to 50%
for EF-Tu(H22Y/T25S) and was, surprisingly, still 10% for EF-Tu(T25A).
In contrast, protection of Phe-tRNAPhe against spontaneous
hydrolysis by the latter two mutants was very low. The intrinsic GTPase
in EF-Tu(H22Y/T25S) and (T25A) was reduced, and the different responses
to ribosomes and kirromycin suggest that stimulation by these two
agents follows different mechanisms. Of the mutants, only EF-Tu(T25A)
forms a more stable complex with EF-Ts than wild type. This implies
that stabilization of the EF-Tu·EF-Ts complex is related to the
inability to bind Mg2+, rather than to a decreased
nucleotide affinity. These results are discussed in the light of the
three-dimensional structure. They emphasize the importance of the
Thr-25-Mg2+ bond, although its absence is compatible with
protein synthesis and thus with an active overall conformation of
EF-Tu.
Elongation factor (EF)1
Tu belongs to the superfamily of guanine nucleotide-binding proteins,
factors involved in numerous cellular processes as carrier of
information or biological components. Common characteristics in the
family are the switching between active and inactive conformations
depending on whether GTP or GDP is bound (1, 2), an intrinsic GTPase
activity that can be stimulated several orders of magnitude by specific
factors (GTPase-activating proteins), and the property that the GDP/GTP exchange can be enhanced by specific exchange factors (guanine nucleotide exchange factors) (reviewed in Refs. 4 and 5). EF-Tu acts as
carrier of aminoacyl-tRNAs (aa-tRNAs) to the ribosome in the elongation
phase of bacterial protein biosynthesis. It has a long history of
functional and structural studies offering many insights into its
functioning (6). Structures of the two basic states of EF-Tu (7-10)
and of the complexes with its guanine nucleotide exchange factor EF-Ts
(11, 12) and with its "load" aa-tRNA (13) are now available. These
provide snapshots of the dynamics of the factor during its cycle, which
involves large interdomain rearrangements. On the other hand, many
functional questions, such as the mechanism of GTP hydrolysis, the
EF-Tu cycle on the ribosome, and the structure-function relationships of many structural elements, still remain unanswered or are awaiting satisfactory answers.
Domain 1 of EF-Tu (G domain) has the guanine nucleotide-binding protein
conserved fold and contains the "fingerprint" conserved sequence
motifs taking care of the interaction with the nucleotide (2, 3). Each
of the first three consensus motifs contains a residue involved in the
coordination network of the essential magnesium ion interacting with
the EF-Tu Thr-25 is central in this coordination of magnesium, whereas
Thr-61 located on the opposite side of the magnesium ion with respect
to Thr-25 is only coordinated in the GTP form (7-10). Moreover, the
homologue of Thr-61 in Ha-Ras p21, Thr-35, was found to only weakly
interact with the ion in the GTP-bound form (17, 18). On the other
hand, mutation of p21 Ser-17 (the homologue of EF-Tu Thr-25) can be
associated with dominant negative phenotypes, emphasizing its
functional importance (19-22).
Because in EF-Tu the structure-function relationships of the residues
involved in magnesium coordination are still mostly unexplored, we have
analyzed in well defined in vitro conditions the effects
associated with the substitution of Thr-25 with two closely related
amino acids, serine and alanine. Serine is the amino acid functionally
most similar to threonine; this substitution thus minimizes the changes
induced in the magnesium ion network. The replacement with alanine
emphasizes the consequences of suppressing the link to the magnesium
ion. A double mutant EF-Tu(H22Y/T25S) isolated during the course of
this study was also characterized and found to display intermediary
properties, thus allowing to distinguish more precisely between
specific effects of the two single substitutions.
Biological Materials--
Ribosomes, EF-G, Phe-tRNA synthetase,
and partially purified tRNAPhe (5 to 11%) were prepared
from E. coli as reported (23, 24). Highly purified
tRNAPhe was also obtained from Sigma.
Construction, Overproduction, and Purification of EF-Tu
Mutants--
To obtain rapid purification, EF-Tu wt and mutants were
overproduced as fusion with glutathione S-transferase (25).
Plasmid pGEX-2TtufA is a derivative of pGEX-2T (25)
containing the E. coli tufA gene coding sequence contiguous
to the thrombin recognition sequence LVPRGS, where serine represents
the first amino acid of EF-Tu (26). The very efficient cleavage by
thrombin after arginine results in EF-Tu containing an additional
N-terminal glycine, as compared with the native cellular EF-Tu, the N
terminus of which is acetylated (27). All mutant proteins and also the control wt used in this study were produced in this way. Mutant T25A
was generated by means of the polymerase chain reaction. Mutant T25S
was obtained by the Unique Site Elimination method (28), using a kit
from Amersham Pharmacia Biotech. Interestingly, when trying to
construct the mutant T25S using either polymerase chain reaction or
Unique Site Elimination methods with an 18 base mutagenic primer, we
systematically obtained a double mutant H22Y/T25S resulting from the
substitution of a flanking base. Use of a longer primer of 25 bases
including codon 22 allowed us to obtain T25S alone.
The EF-Tu mutants were overproduced in E. coli DH5 Functional Assays--
Poly(Phe) synthesis was determined
kinetically from the formation of hot trichloroacetic acid-insoluble
material (29), and the GTPase activity was determined from liberation
of [32P]Pi using the molybdate method (30).
Nucleotide dissociation rates and affinities (15, 31-33), and
protection of aa-tRNA by EF-Tu against spontaneous hydrolysis (34) were
measured as described. Details are reported in the legends to the figures.
Production and Purification of EF-Tu Mutants--
Glutathione
S-transferase-fused EF-Tu was expressed in E. coli DH5
The presence of an additional glycine at the N-terminal extremity in
EF-Tu wt did not modify its function in any of the activities tested in
this work, including poly(Phe) synthesis.
Interaction with GDP and GTP--
As shown in Table
I, the single substitution T25S decreases
the affinity for GDP already by a factor three, due to an increase in
dissociation rate, whereas the association rate is comparable to wt.
The T25A substitution and the addition of substitution H22Y to T25S
have more dramatic effects: dissociation rates are 2 orders of
magnitude higher than wt, and association rates decreased. Thus the
affinity constants have µM instead of nM
values, a difference of 3 orders of magnitude. The fast GDP
dissociation causes a technical problem concerning the evaluation of
the GDP binding capacity for mutants H22Y/T25S and T25A. The half-lives
of 1 min and 25 s, respectively, are of the order of the filter
washing time. Consequently the loss of nucleotide during filter washing
is considerable; compared with wt around 50-60% of GDP is retained on
filter by the double mutant and EF-Tu(T25A) shows more variation, but
still retains 25-50%. Taking this and the order of the dissociation reaction into account, we estimated that the actual GDP binding capacity of these mutants should not differ much from that of EF-Tu wt
(T25S proved identical). Consequently, we did not apply a correction
factor in evaluating experiments in the other sections. For the
properties presented in this section, a standardized washing procedure
reduced experimental variation. Because the calculation of the
constants in this section is based on relative experimental values at
different time points or nucleotide concentrations, this technical
difficulty does not affect the values in Table I.
A related reduction of filter retention compared with the theoretical
value is already observed for EF-Tu(wt)·GTP filter
binding,2 and is worse for
the mutants. For EF-Tu(T25A), GTP retention by the filter-bound protein
was even immeasurable. For the T25S single and double substitutions,
GTP dissociation rates turned out to be 3-4-fold higher than for wt.
Interestingly, although for the double mutant the affinity is nearly
6-fold lower and the association rate is not much changed compared with
wt, for the T25S single substitution the affinity is actually slightly higher than for EF-Tu wt, and therefore the association rate is about
7-fold higher.
Activity in Poly(Phe) Synthesis--
The most complete indication
about the health of an EF-Tu mutant is its ability to support protein
biosynthesis. We tested this activity by poly(U)-directed poly(Phe)
synthesis at a magnesium ion concentration of 10 mM to
ensure that the differences seen were not the result of insufficient
saturation of EF-Tu with aa-tRNA, or insufficient binding to the
ribosomal A site (see next paragraph); the GTP concentration was 400 µM, far above the affinity values measured at 0 °C, to
convert the EF-Tu to the GTP form. As shown in Fig.
1, EF-Tu(T25S) could synthesize poly(Phe)
as efficiently as wt. The double mutant EF-Tu(H22Y/T25S) had a lower
activity, about half that of EF-Tu wt. Surprisingly, EF-Tu(T25A), also, showed an activity that was significantly above the background level
and was ~10% that of the EF-Tu wt elongation rate.
Enzymatic Binding of aa-tRNA to Programmed Ribosomes--
The
enzymatic binding of Phe-tRNAPhe to poly(U)-programmed
ribosomes shows a modified dependence on Mg2+ concentration
only for EF-Tu(T25A) and the double mutant, that of EF-Tu(T25S) being
the same as that of EF-Tu wt (Fig. 2).
EF-Tu(T25A) needed considerably (5-6 mM) more magnesium
ion than EF-Tu wt to obtain 50% of the maximum binding of
Phe-tRNAPhe; moreover, it was unable to reach the same
plateau of binding as EF-Tu wt at any of the Mg2+
concentrations used, its maximum level corresponding to about 75% the
control. In contrast, the double mutant displayed the same plateau
level as EF-Tu wt, even though it needed approximately 3 mM
more magnesium. The optimum enzymatic activity, defined as the highest
ratio of enzymatic versus nonenzymatic binding occurred between 8 and 11 mM Mg2+. This prompted our
choice of a standard 10 mM magnesium ion concentration for
the other assays.
Ester-bond Protection of aa-tRNA against Spontaneous
Hydrolysis--
The ability to participate in poly(Phe) synthesis and
enzymatic binding, described in the previous section, established that the mutants have conserved either full (EF-Tu(T25S)) or at least partial activity (EF-Tu(H22Y/T25S) and EF-Tu(T25A)). On the other hand,
the ability of EF-Tu to protect the ester-bond of aa-tRNA from
spontaneous hydrolysis was strongly affected with the latter two
mutants, as shown in Fig. 3, at 10 mM Mg2+. In fact EF-Tu(T25S) induced the same
protection as EF-Tu wt, whereas with the other two mutants, the
half-life of Phe-tRNAPhe decreased from several hours to
approximately 20 min, i.e. to virtually the same level as
obtained with Phe-tRNAPhe in the absence of EF-Tu. This was
surprising, especially in the case of the double mutant that was shown
above to sustain a high rate of poly(Phe) synthesis and, at 10 mM Mg2+, nearly the same enzymatic binding of
aa-tRNA to the ribosome as EF-Tu wt.
Intrinsic GTPase Activity. Effect of aa-tRNA, Programmed Ribosomes
and Kirromycin--
For measuring the intrinsic GTPase activity of
these mutants, high GTP concentrations were used to saturate the EF-Tu
with nucleotide, but for EF-Tu(T25A), this may not have been sufficient to ensure complete saturation. The intrinsic GTPase activity of the
mutants versus that of EF-Tu wt was variously affected (Fig. 4A): for EF-Tu(T25S), it was
comparable to that of EF-Tu wt and for the double mutant it was
markedly reduced (~25%), whereas the rate of EF-Tu(T25A) was so low
that it was difficult to determine. Interestingly, the presence of
Phe-tRNAPhe, which at magnesium concentrations
Kirromycin was able to enhance the GTPase activity of EF-Tu(T25S) and
EF-Tu(H22Y/T25S), but not of EF-Tu(T25A), on which it was virtually
inactive (Fig. 4B). Nevertheless, in poly(Phe) synthesis, the 50% inhibitory concentration for this mutant is even slightly lower (approximately 2 times) than for wt (not shown). Interestingly, kirromycin could compensate entirely the negative influence of the
double mutation on the GTPase.
The presence of the ribosome, whether programmed with poly(U) or not
(the latter not shown), markedly enhanced the GTPase activity of EF-Tu.
In the absence of EF-Ts, the turnover rate of GTP hydrolysis of
EF-Tu(T25S) is twice that of EF-Tu wt, as shown in Fig. 4C.
This reflects the higher intrinsic GDP/GTP exchange rate of this
mutant; in the presence of 50 nM EF-Ts, where the hydrolysis rate becomes limiting again, this difference disappears. The
intrinsic GDP dissociation rate of the double mutant and that of
EF-Tu(T25A) are sufficiently high that the turnover GTPase rate is not
limited by GDP/GTP exchange (not shown). The double mutant shows the
same rate as wt in the absence of EF-Ts, but if compared with wt under
conditions in which the catalysis is rate-limiting (in the presence of
EF-Ts), it is lower by a factor of 5. Interestingly, EF-Tu(T25A)
displays a clear GTPase in the presence of ribosomes, approximately 3%
of the ribosome-stimulated wt rate (when the background rate is
subtracted). The turnover rates of wt and the other mutants in the
presence of ribosomes are 25-35 times their intrinsic GTPase rates.
That of EF-Tu(T25A) is thus stimulated by ribosome as least as much.
Dominant Negative-like Properties of the Two Mutants--
In Ras
proteins, mutants of Ser-17, the residue equivalent to EF-Tu Thr-25,
are known to cause a dominant negative phenotype, meaning that the
mutant competes effectively with wt Ras for binding to its specific
exchange factor, sequestering it and preventing activation of Ras. This
was found to be associated with a strong decrease in the affinity of
the mutants for GDP, and even more for GTP. To examine whether the same
also holds for EF-Tu Thr-25 mutants, the stimulation by EF-Ts of the
dissociation of radiolabeled GDP from EF-Tu wt was followed in the
presence of increasing concentrations of nonlabeled EF-Tu·GDP. As
shown in Fig. 5, the inhibition of the
stimulation of EF-Ts on the EF-Tu(wt)·[3H]GDP
dissociation as a function of the concentration of added nonlabeled
EF-Tu revealed a competitive inhibitory effect This work analyzes the effect induced on EF-Tu functions by
substituting threonine 25, a strictly conserved The obtained results confirm the important structural role of this
residue in the magnesium-substrate coordination, yet they show subtle
effects not readily predictable from the current structural models. As
for the nucleotide binding, the decrease in affinity of EF-Tu(T25A) for
GDP by 3 orders of magnitude emphasizes the central role that the
magnesium ion plays in the binding of the nucleotide to EF-Tu.
EF-Tu(T25S) shows a 3-fold increase in dissociation rate, causing a
subtle modification of the GDP affinity. This could be explained in
terms of modification of the effector loop structure. In fact, two
effector loop residues, Phe-46 and Ile-49 (Tyr-47 and Ile-50,
respectively, in Thermus thermophilus) at both edges of the
one-turn helix
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ABSTRACT
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- and
-phosphates of the nucleotide. In Escherichia
coli EF-Tu, these residues are Thr-25 and Thr-61, which bind to
the magnesium ion directly via the OH-oxygen, and Asp-80 and Asp-50,
which hydrogen-bond each to one of the water molecules, completing the
coordination sphere of magnesium. In other guanine nucleotide-binding
proteins, threonine from the first consensus element is often
substituted by a serine, which can coordinate similarly to magnesium.
The important role of this metal ion in the nucleotide binding is
emphasized by the observation that its chelation decreases the affinity
for GDP and GTP by at least 3 orders of magnitude (14). Significant
amounts of free magnesium are not necessary for GTPase activity, and
the tightly bound magnesium ion must be extracted with a large excess
of EDTA before activity is lost (15, 16).
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grown
in LB medium at 30 °C to 0.5-0.8 A600, after
which induction with 0.08 mM
isopropyl-
-D-thiogalactopyranoside took place at
22 °C with incubations up to 40 h. Cells were disrupted by
sonication in 50 mM Tris-HCl, pH 7.5, 110 mM
KCl, 10 mM MgCl2, 7 mM ME and 10%
glycerol (Buffer A). After centrifugation at 30,000 × g for 20 min, the supernatant was applied to a
glutathione-Sepharose 4B column (Amersham Pharmacia Biotech; 2.5 ml of
resin /g of cells), which was then extensively washed at 4 °C with
Buffer A containing 30-150 µM GDP, to remove bound EF-Ts
as far as possible. Glutathione S-transferase-EF-Tu was
cleaved in situ and eluted at 4 °C with Buffer A
supplemented with 30 µM GDP, 2.5 mM
CaCl2, and 1 unit/ml thrombin at a low flow rate
(approximately 0.3 ml/min) until no more protein was released. Thrombin
in the eluent was inactivated by adding 0.1 mM
phenylmethylsulfonyl fluoride. The obtained EF-Tu was further purified
to remove residual EF-Ts, using either Q Sepharose chromatography or
Superdex 75 gel filtration. For the first method, the eluate from the
glutathione affinity column was brought to a final GDP concentration of
250 µM and applied to a Q Sepharose FF column (6 ml).
This was washed with Buffer A containing 250 µM GDP to
induce the dissociation of EF-Tu·EF-Ts, and 120 mM KCl,
at which concentration EF-Ts cannot bind to the resin. EF-Tu was eluted
with a linear KCl gradient to 400 mM. For the second
method, the affinity column eluate was concentrated by ultrafiltration
(Amicon CentriFlo 25) to <2 ml before loading on a Amersham Pharmacia
Biotech Superdex 75 column, which was eluted with Buffer A containing
40 µM GDP. Pooled peak fractions from Q Sepharose or
Superdex columns were concentrated by ultrafiltration, dialyzed against
50 mM Tris-HCl, pH 7.5, 60 mM
NH4Cl, 10 mM MgCl2, 1 mM dithiothreitol, 55% glycerol, 20 µM GDP
and stored at
30 °C. Protein concentrations were determined by the
Bio-Rad protein assay, using bovine serum albumin as standard.
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cells grown in LB medium at 30 °C to 0.5-0.8
A600. At this cell concentration and
temperature, induction by 0.08 mM isopropyl-
-D-thiogalactopyranoside for 6-8 h resulted
in high level overproduction. However, whereas overproduced glutathione S-transferase-EF-Tu wt and T25S displayed good solubility
(70-80%), yielding up to 2 mg of thrombin-cleaved EF-Tu/g cells (wet
weight), mutants T25A and H22Y/T25S were about 10% soluble. Longer
induction times (up to 40 h) at lower temperature during the
induction phase (22 °C) somewhat increased the solubility. On the
glutathione affinity column, some EF-Ts is always retained as a complex
with EF-Tu, making a second purification step necessary, either by Q
Sepharose chromatography or Superdex 75 gel filtration (see under
"Materials and Methods"). This was particularly the case for the
Ala mutant, which complexes very tightly with EF-Ts (75% EF-Tu·EF-Ts
complex was still present after the first affinity step).
Nucleotide interaction parameters
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Fig. 1.
Poly(U)-directed poly(Phe) synthesis.
Phe incorporated by EF-Tu wt ( ), EF-Tu(T25A) (
), EF-Tu(H22Y/T25S)
(
), or EF-Tu(T25S) (
) or in absence of EF-Tu (×). Final reaction
mixture (60 µl) contained 50 mM Hepes-KOH, pH 7.5, 70 mM NH4Cl, 10 mM MgCl2,
and 7 mM ME (Buffer B) with 1.5 mM ATP, 0.4 mM GTP, 3.8 mM PEP, 30 µg/ml pyruvate kinase
(EC 2.7.1.40), 2 units/ml myokinase (EC 2.7.4.3), 50 nM
EF-Ts, 0.8 µM EF-G, 20 µM
[14C]Phe (specific activity, 120 dpm/pmol), 3 µM tRNAPhe, 26 nM Phe-tRNA
synthetase (EC 6.1.1.20), 1.5 µM ribosomes with
tRNAPhe-preloaded P-site, 100 µg/ml poly(U), and 0.5 µM EF-Tu, as indicated. The reaction was started by
adding together a mix containing ribosomes, poly(U), and
tRNAPhe in Buffer B and a mix containing all other factors
and components, which had been separately incubated at 30 °C for 15 min. Incubation was continued at 30 °C, 6-µl samples were
withdrawn at the indicated times and spotted on glass fiber filters,
and hot trichloroacetic acid-insoluble radioactivity determined.
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Fig. 2.
Enzymatic binding of Phe-tRNAPhe
to the ribosomal A site as function of [Mg2+].
[14C]Phe-tRNAPhe bound to the ribosomal A
site by EF-Tu wt ( ), EF-Tu(T25A) (
), EF-Tu(H22Y/T25S) (
), or
EF-Tu(T25S) (
) or in absence of EF-Tu (+) after a 5 min incubation
at various MgCl2 concentrations. The reaction was started
by adding together a mix containing preformed
EF-Tu·GTP·[14C]Phe-tRNAPhe ternary complexes
(mix I) and a mix containing poly(U)-programmed ribosomes with
tRNAPhe preloaded in the A site (mix II). After 5 min at
30 °C, the reaction (50 µl) was filtered through nitrocellulose.
Filters were immediately washed twice with 2 ml of ice-cold 50 mM Tris-HCl, pH 7.6, 60 mM NH4Cl,
and 10 mM MgCl2, and the retained radioactivity
was counted. Mix I (42 µl) consisted of 8 µl of a preincubated
tRNAPhe charging reaction and other components to give a
final composition of 50 mM Hepes-KOH, pH 7.5, 70 mM NH4Cl, MgCl2 at variable
concentration, 14 mM ME, 1.2 mM ATP, 0.24 mM GTP, 2.4 mM PEP, 30 µg/ml pyruvate kinase,
[14C]Phe-tRNAPhe (formed from 0.9 µM tRNAPhe and 1.0 µM
[14C]Phe (specific activity, 1105 dpm/pmol) by 8 nM Phe-tRNA synthetase), and 0.54 µM of the
respective EF-Tu. Mix II (8 µl) contained 50 mM
Hepes-KOH, pH 7.5, 70 mM NH4Cl, 8 mM MgCl2, 14 mM ME, 500 µg/ml
poly(U), 1.7 µM tRNAPhe, and 1.3 µM ribosomes. Both mixes were incubated separately at
30 °C for 15 min before starting the reaction.
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Fig. 3.
Protection against spontaneous hydrolysis of
the ester bond of Phe-tRNAPhe by EF-Tu. The
concentration of [14C]Phe-tRNAPhe in solution was
followed in time in the presence of EF-Tu wt ( ), EF-Tu(T25A) (
),
EF-Tu(H22Y/T25S) (
), of EF-Tu(T25S) (
) or in absence of EF-Tu
(×). The inset shows longer incubation times for EF-Tu wt
and EF-Tu(T25S). Final reaction mixture (62 µl) contained 25 mM Hepes-KOH, pH 7.5, 60 mM NH4Cl,
10 mM MgCl2, 7 mM ME, 0.2 mM GTP, 1 mM PEP, 30 µg/ml pyruvate kinase,
1.7 µM [14C]Phe-tRNAPhe
(specific activity, 405 dpm/pmol), and 3 µM EF-Tu as
indicated. The reaction (at 30 °C) was started by adding 15 µl of
buffer containing the [14C]Phe-tRNAPhe to 47 µl of mix with the other components, which had been preincubated at
30 °C for 15 min to convert EF-Tu·GDP to EF-Tu·GTP. At the
indicated times, 8-µl samples were taken and spotted onto glass fiber
filters, and cold trichloroacetic acid-insoluble material on the
filters was measured.
10
mM is known to hinder the hydrolysis of the EF-Tu wt-bound
GTP (23, 35-37), decreased the activity of EF-Tu(T25S) by 60%, as for
EF-Tu wt, whereas EF-Tu(H22Y/T25S) did not always show a clear
reduction, and for EF-Tu(T25A) it gave low but measurable rates with a
good correlation coefficient, at ~5% of the wt intrinsic rate.
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Fig. 4.
GTPase activity: intrinsic and in the
presence of Phe-tRNAPhe (A), kirromycin
(B), and ribosomes (C).
Liberation of [32P]phosphate from
[ -32P]GTP by EF-Tu wt (
and
), EF-Tu(T25A) (
and
), EF-Tu(H22Y/T25S) (
and
), and EF-Tu(T25S) (
and
)
in the various conditions. A, in absence (open
symbols) or presence (filled symbols) of 3 µM [14C]Phe-tRNAPhe (specific
activity, 150 dpm/pmol). B, in absence (open
symbols) or presence (filled symbols) of 50 µM kirromycin. C, in presence of 4 µM ribosomes, with only EF-Tu (open symbols)
or with additionally 50 nM EF-Ts (filled
symbols), or in absence of EF-Tu (×). Final reaction conditions
were 50 mM Hepes-KOH, pH 7.5, 100 mM
NH4Cl, 10 mM MgCl2, 7 mM ME, 1 mM ATP, 50 µM
[
-32P]GTP (specific activity, 740-1500 cpm/pmol), 1 mM PEP, 10 µg/ml pyruvate kinase, 2 µM of
the respective EF-Tu. EF-Tu·GTP was preformed by incubating this mix
without the [
-32P]GTP for 10 min at 30 °C.
Reactions were started after further incubation with
[
-32P]GTP on ice for 10 min, by adding the stimulatory
component and shifting the reaction to 30 °C. Appropriate-sized
samples were taken at indicated times and quenched in 1 M
HClO4/3 mM KH2PO4, and
free [32P]phosphate was determined by the
molybdate/isopropyl acetate method (23).
i.e. an
effect much stronger than when the "competitor" is EF-Tu wt
only in the case of EF-Tu(T25A). This is very likely due to a sequestration of EF-Ts resulting from the formation of a stable complex with EF-Tu(T25A), as was observed also during the purification of this mutant after overexpression in E. coli. Interestingly, no
dominant inhibitory effect could be observed with EF-Tu(T25S) and
EF-Tu(H22Y/T25S), even though for the double mutant the nucleotide
affinity is affected to almost the same extent as for EF-Tu(T25A).
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Fig. 5.
Inhibition of EF-Ts stimulation of
EF-Tu(wt)·[3H]GDP dissociation by external
EF-Tu·GDP. This figure shows how the stimulation of
EF-Tu(wt)·[3H]GDP dissociation by EF-Ts is inhibited by
the presence of increasing concentrations of competitor EF-Tu in the
(unlabeled) GDP form: EF-Tu wt ( ), EF-Tu(T25A) (
),
EF-Tu(H22Y/T25S) (
), or EF-Tu(T25S) (
). The inhibition is defined
as the decrease of the ratio of the observed [3H]GDP
dissociation rate (kobs) and the
EF-Ts-stimulated rate in the absence of competitor
(kstim), both corrected for the intrinsic
[3H]GDP dissociation rate ki
(i.e. in absence of EF-Ts). Values plotted are thus (1
(kobs
ki)/(kstim
ki)) × 100%. Reaction conditions were 50 mM Hepes-KOH, pH 7.5, 60 mM NH4Cl,
10 mM MgCl2, 1 mM dithiothreitol,
30 nM EF-Tu(wt)·[3H]GDP (specific activity,
5800 dpm/pmol), 9 nM EF-Ts, 20 µM GDP, and
indicated concentrations of various competitor EF-Tu·GDP. The
reaction was started by adding a mix containing EF-Ts, competitor
EF-Tu, and excess cold GDP to the preformed
EF-Tu(wt)·[3H]GDP on ice. Samples were filtered through
nitrocellulose filters as for Fig. 2.
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-helix 1 residue that flanks at the C-terminal side the P-loop (L1), and represents an
essential element in the network coordinating the nucleotide-bound magnesium ion. Previous work of Hwang et al. (38) reported
that substitution of this residue by isoleucine abolishes the
interaction with the nucleotide. We decided to extend the analysis of
the function of this crucial residue by introducing two other
substitutions in the hope of correlating functional data with the
recent impressive development of our knowledge of the three-dimensional
structure of EF-Tu and its complexes. We also intended to compare the
effects of this mutation with those reported for other guanine
nucleotide-binding proteins on substituting the homologous residue.
1' make hydrophobic contacts with the Thr-25 side
chain methyl group, forming a kind of "hydrophobic clamp"
(conserved motif (Y/F)XX(I/V)) around it (Fig.
6, A and B). It
represents the major interaction of this part of the effector region,
in between the hypervariable first part and the conserved second part.
Loss of this stabilizing hydrophobic interaction could unfavorably
alter the structure of the effector loop, possibly affecting the
water-mediated bond of Asp-50 with the Mg2+. In eukaryal
and archeal EF1
s, the conserved Thr at position 25 is replaced by a
conserved Ser that lacks the
-carbon methyl group. Notably, in these
factors the hypervariable part of the effector loop has in place of the
hydrophobic clamp motif of prokaryal EF-Tu, a conserved very
hydrophobic motif containing several bulky aromatic
residues.3 This might
indicate a steric compensation for the absence of the methyl group at
position 25.
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Fig. 6.
Details of structural elements discussed in
the text. A and B, close-up of the contacts
between the effector region of EF-Tu, Thr-25, the nucleotide, and the
magnesium ion. A, E. coli EF-Tu in the GDP-form
(9); B, T. thermophilus EF-Tu in the GTP form (7). The
effector region is highlighted in pale blue, with residues
forming the hydrophobic clamp shown as wireframe plus
transparent blue Van der Waals surface, surrounding the Thr-25 side chain methyl group,
indicated similarly in yellow. Magnesium (dark
green) and the nucleotide are also visualized. C,
close-up of the P-loop with residue His-22 and loop L6-helix 3.
Yellow, E. coli EF-Tu in the GDP form (9);
green, structure of EF-Tu from the E. coli
EF-Tu·EF-Ts complex (structures were aligned by least-square fitting
of residues 9-35, 66-79, 97-109, 129-169, and 183-199 (E. coli numbering)). Note the displacement of the top of helix
3
in the EF-Tu·EF-Ts structure relative to EF-Tu·GDP. The two loops
L1 (P-loop) and L6 are represented as a backbone wireframe, with the
side chains of His-22 and Met-112 of EF-Tu·GDP shown as well.
Superimposed on His-22 is a tyrosine (purple) in the same
orientation. Its terminal OH group is surrounded by a transparent Van
der Waals surface, as are atoms from Ala-110 and Met-112 in loop L6 in
which it would clash. Representations were made using Molscript and
Raster3D (44, 45).
The surprising effect of the supplementary mutation H22Y on the
nucleotide affinity of EF-Tu(T25S) is more difficult to explain. Residue His-22 is part of the P-loop pointing toward L6 preceding helix
3. Substitution by the approximately 1.5 Å longer tyrosine in the
same orientation would cause a steric collision with the
-carbon of
Met-112. Its accommodation would thus imply a displacement of L6, and
consequently perhaps also of helix
3 (Fig. 6C).
Noteworthy, in the EF-Tu·EF-Ts complex crystal structure, this helix
has been displaced in the same direction by approximately 1.5 Å as a
consequence of the intrusion of EF-Ts Phe-81 in between EF-Tu helices
2 and
3. Thus, such a displacement might cause modifications
similar to the sequence of interactions (from His-118 via Gln-114 to
the P-loop) that Wang et al. (12) propose to lead to release
of the nucleotide. Another possible effect of substitution H22Y could be the deformation of the P-loop, hindering nucleotide accommodation. The slightly higher magnesium requirement for enzymatic binding indicates that some perturbation indeed occurs at the level of the
magnesium binding elements in the double mutant and not with the single
substitution T25S.
A related observation deserves some attention. In Ras proteins, mutations of the position equivalent to EF-Tu Thr-25 are known to be associated with dominant negative phenotypes (19-22). This has been shown in vitro for Ras2p(S24N) to be associated with increased stability of the complex of Ras2p with its exchange factor (22, 39), thus leading to sequestration of the guanine nucleotide exchange factor. Interestingly, here we describe two mutants of EF-Tu (EF-Tu(H22Y/T25S) and EF-Tu(T25A)) that have a comparable decrease in affinity for GDP, whereas only one, EF-Tu(T25A), shows dominant negative-like behavior through increased stability of the complex with EF-Ts. Nevertheless the stimulation by EF-Ts of GDP dissociation on EF-Tu(H22Y/T25S) is not less efficient than with wt (not shown). This indicates that the interaction of the magnesium ion with Thr-25 plays a central role in the mechanism of dissociation of the EF-Tu·EF-Ts complex. In the crystal structure of the complex, the phosphate cavity is not completely open due to a peptide flip in the P-loop. If the stable interaction Mg2+-Thr-25(OH) cannot be formed, the insertion of the nucleotide into the binding site and the release of EF-Ts has apparently a much lower chance of succeeding. This observation may also have implications for the reverse process, the dissociation of EF-Tu·nucleotide by EF-Ts, in which breaking of the Mg2+-Thr-25(OH) bond could represent the major energetic barrier. This would emphasize the importance of the displacement of magnesium in the mechanism of stimulation of EF-Tu·GDP dissociation by EF-Ts, in agreement with the model of Kawashima et al. (11).
Concerning the influence of the various mutations on the GTPase
activity, the most striking observation is the different effects on the
stimulation of the EF-Tu GTPase by kirromycin and ribosomes. The
negative influence of the double mutation on the intrinsic GTPase can
be completely compensated by kirromycin, whereas the GTPase of
EF-Tu(T25A) is completely insensitive to the antibiotic, although it
still interacts. In contrast, ribosomes are shown here to stimulate all
mutants and wt comparably. This is probably related to a different
mechanism of stimulation of the catalysis by the two agents, which are
known to have an additive effect (15). The ribosome stimulation may be
somehow related to that proposed for the GTPase-activating
protein stimulation of small GTPases, even though some caution in
comparing the two systems is in order (6). Our findings suggest that
the enhancing effect of the ribosome is not critically dependent on the
tight interaction of the magnesium ion with the core of EF-Tu, although
the magnesium ion is essential for achieving the correct binding of
nucleotide and the intrinsic GTPase. This is in evident contrast with
the effect of the antibiotic kirromycin that we show here to stimulate the GTPase only if the Mg2+-25(OH) bond is intact, thus
indicating a different mechanism of action from that of the ribosome.
The location and mode of binding of the antibiotic to EF-Tu are not yet
precisely defined. Whereas resistance mutations were mapped to the
interface of domains 1 and 3 (40, 41), the known competition between
EF-Ts and kirromycin for binding to EF-Tu could rather implicate
elements from the EF-Tu·EF-Ts contact area (L1(P-loop), L6-3, and
4 (11)) in kirromycin binding (6). Consequently, kirromycin could
have a direct or indirect interaction with the GTPase center, perhaps acting through the P-loop. There it might strengthen the hydrogen bond
of the Asp-21 main chain NH toward the
-bridging oxygen, which
was suggested for p21 to play a major role in the intrinsic GTPase
(42).
Concerning the interaction with tRNA, we have observed various effects. First, the activities in poly(Phe) and enzymatic binding indicate that EF-Tu(T25S) has conserved full ability to bind aa-tRNA, that also the double mutant at 10 mM Mg2+ is like wt, and that even EF-Tu(T25A) has conserved at least partial activity. In surprising contrast is the complete lack of protection against spontaneous hydrolysis of the aa-tRNA ester bond by the latter two mutants. We interpret this to indicate that whereas the overall binding of aa-tRNA may not be dramatically affected, the interaction with the aminoacyl end of the tRNA is anomalous for these mutants. Interestingly, this recalls the situation observed for wt EF-Tu in the presence of the antibiotic enacyloxin IIa (43). A combination of factors probably contributes to the impaired rate of poly(Phe) synthesis with two of the three mutants: the anomalous ternary complex, the reduced efficiency of the GTPase center, and in the case of EF-Tu(T25A) also the more stable complex with EF-Ts.
To conclude, our results underline the importance of the magnesium
binding by Thr-25 in the functioning of EF-Tu, whereas on the other
hand the ability of EF-Tu to participate in protein synthesis proves
conserved to some extent even in the absence of this bond. This shows
that modification of the magnesium-nucleotide binding network, although
drastic, as in the case of substitution of the key component Thr-25 by
Ala, is still compatible with an active overall conformation of the
EF-Tu molecule. It also emphasizes that although the
Mg2+-Thr-25(OH) bond contributes greatly to the stability
of nucleotide binding, other interactions are important too.
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ACKNOWLEDGEMENTS |
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Drs. K. Harmark and A. Weijland have done in our group some preliminary work on Thr-25 mutants using a different vector and purification system. We thank Dr. R. Hilgenfeld for providing coordinates of EF-Tu crystal structures and Drs. J. B. Créchet, E. Jacquet, and A. Weijland for comments on the manuscript.
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FOOTNOTES |
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* This work was carried out in the framework of Contract ERB-CHRXCT 940510 (Program Human Capital and Mobility of the E.C.) and was supported by grants from the Ligue Nationale Française Contre le Cancer, Fédération Nationale des Centers de Lutte Contre le Cancer, and Association pour la Recherche sur le Cancer (Grant 6377).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 33-1-6933-41-80;
Fax: 33-1-6933-48-40; E-mail: andrea{at}poly.polytechnique.fr.
2 A. Parmeggiani, unpublished observation.
3 I. M. Krab, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: EF, elongation factor; ME, 2-mercaptoethanol; PEP, phosphoenolpyruvate; wt, wild type; aa-tRNA, aminoacyl-tRNA.
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REFERENCES |
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