©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphorylation of Elongation Factor Tu Prevents Ternary Complex Formation (*)

Christian Alexander (1), Nese Bilgin (2), Carsten Lindschau (3), Jeroen R. Mesters (4) (5), Barend Kraal (4), Rolf Hilgenfeld (5), Volker A. Erdmann (1), Corinna Lippmann (1)(§)

From the (1)Institut für Biochemie, Freie Universität Berlin, Thielallee 63, D-14 195 Berlin (Dahlem), Germany, the (2)Institutionen för molekylärbiologi, Uppsala Universitet, BMC, Box 590, S-751 24 Uppsala, Sweden, the (3)Franz-Volhard-Klinik am Max-Delbrück-Centrum, Humboldt Universität Berlin, Wiltbergstr. 50, D-13 122 Berlin, Germany, the (4)Gorlaeus Laboratories, P. O. Box 9502, NL-2300 RA Leiden, The Netherlands, and the (5)Department of Structural Biology and Crystallography, Institute of Molecular Biotechnology, P. O. Box 100813, D-07708 Jena, Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The elongation factor Tu (EF-Tu) is a member of the GTP/GDP-binding proteins and interacts with various partners during the elongation cycle of protein biosynthesis thereby mediating the correct binding of aminoacylated transfer RNA (aa-tRNA) to the acceptor site (A-site) of the ribosome. After GTP hydrolysis EF-Tu is released in its GDP-bound state. In vivo, EF-Tu is post-translationally modified by phosphorylation. Here we report that the phosphorylation of EF-Tu by a ribosome associated kinase activity is drastically enhanced by EF-Ts. The antibiotic kirromycin, known to block EF-Tu function, inhibits the modification. This effect is specific, since kirromycin-resistant mutants do become phosphorylated in the presence of the antibiotic. On the other hand, phosphorylated wild-type EF-Tu does not bind kirromycin. Most interestingly, the phosphorylation of EF-Tu abolishes its ability to bind aa-tRNA. In the GTP conformation the site of modification is located at the interface between domains 1 and 3 and is involved in a strong interdomain hydrogen bond. Introduction of a charged phosphate group at this position will change the interaction between the domains, leading to an opening of the molecule reminiscent of the GDP conformation. A model for the function of EF-Tu phosphorylation in protein biosynthesis is presented.


INTRODUCTION

The bacterial peptide chain elongation factor Tu belongs to the guanosine triphosphatase superfamily of proteins which act as molecular switches with active and inactive forms depending on the phosphorylation state of the bound guanine nucleotide (Bourne et al., 1991). In prokaryotes, EF-Tu()is the most abundant protein. The active EF-TuGTP complex catalyzes the binding of aminoacyl-tRNA to the programmed ribosome. Activity is terminated after the hydrolysis of guanosine triphosphate (GTP), and the resulting guanosine diphosphate (GDP) has to be replaced by GTP in the elongation factor Ts-mediated guanine-nucleotide exchange reaction. EF-Ts is an essential protein for the growth of Escherichia coli and its expression is tightly regulated (Hwang et al., 1989) but only little is known about the molecular mechanism of its interaction with EF-Tu (Hwang et al., 1992).

Protein biosynthesis is the target of numerous antibiotics (Nierhaus, 1982). The antibiotic kirromycin binds with high affinity to EF-Tu forcing this protein into a conformation that is unable to maintain its proper function during the translational elongation cycle. The affinity of the EF-TuGTPkirromycin complex toward aa-tRNA is drastically reduced (Abrahams et al., 1991) while even the GDP form of EF-Tu promotes binding of aa-tRNA to the ribosomal A-site in presence of the antibiotic (Wolf et al., 1974). The inhibitory effect of kirromycin on translation is due to the fact that the release of EF-TuGDP from the acceptor site of the ribosome is prevented (Wolf et al., 1977). Crystallographic studies have shown that EF-Tu consists of three distinct domains: domain 1 (residues 1-200), domain 2 (residues 209-299), and domain 3 (residues 300-393) (Kjeldgaard and Nyborg, 1992; Berchtold et al., 1993; Kjeldgaard et al., 1993). Analysis of kirromycin-resistant mutants (Zeef and Bosch, 1993) revealed that the antibiotic may interact at the interface of domains 1 and 3 when EF-Tu is in the GTP bound state (Mesters et al., 1994).

It is known of the E. coli EF-Tu that the protein is N terminally acetylated (Arai et al., 1980; Jones et al., 1980) and that lysine 56 can be mono- or dimethylated in vivo (L'Italien and Laursen, 1979). Recently we have shown that EF-Tu from E. coli and Thermus thermophilus are post-translationally modified by phosphorylation in vivo (Lippmann et al., 1991, 1993). Phosphorylation of the eukaryotic EF-1 (the eukaryotic counterpart of EF-Tu) was also described, but the site of modification was not determined (Venema et al., 1991a, 1991b). We identified the site of phosphorylation in E. coli EF-Tu as threonine 382 (Lippmann et al., 1993). This residue is strictly conserved in all known EF-Tu and EF-1 sequences. The modification can be reversed by the action of a phosphatase. Here we report the influence of the phosphorylation on the interaction of EF-Tu with EF-Ts, kirromycin, and aa-tRNA.


MATERIALS AND METHODS

Standard chemicals were purchased from Merck (Darmstadt, Germany) and were of the highest quality available. Radiochemicals and x-ray films were obtained from Amersham (Braunschweig, Germany). Kirromycin was a gift from A. Parmeggiani (Palaiseau, France), other antibiotics were from Serva (Heidelberg, Germany) and nucleotides from Boehringer (Mannheim, Germany). PhastGels and buffer strips were purchased from Pharmacia (Freiburg, Germany). EF-TuGDP from E. coli was isolated as described by Leberman et al. (1980) and further purified by crystallization (Lippmann et al., 1988).

In Vitro Phosphorylation Assay

In vitro phosphorylation of EF-Tu was performed in a simplified assay system using cleared whole cell lysate (S15) as source of EF-Tu-specific protein kinase activity. The S15 results in the same phosphorylation effect as the crude 70 S ribosomes preparations described (Lippmann et al., 1993). Frozen cells from mid-log phase (E. coli MRE 600) where resuspended in assay buffer (50 mM TrisHCl, pH 7.4, 10 mM MgCl, 15 mM dithioerythritol, 1 mM phenylmethylsulfonyl fluoride) and lysed by sonification. Cell debris were removed by centrifugation (15,000 g, 20 min, 4 °C) and the absorption at 260 nm of the supernatant (``S15'') was determined. In vitro phosphorylation assays (final volume 10 µl) were performed in the same buffer containing 5 µM EF-Tu, 150 µM [-P]ATP (specific activity 250 GBq mmol), and 0.01 (or 0.25) A of S15 extract at 30 °C. Additional components tested for influence on EF-Tu phosphorylation were present as indicated in the corresponding figures. Reactions were terminated by addition of sample buffer (Laemmli) and boiling for 5 min and subjected to SDS-PAGE (12%). For removal of nucleic acids (RNA + DNA), the gels were treated with 7% (v/v) trichloroacetic acid (30 min, 95 °C) prior to Coomassie staining. After drying, gels were exposed to x-ray films.

Native Gel Electrophoresis with the PhastSystem

Native buffer strips for the PhastSystem (Pharmacia) were equilibrated in 8 mM TricineNaOH, pH 8.2, for more than 1 h. Separation was performed in 20% homogeneous polyacrylamide PhastGel in a PhastSystem (Pharmacia) under the following running conditions. Sample application at step 2 from 0 V-h (down) to 2 V-h (up); separation step 1: 400 V, 10 mA, 2.5 W at 4 °C for 10 V-h; separation step 2: 400 V, 1 mA, 2.5 W at 4 °C for 2 V-h; separation step 3: 400 V, 10 mA, 2.5 W at 4 °C for 288 V-h. Note that only one gel could be run at a time because two gels exceeded the cooling capacity of the system.

Native Electrophoresis in the Presence of GTP

After phosphorylation of EF-Tu by S15, samples were desalted and converted to EF-TuGTP. In the presence of 100 pmol of EF-Tu, tRNA was added (as indicated in Fig. 6) in a 50-µl reaction volume in polymix buffer (5 mM Mg-acetate, 0.5 mM CaCl, 95 mM potassium chloride, 5 mM NHCl, 8 mM putrescine, 1 mM spermidine, 5 mM potassium phosphate, 1 mM dithioerythritol, pH 7.5) containing 1 mM ATP, 10 mM phosphoenolpyruvate, 1 mM GTP, 5 µg of pyruvate kinase, 0.3 µg of myokinase, and 10 units of Phe-tRNA synthetase. Complex formation was at 37 °C for 10 min. Samples were cooled on ice and 0.1 volume of 50% glycerol was added. Samples were separated on 5% acrylamide gels at 4 °C with circulation of the buffer (50 mM TrisHCl, 10 mM Mg-acetate, 65 mM NH acetate, 1 mM EDTA, 1 mM dithioerythritol, 10 µM GTP, pH 6.8).


Figure 6: Kirromycin resistance overcomes the phosphorylation inhibition of the antibiotic. Analysis of EF-Tu phosphorylation by different S15 preparations was performed in the absence (-) or presence (+) of 50 µM kirromycin. Upper panel, phosphorylation analysis was performed with 0.25 A S15 from the mentioned strain without addition of EF-Tu, using the endogenous EF-Tu as substrate. As could be inferred from the figure, the antibiotic efficiently blocks phosphorylation in the wild-type strain MRE 600, but did not show any effect on the modification of mutant EF-TuA from the PM455 strain. Lower panel, catalytic amounts of S15 from the strains indicated were analyzed for their ability to phosphorylate wild-type EF-Tu. The kinase of the mutant strain is not able to modify the wild-type EF-Tu. A, displays Coomassie staining; B, autoradiography.




RESULTS

To investigate the influence of different substances on EF-Tu phosphorylation and the binding properties of the modified factor, we established a simplified in vitro system as described under ``Materials and Methods.''

In short, cells were disrupted by sonification, the lysate cleared by centrifugation (S15) and in some experiments the S15 separated into crude 70 S ribosomes (P100) and supernatant fraction (S100) by a short high-speed centrifugation. The assays consisted of freshly prepared subcellular fractions (S15, P100, S100) as source of the kinase and addition of [-P]ATP and EF-Tu as substrates. The extent of modification was monitored by SDS-PAGE followed by autoradiography. In this system we analyzed the effect of EF-Ts. Addition of EF-Ts resulted in an increase in EF-Tu phosphorylation, which was most significant in the crude ribosomal fraction (Fig. 1, A and B). Neither EF-Ts nor EF-Tu, nor the combination of both, displayed any incorporation of radioactivity in the absence of S15, excluding EF-Ts itself as the kinase. The stoichiometry of the EF-Ts activation was monitored with S15. Maximal enhancement was reached at an EF-Ts concentration nearly equimolar to that of EF-Tu (Fig. 1C). Apparently, interaction with EF-Ts induces a conformational change in EF-Tu exposing the phosphorylation site to the kinase.


Figure 1: Exchange factor EF-Ts enhances EF-Tu phosphorylation. Crude cellular fractions were prepared as described elsewhere (Lippmann et al., 1993). From each fraction 0.01 A aliquots were analyzed for their ability to phosphorylate EF-Tu in the absence or presence of EF-Ts in an equimolar concentration to EF-Tu. Samples were separated on 12% SDS-PAGE. The enhancement of the phosphorylation by EF-Ts is manifold stronger in the crude 70 S ribosome preparation than in S15, indicating an association of the kinase activity with these translational components. Addition of EF-Ts to EF-Tu alone in the presence of [-P]ATP leads to no incorporation of radioactivity into EF-Tu (right lane). A, Coomassie Blue staining of the SDS-PAGE; B, autoradiography. C, concentrations of EF-Tu (5.4 µM) and S15 (0.01 A) were kept constant and increasing amounts of EF-Ts were added. Quantifications were done by densitometry of the autoradiogram (inset), corrected by estimation of protein quantities from densitometry of the Coomassie-stained gel.



We further examined the influence of various antibiotics acting on protein biosynthesis in the in vitro phosphorylation system. Using S15 as source for kinase, the presence of 20- to 100-fold excess over EF-Tu of puromycin, chloramphenicol, or streptomycin resulted neither in significant enhancement nor in reduction of EF-Tu modification (data not shown), but kirromycin abolished the phosphorylation completely (Fig. 2).


Figure 2: Inhibition of EF-Tu phosphorylation by kirromycin. Wild-type EF-TuGDP from E. coli was incubated for 20 min with catalytic amounts (0.01 A) of S15 from E. coli MRE 600 in the presence of [-P]ATP and separated by SDS-PAGE as described under ``Materials and Methods.'' The inset shows an example (molecular mass from the marker are 94, 67, 43, and 29 kDa). The graph displays data derived from densitometry of autoradiograms, corrected for protein content by densitometry of the Coomassie-stained gel. The dotted line indicates the EF-Tu concentration under assay conditions.



Therefore, we analyzed the influence of this antibiotic on EF-Tu modification in more detail. Addition of kirromycin in increasing concentrations led to a sharp reduction in phosphorylation under conditions of 1:1 binding (Fig. 2). To clarify whether the inhibition is an effect of binding to EF-Tu and not due to a dephosphorylation by kirromycin (or stimulation of a highly active phosphatase) we used a native gel electrophoresis system under nonequilibrium conditions using commercially available mini gels (PhastGel; Pharmacia). After optimizing the buffer and running conditions EF-Tukirromycin complexes could be separated from free EF-Tu. Results are shown in Fig. 3, upper panel, lane 4. As can be concluded from the Coomassie staining, the nonphosphorylated EF-Tu displayed normal kirromycin binding properties, leading to a higher migration velocity than the free EF-Tu, whereas the phosphorylated portion did not participate in this interaction, migrating to the same position as the control (Fig. 3, lane 1). From the intensity of Coomassie staining of the nonphosphorylated complexed EF-Tu and the free phosphorylated protein the extent of modification was estimated to approximately 7% (lane 4). This is the first demonstration of a wild-type form of EF-Tu that is unable to recognize kirromycin.


Figure 3: Phosphorylated EF-Tu is unable to bind kirromycin, but is still capable to complex EF-Ts. Complexes were formed at 0 °C after phosphorylation of EF-Tu by S15 extract and separated by native PAGE (see ``Materials and Methods'') on 20% homogeneous PhastGels as shown in the upper panel and SDS-PAGE (12%; lower panel). Lane 1, without any addition; lane 2, addition of EF-Ts (1:1 molar ratio to EF-Tu); lane 3, addition of EF-Ts and kirromycin (1:2:2 molar ratio of EF-Tu:EF-Ts:kirromycin); lane 4, addition of 10-fold molar excess of kirromycin (over EF-Tu); lane 5, addition of 5-fold molar excess of EF-Ts (over EF-Tu). Note: in lane 5 the running behavior of free EF-Ts is displayed. The EF-Ts migrates as a doublet (only under native conditions; compare upper and lower panel), the upper band may indicate a dimer. Both bands disappeared in the presence of saturating amounts of EF-Tu, confirming the identity as EF-Ts (lane 2). The phosphorylated EF-Tu is clearly capable of binding to EF-Ts (lanes 2, 3, and 5), but not to kirromycin (lanes 3 and 4).



The native gel system enabled us to investigate also the interaction behavior of the modified elongation factor Tu with its exchange factor EF-Ts. In these experiments the ability of the phosphorylated EF-Tu to complex EF-Ts could be demonstrated (Fig. 3, lanes 2, 3, and 5). The phosphorylated part seems to have a higher affinity for EF-Ts as can be concluded from data where EF-Tu was in excess over EF-Ts (not shown). Under the running conditions employed, free EF-Ts migrated as two separate bands (Fig. 3, lane 5) which both were complexed in the presence of saturating amounts of EF-Tu (Fig. 3, lane 2).

We used another method to analyze the ternary complex formation. After phosphorylation of EF-Tu the samples were desalted, GDP was converted to GTP by phosphoenolpyruvate and pyruvate kinase in the presence of aa-tRNA as described under ``Materials and Methods.'' Samples were then separated by native gel electrophoresis in the presence of GTP. Under these conditions EF-Tu was able to form a ternary complex (Fig. 4, left) but autoradiography revealed the absence of the phosphorylated part in the complex (Fig. 4, right). Due to additional steps the degree of phosphorylation decreased because the phosphatase was still present in the preparation until the electrophoresis.


Figure 4: Phosphorylated EF-Tu is unable to bind aa-tRNA. After phosphorylation of EF-Tu by S15 extract samples were desalted and converted to EF-TuGTP as described under ``Materials and Methods.'' Analysis of 100 pmol of EF-Tu for aa-tRNA binding was performed in a 5% PAGE under native conditions with circulating buffer. After fixation in 5% acetic acid and 50% methanol the gel was boiled in 5% trichloroacetic acid for 30 min to remove all nucleic acids. As displayed by Coomassie staining (left) the EF-Tu is able to bind aa-tRNA, but on the autoradiogram (right) no phosphorylated EF-Tu could be detected at the position of the ternary complex.



To further analyze the inability of the post-translationally modified EF-Tu to bind kirromycin and aa-tRNA, we employed a method which is especially suitable to detect weak interactions under equilibrium conditions, namely zone-interference gel electrophoresis (Abrahams et al., 1988). The result confirmed the absence of phosphorylated EF-Tu in complex with kirromycin and aa-tRNA (Fig. 5). From the ligand concentration in the zones one can deduce, that the K values for eventual EF-Tu complexes with kirromycin and aa-tRNA should at least be higher than 25 µM.


Figure 5: EF-Tu phosphorylation even prevents weak interactions with kirromycin and aa-tRNA. Zone-interference gel electrophoresis (Abrahams et al., 1988) was applied to examine the binding of phosphorylated EF-Tu (pEF-Tu) to kirromycin. An EF-Tu preparation phosphorylated by S15 as described under ``Materials and Methods'' was analyzed, using samples and zones with concentrations of kirromycin and aa-tRNA as indicated. The preparation displayed a normal binding of kirromycin and aa-tRNA, except the phosphorylated part which did not show any interaction migrating to the same position as the control. Panel A, Coomassie Blue staining; panel B, autoradiography.



In the three-dimensional structure the mutation A375T leading to kirromycin resistance is close to the site of phosphorylation (Kjeldgaard and Nyborg, 1992; Berchtold et al., 1993; Mesters et al., 1994). The kirromycin-resistant EF-Tu mutants are able to promote polypeptide chain growth even in the presence of high concentrations of the antibiotic, whereas in wild-type cells EF-TuGDPkirromycin sticks to the ribosome. Because kirromycin also blocks phosphorylation we investigated the modification of a mutated EF-Tu. We used the E. coli strain PM455 carrying a mutant tufA gene coding for kirromycin-resistant EF-TuA (EF-Tu(Ala-Thr)) and an inactivated tufB gene (Mu insertion) (Van der Meide et al., 1982). As displayed in Fig. 6(upper panel), the mutant EF-TuA was modified to the same extent both in the presence and absence of kirromycin and showed an even stronger degree of phosphorylation than wild-type EF-Tu of an equally prepared S15 extract from E. coli MRE 600. Surprisingly, catalytic amounts of the S15 preparation from the mutant strain appeared to be unable to mediate phosphorylation of wild-type EF-Tu (Fig. 6, lower panel). Identical results were obtained with another strain (LZ13; Zeef and Bosch, 1993) carrying the same mutations concerning the EF-Tu genes (data not shown).

The data on the influence of kirromycin on the phosphorylation and the inability of the modified EF-Tu to interact with the antibiotic suggest a specific step in protein biosynthesis as function for the in vivo phosphorylation. Kirromycin prevents the release of EF-TuGDP from the ribosome after GTP hydrolysis. To analyze whether this is the point where phosphorylation occurs, we performed the following experiment. Nucleotide-free EF-Tu was saturated with GTP and used as substrate in the phosphorylation assay. Because the nucleotide-free form of E. coli EF-Tu is hard to prepare we changed to T. thermophilus. S15 was also prepared from T. thermophilus as described for E. coli. The phosphorylation reaction was studied at 50 °C as a function of time. Under these conditions one may expect that the EF-TuGTP will rapidly be converted into EF-TuGDP (Limmer et al., 1992). The result is shown in Fig. 7. At the time points indicated aliquots were removed and analyzed on SDS-PAGE followed by autoradiography. One can see that at the beginning where the ribosome-stimulated transition rate from GTP to GDP complex is fast, the degree of phosphorylation is also enhanced. In addition, the experiment shows that the reaction is reversible. Despite the excess of ATP and the permanent presence of EF-Ts, the degree of phosphorylation diminishes when the amount of GTP becomes exhausted.


Figure 7: Time course of phosphorylation of EF-TuGTP. Nucleotide-free EF-Tu from T. thermophilus HB8 was saturated by a 4-fold molar excess (300 µM) of GTP prior to phosphorylation by S15 from T. thermophilus HB8 prepared essentially as described for E. coli. The reaction was started by addition of S15 and incubation at 50 °C. At the time points indicated aliquots were removed and the reaction terminated by boiling with sample buffer. Only the relevant portion of the autoradiogram is shown.



In summary, we could demonstrate that phosphorylation of EF-Tu can be inhibited by kirromycin and enhanced by EF-Ts. The modification abolishes the complex formation between EF-Tu and kirromycin as well as aa-tRNA. Kirromycin-resistant cells display a recognition between EF-Tu and kinase different from that in the case of sensitive cells. The reversible phosphorylation of EF-Tu is correlated with the transition from EF-TuGTP to EF-TuGDP.


DISCUSSION

The stimulation of EF-Tu phosphorylation by EF-Ts, another component of the translational machinery, strongly supports a function of this post-translational modification in protein biosynthesis. The interaction between EF-Tu and EF-Ts could lead to a structural rearrangement of EF-Tu facilitating the access of the kinase to the site of phosphorylation. Inhibition of the phosphorylation by kirromycin is also a direct EF-Tu-correlated effect and not mediated by inhibition of the kinase as can be concluded from experiments in which EF-TuA was phosphorylated by wild-type S15 (MRE 600) in the presence of kirromycin (data not shown). The inability of the mutant strain kinase to modify the wild-type EF-Tu underlines the importance of the phosphorylation for proper function. The small structural aberration by the single amino acid replacement in EF-TuA seems to be compensated by a parallel mutation in the kinase.

We have attempted to analyze whether EF-Tu is accessible for the kinase in the ternary complex. The analysis was complicated by the fact that another kinase has to be added to the system to ensure complete conversion of GDP to GTP. Therefore we used the nucleotide-free EF-Tu from T. thermophilus and complexed it with GTP and aa-tRNA (data not shown). The presence of aa-tRNA reduced the phosphorylation slightly as compared to GTP alone (in Fig. 7, last lane), but after addition of an substantial amount of EF-Ts which ensures complete ternary complex formation no phosphorylated EF-Tu could be observed after 15 min reaction (data not shown). This led us to the assumption that in the ternary complex EF-Tu is not accessible for the kinase. In a complementary way, phosphorylated EF-Tu is not able to bind aa-tRNA.

Only limited knowledge exists about the EF-TuEF-Ts interaction. Binding experiments suggests involvement of domains 2 and 3 of EF-Tu (Peter et al., 1990), whereas mutant analysis refer to domain 1 (Hwang et al., 1992). Recent results point to an extended EF-Ts interaction during the activity cycle of EF-Tu, going from nucleotide exchange up to a quaternary complex of EF-TuGTPaa-tRNAEF-Ts that enters the ribosome (Bubunenko et al., 1992; Schwartzbach and Spremulli, 1991). Ehrenberg and co-workers (Ehrenberg et al., 1990; Scoble et al., 1994) and Weijland and Parmeggiani(1993) have shown that 2 molecules of GTP are hydrolyzed per peptide bond formation by the action of EF-Tu. The presence of EF-Ts in a complex on the translating ribosome would allow a direct exchange of the GDP hydrolyzed. We have found that the major kinase activity is associated with a crude 70 S preparation (Lippmann et al.(1993) and Fig. 1) and that EF-Tu phosphorylation is enhanced by the addition of EF-Ts.

The inhibition of phosphorylation by kirromycin implies an antagonistic conformational change. On the other hand, the antibiotic can be present in complexes of EF-Tu with GDP, GTP, and aa-tRNA, but not in complex with phosphorylated EF-Tu. This suggests that the modified factor adopts a new conformation different from the GDP/GTP/aa-tRNA complexed form. This may occur after the GTP hydrolysis when EF-TuGDP(EF-Ts) is still on or close to the translating ribosome. Although the kinase can use GTP as well as ATP as substrate we can rule out that the -phosphate of the GTP complexed to EF-Tu is the source for phosphorylation.()

The inability of the phosphorylated EF-Tu to bind aa-tRNA points to a similar behavior on the ribosome and suggests the following model. After binding of the ternary complex to the A-site of the ribosome the EF-Tu affinity to aa-tRNA is changed by GTP hydrolysis, which opens the EF-Tu complex for the kinase. The phosphorylation will then loosen the binding of EF-Tu to the ribosomal complex, thus facilitating EF-Tu to leave the site of translation.

As stated in the first report on the phosphorylation of EF-Tu (Lippmann et al., 1993) the degree of phosphorylation in vivo is low, approximately 5%. In our in vitro studies we reached a higher rate only with the enhancement by EF-Ts. Preliminary experiments with a partially purified kinase activity did not considerably increase the extent of phosphorylation. This suggests that the limiting step in phosphorylation is not the kinase itself but the substrate. The degree of in vivo modified EF-Tu is in good correlation with the fraction which is active on the ribosome at a given time point.

Assuming that the affinity of EF-Tu from E. coli for GDP is 100-fold higher than for GTP and that some GTP is still present in the S15 preparation used for the in vitro experiments this number of about 5% within the experimental error also correlates with the fraction initially complexed to GTP. Saturating amounts of GTP could not be used in the assay because, as stated above, the kinase can use GTP as well as ATP as substrate and therefore the specific activity drops and the analysis will be impaired by a loss of the signal.

These in vivo and in vitro data let us conclude that the fraction of EF-Tu which changes its conformation from GTP to GDP may be the substrate for the kinase. This is substantiated by the experiment in which the time course of phosphorylation of EF-TuGTP was followed. Under the conditions employed the transition rate from GTP to GDP complex is initially high until the majority of the GTP is hydrolyzed. The result (Fig. 7) clearly demonstrated that this is in correlation with the degree of phosphorylation.

A comparison of the three-dimensional structures of the complexes of EF-Tu with GDP (Kjeldgaard and Nyborg, 1992) and with the GTP analogue, Gpp(NH)p (Berchtold et al., 1993; Kjeldgaard et al., 1993) supports this view. Upon transition from the active GTP form to the inactive GDP complex, the phosphorylation site changes position by as much as 21.8 Å (see purple balls in Fig. 8). In the active GTP form, Thr-382 is located almost inaccessible, in the interface between domain 1 and 3 of EF-Tu; its side chain hydroxyl group donates a 2.70-Å hydrogen bond to Glu-117, across this interface (Fig. 9). It is thought that the aa-tRNA will bind to the cleft identified between domain 1 and 2 (Berchtold et al., 1993; Förster et al., 1993), possibly locating the aminoacylated CCA-end close to the phosphorylation site. During binding of the ternary complex to the ribosome conformational changes of components occur (Rodnina et al., 1993), possibly of the aa-tRNA after correct alignment to the codon (Bertram et al., 1983) or of EF-Tu. One or both events may weaken the interaction between domains 1 and 3, making the site of phosphorylation accessible for the kinase. Introduction of the negatively charged phosphate group will lead to the repulsion of Glu-117 and result in complete disruption of the interface. Such a mechanism could accelerate the switch to the ``open'' GDP form, which falls off from the ribosome.


Figure 8: Structure of EF-TuGDP and EF-TuGpp(NH)p. Ribbon model of EF-Tu in complex with GDP (top) and with Gpp(NH)p (bottom). Domain 1 is shown in orange, domain 2 in yellow, and domain 3 in blue. The nucleotide in domain 1 is displayed in green and the white sphere indicates the Mg ion. The site of phosphorylation is indicated by a purple sphere in domain 3. The switch between the two states moves the phosphorylation site by approximately 21 Å.




Figure 9: The phosphorylation site of EF-Tu in the structure of the Gpp(NH)p complex. Stereo illustration of the interaction between domain 1 (orange ribbon) and domain 3 (blue ribbon) around the site of phosphorylation. Residues are numbered according to E. coli EF-Tu. Dashed lines shows hydrogen bonds, red crosses represent positions of water molecules.



The inhibition of phosphorylation of Thr-382 by kirromycin is also in agreement with the structural data. Mesters et al.(1994) have shown that the presumable binding site for kirromycin on the E. coli EF-Tu is along the interface between domains 1 and 3, since all the known resistance mutations are located here. Two of these, Gln-124 and Thr-375, are spatially close to Thr-382 (see Fig. 9). Thus, it may be that the inhibition of phosphorylation by the antibiotic provides an explanation for the immobilization of EF-TuGDPkirromycin on the ribosome.

In any case, the phosphorylation of EF-Tu appears to be a novel mechanism for a reversible modification of the interaction behavior of the factor and thus for the regulation of protein synthesis in general.


FOOTNOTES

*
This work was supported in part by grants from the Deutsche Forschungsgemeinschaft (DFG, Li 611/1-1) (to C. L.), the Bundesministerium für Forschung und Technologie (BMFT) (to V. A. E.), the European Community (``human capital and mobility,'' No. ERB CHRX-CT94-0510) (to B. K., R. H., and V. A. E.), and the Swedish Natural Science Research Council (to N. B.). C. A., N. B., Ca. L., and J. R. M. contributed equally to this work. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 49-30-838-6003; Fax: 49-30-838-6403.

The abbreviations used are: EF, elongation factor; aa-tRNA, aminoacylated transfer RNA; PAGE, polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; V-h, volt hour; Gpp(NH)p, guanosine 5`-(,-imino)triphosphate.

C. Lindschau, C. Alexander, V. A. Erdmann, and C. Lippmann, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank H. Ebeling for photography and W. Rossner for carefully reading the manuscript. The generous gifts of kirromycin from A. Parmeggiani and of EF-Ts from S. Lorenz are gratefully acknowledged. We thank M. Sprinzl for providing us nucleotide-free EF-Tu and for fruitful discussions and critical comments.


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