The Importance of Structural Transitions of the Switch II Region for the Functions of Elongation Factor Tu on the Ribosome*

Charlotte KnudsenDagger §, Hans-Joachim Wieden, and Marina V. Rodnina||

From the Dagger  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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha 2 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 alpha 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha 1 and strand beta 2 and varies in length, sequence, and structure among different GTPases, and the more conserved switch II region, which consists of helix alpha 2 and the flanking loops. The N-terminal flanking loop contains the consensus element DXXG, which is involved in binding of the gamma -phosphate, the Mg2+ ion, and a water molecule in the vicinity of the gamma -phosphate. Many of the family members, e.g. EF-Tu, Galpha i1, and small Ras-like GTPases, also contain one or more glycines located in the C-terminal loop flanking the switch II region.

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 gamma -phosphate induces the transition starting from Gly83 (E. coli numbering) at the N terminus of helix alpha 2 by breaking the hydrogen bond between the main chain amide nitrogen and the gamma -phosphate. As a consequence, the peptide bond between Pro82 and Gly83 flips by 150°, thereby causing a rotation of helix alpha 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 alpha  to beta  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 alpha 2 of other G proteins, such as Ras (9) and Galpha i1 (10).

The structure-function relations of the two glycines, Gly83 and Gly94, flanking helix alpha 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 alpha 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 [gamma 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.

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 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-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

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 k-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).


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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 k-1, 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 k-1 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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Fig. 2.   Initial binding of ternary complexes to the ribosome. Time courses were measured with 0.3 µM ternary complexes and varying amounts of nonprogrammed ribosomes. kapp values were determined by exponential fitting ("Experimental Procedures") and are plotted relative to the ribosome concentration. Open circles, wild type EF-Tu; closed squares, EF-Tu(G94A); open triangles, EF-Tu(G83A); closed triangles, EF-Tu(G83/94A). The values of k1 and k-1 calculated from the plots are summarized in Table I.

                              
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Table I
Effect of the G94A and G83A mutations in EF-Tu on the elemental rate constants of factor-dependent A site binding of aa-tRNA

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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Fig. 3.   Effect of the G94A mutation on the kinetics of ternary complex binding to the A site. The fluorescence of Phe-tRNAPhe(Prf16/17) was monitored in short (A) and long (B) time windows. Poly(U)-programmed ribosomes with AcPhe-tRNAPhe in the P site (0.5 µM) were mixed with ternary complexes (0.3 µM) prepared using a 4-fold excess of EF-Tu·GDP over Phe-tRNAPhe(Prf16/17). Exponential fitting of the data required multiple exponential terms with similar values of apparent rate constants and was therefore not applied, except for the well defined slow step observed with EF-Tu(G94A). kapp = 0.07 ± 0.02/s. WT, wild type.

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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Fig. 4.   Effect of the G83A mutation on the kinetics of ternary complex binding to the A site. The fluorescence of Phe-tRNAPhe(Prf16/17) was monitored. A, time courses were measured with 0.5 µM poly(U)-programmed ribosomes, and 0.3 µM ternary complex was prepared using a 4-fold excess of EF-Tu·GDP over Phe-tRNAPhe(Prf16/17). The values of the apparent rate constants calculated by two-exponential fitting were kfast = 26 ± 2/s and kslow = 0.16 ± 0.01/s for EF-Tu(G83A) and kfast = 12 ± 2/s and kslow = 0.06 ± 0.02/s for EF-Tu(G83/94A). B, concentration dependence of the apparent rate constant of the fast step. Open triangles, EF-Tu(G83A); closed triangles, EF-Tu(G83/94A). The apparent rate constant of the slow step was independent of the ribosome concentration (not shown).

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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Fig. 5.   Conformational changes of EF-Tu and EF-Tu mutants upon binding of the ternary complex to the A site. Time courses monitored by mant-GTP fluorescence with wild type EF-Tu and EF-Tu(G94A) displayed in short (A) and long (B) time windows or with EF-Tu(G83A)and EF-Tu(G83A/G94A) (C). Phe-tRNAPhe was present in a 4-fold excess over EF-Tu·mant-GTP, resulting in a maximal possible concentration of ternary complex of 0.3 µM, which was mixed with 0.5 µM poly(U)-programmed ribosomes. The values of the apparent rate constants determined by two-exponential fitting were kfast = 33 ± 2/s and kslow = 2.7 ± 0.1/s for wild type EF-Tu and kfast = 20 ± 2/s and kslow = 0.06 ± 0.02/s for EF-Tu(G94A). The time courses obtained with EF-Tu(G83A) and EF-Tu(G83/94A) were fitted to a single exponential function, yielding kapp = 10 ± 2/s and 5 ± 1/s, respectively. WT, wild type.

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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Fig. 6.   Effects of mutations in EF-Tu on GTP hydrolysis (A) and peptide bond formation (B). The concentrations after mixing were 0.3 µM ternary complex and 0.5 µM programmed ribosomes. Open circles, wild type EF-Tu; closed squares, EF-Tu(G94A); open triangles, EF-Tu(G83A); closed triangles, EF-Tu(G83/94A). The apparent rate constants of GTP hydrolysis were 27 ± 3/s and 16 ± 2/s for the wild type EF-Tu and EF-Tu(G94A), respectively. The apparent rate constants of peptide bond formation were 0.9 ± 0.1/s, 0.07 ± 0.01/s, 0.19 ± 0.02/s, and 0.06 ± 0.01/s for wild type EF-Tu, EF-Tu(G94A), EF-Tu(G83A), and EF-Tu(G83/94A), respectively. WT, wild type.

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 k-1 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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Fig. 7.   Results of the global fit for the mutant EF-Tu(G94A). A, conformational changes of aa-tRNA monitored by changes in proflavin fluorescence at varying ribosome concentrations (from bottom to top, 0.3, 0.5, 1.0, 1.5, and 2.0 µM). B, rearrangements in EF-Tu monitored by changes in fluorescence of mant-GTP at the same conditions as in A (4-fold excess of EF-Tu·mant-GTP over aa-tRNA) measured with 0.5 µM ribosomes. C, time courses of GTP hydrolysis (squares) and peptide bond formation (triangles) measured with 0.5 µM ribosomes. The smooth lines are calculated from the elemental rate constants (Table I).

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.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Role of Helix alpha 2 in EF-Tu Binding to the Ribosome-- The replacement of glycine 83 at the N terminus of helix alpha 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 alpha 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 alpha 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 alpha 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 Galpha s, G226A, abolishes the activation of the effector adenylyl cyclase, and the mutant Galpha s is unable to undergo the conformational transition required for the GTP-dependent dissociation from the beta gamma subunits. Furthermore, the mutation lowers the apparent affinity of Galpha 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 gamma -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 alpha 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 alpha 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 alpha 2 impairs the proper positioning of EF-Tu for GTPase activation on the ribosome.

The mutation in the alpha  subunit of the heterotrimeric G protein Galpha 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 Galpha 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 Galpha i1 in the GTP form, Galpha i1·GTPgamma S, and in the transition state mimicked by the Gialpha 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 alpha 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 alpha 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 gamma -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 alpha 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 alpha 2; in the structure of EF-Tu from T. aquaticus (7), the corresponding Gly95 has phi  and psi  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 alpha 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 phi  and psi  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 alpha 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 Galpha i1(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 alpha 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 gamma -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.

    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.

    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

    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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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