THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 12, Issue of April 25, pp. 6744-6749,199O Printed in U.S. A.

Substitution of Proline 82 by Threonine Induces Autophosphorylating Activity in GTP-binding Domain of Elongation Factor Tu*

(Received for publication, November 14, 1989)

Robbert H. Cool& Michael Jensenl, JiS Jonikn, Brian F. C. Clark& and Andrea ParmeggianiS From the jLaboratoire de Biochimie, Ecole Polytechnique, URA 240 du Centre National de la Rech.erche Scientifique, F-91128 Palaiseau, France, the SDivision of Biostructural Chemistry, Department of Chemistry, Aarhus University, DK-8000 Aarhus C, Denmark, and the Vnstitute of Molecular Genetics, Czechoslovak Academy of Sciences, CS-16637 Prague, Czechoslovakia

Mutation of Pro*’ into Thr, a residue situated in the second element (D80CPG83) of the consensus sequence proposed to interact with GTP/GDP in GTP-binding proteins was introduced via site-directed mutagenesis in the isolated guanine nucleotide-binding domain (G domain) of elongation factor Tu. G domainPT82 dis- plays virtually no GTPase activity. As a major change, the apparent inhibition of the GTPase reaction is as- sociated with the appearance of autophosphorylating activity, as in ras product p21 in the case of mutation Ala6’ + Thr, corresponding to 82 in elongation factor Tu. Dependence of this reaction on mono- and divalent cation concentration and on pH is essentially the same as for the GTPase of wild-type G domain. The autoki- nase reaction follows an apparent first order rate, suggesting an intermolecular mechanism. Analysis of amino acid and peptide composition of the 32P-labeled G domainPT82, as well as Edman degradation of the tryptic peptide containing the covalently bound 32P, shows that Thrs2 is the phosphorylated residue. Taken together, these results point out that Thrs2 is in close proximity to the -y-phosphate of GTP, as in the case of Thr” in ~21. These results are in agreement with the observations derived from x-ray diffraction analysis that the tertiary structure of the GTP-binding domain of elongation factor Tu and that of p21 are similar.

Elongation Factor Tu (EF-Tu)’ from Escherichia coli, the carrier of AA-tRNA, where AA represents any amino acid, to the ribosome in the elongation cycle of bacterial protein biosynthesis (1, 2), is one of the best characterized guanine nucleotide-binding proteins, a class of proteins playing a crucial role in the signal transmission of the cell (3-6). These proteins display an intrinsic GTPase, a pivotal reaction, since it converts the active form induced by GTP and needed for the interaction with ligands, into the inactive form induced by GDP. Comparison of the primary structures of these proteins has revealed different regions of homology (7-10)

* This work was supported in part by Grants BAP-0066-F (to A. P.) and BAP-0058-DK (to B. F. C. C.) from the Biotechnological Action Program of the Commission of the European Community, Association pour la Recherche Contre le Cancer Grant A.R.C.-6377 (to A. P.), Danish Natural Science Research Council FTU Project (to B. F. C. C.). and Danish Biotechnoloav Research Program (via BIOREG, to’h. F. C. C.). The costs of puciication of this a&le were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ The abbreviations used are: EF-Tu, elongation factor Tu from E. coli; GTPase, guanosine 5’-triphosphatase; G domain, isolated GTP- binding domain of EF-Tu; SDS-PAGE, sodium dodecyl sulfate-poly- acrylamide gel electrophoresis.

and has led to the identification of a consensus sequence related to the guanine nucleotide-binding site (11, 12). This sequence is constituted by three elements with defined spacing and in most cases is located in the N-terminal part of the protein.

X-ray analysis of a nicked EF-Tu. GDP complex refined to a resolution of 2.6 A revealed a structure which shows three distinct domains: the N-terminal, the middle, and the C- terminal domain (13-16). The three-dimensional structure of the N-terminal domain of EF-Tu, comprising the first 199 amino acids, has been used in protein engineering studies to predict areas where substitution should affect the function of EF-Tu in respect of both specificity and phosphate hydrolysis. In addition, a combination of molecular graphics operations and sequence homology studies has enabled the model build- ing of other GTP/GDP-binding proteins into the EF-Tu structure, in particular the ras protooncogenic p21 (11). To study further the analogy between EF-Tu-like structures and ra.s proteins and to search for possible common functions we have isolated the N-terminal domain (G domain) and related mutants of EF-Tu by genetic engineering methods (17-20). The G domain binds GTP and GDP and catalyzes the hy- drolysis of GTP. However, these activities are no longer under control of the allosteric mechanisms regulating the function of intact EF-Tu (17,18). Therefore the G domain can be used as a simplified model for investigating the interaction with the substrate and the catalytic activity.

Because of our interest in the phosphate-binding site of EF-Tu and the finding that certain oncogenic mutants of p21 could be autophosphorylated at Thr5’ (21-23), we constructed a mutant of the G domain of EF-Tu with Thr instead of Pro”, the equivalent position to 59 in ~21. Here we report the properties of this mutant G domainPT82, stressing the iind- ing that it can be autophosphorylated at Thrs’.

MATERIALS AND METHODS

Biological Components-The plasmids pEMBLS+ and pCP40 and the E. cili strain N4830 were kindly provided by R. Cortese (EMBL, Heidelberg). W. Fiers (State Universitv of Ghent. Ghent), and 0.

I ,

Fasano (EMBL, Heidelberg), respectively. Site-directed Mutagenesis, Overproduction, and Purification of G

DomainPT’82-For mutagenesis we used the pEMBLS+ vector, con- taining the cloned truncated tufA gene coding for the N-terminal domain of EF-Tu, as described earlier (17). The mutagenic primer (5’~CGTAGACTGCACGGGGCACG-3’), phosphorylated at its 5’ end, was hybridized to a gapped duplex formed by single stranded pEMBL9’tufA(A610-1180) annealed with purified large fragment of EcoRI/BclI-digested pEMBLS+tufA(A610-1180), leaving a stretch of single stranded DNA from the 5’ end of tufA up to the triplet coding for Met”. The residual DNA gaps were filled in with Klenow enzyme and T4 ligase (Pharmacia LKB Biotechnology Inc). After transfor- mation of E. coli strain 71/l& the colonies carrying the mutated plasmids were selected by cell colony hybridization and DNA sequenc-

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Autophosphorylution of Thr ” in EF-Tu

ing as already described (17). The mutated gene was cloned under control of XPL in the “runaway” vector pCP40 (24). For overpro- duction we used strain N4830 (25), carrying a chromosomic thermo- sensitive XPL repressor. After determination of the optimum condi- tions for overproduction of soluble G domain, a culture of cells was grown at 28 “C in a 20-liter fermentor (Biolafitte) up to a cell density of O&& units, at which point the temperature was rapidly increased to 42 “C. After an additional incubation of 1.5 h, the cells were harvested, sonicated in 10 ml of sonication buffer (50 mM imidazole acetate, pH 7.7,100 mM KCl, 5 mM MgCl*, 7 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride (Sigma), and 15% glycerol) per g of wet cells and centrifugated for 40 min at 20,000 x g as described (17). Purification of the overproduced G domain was carried out in four steps: ionic chromatography on Q-Sepharose (gradient of 80-500 mM KC1 in 50 mM Tris-HCI, pH 7.7, 10 mM MgC12, 7 mM 2- mercantoethanol. and 15% glycerol), filtration on Superose 12 Pre- parative Grade (repeated twice, using a buffer of 50 mM Tris-HCl, pH 7.7, 50 mM KCl, 10 mM MgCl*, 7 mM P-mercaptoethanol, 15% glycerol, and 20 MM GDP), and ionic chromatography on Mono-Q (gradient of 180-230 mM KC1 in 50 mM Tris-HCl, pH 7.7, 10 mM M&I,. 7 mM 2-mercantoethanol, 15% alvcerol, and 20 UM GDP), . -_ using the fast protein liquid chromatography system of Pharmacia.

The standard buffer contained 50 mM imidazole acetate, pH 7.6- 7.7, 50 mM NH&l, 10 mM MgC12, and 1 mM dithiothreitol. Protein concentrations were determined by the method of Bradford (26), using bovine serum albumin as a standard. Apparent dissociation constants and dissociation rates were determined in standard buffer as described (18).

Determination of the GTPase Activity-GTPase activities were measured by incubation with [y-32P]GTP at 30 “C. The reaction was terminated by the addition of an equal volume of a solution containing 1 M HClO, and 3 mM KHIPOl (0 “C). The liberated3’Pi was measured after the charcoal- or isopropylacetate:molybdate method (27, 28).

Autophosphorylution-To test autophosphorylating activity in the cell extract, small cultures of cells overproducing EF-Tu, G domain, or G domainPT82 were grown. The cytosolic proteins were extracted bv sonication of the cells and centrifuged in order to eliminate the cell debris. The resulting mixture of soluble proteins was incubated with ~Y-~‘P~GTP at 30 “C. Samnles were taken at different time inter&s, whereafter the proteins were precipitated with cold 10% trichloroacetic acid and analyzed on SDS-PAGE by dissolving the pellet in SDS loading buffer (30 mM Tris-HCI, pH 6.7, 1% SDS, 5% glycerol, and 0.05% brom-phenol blue). After boiling for 3 min, the sample was loaded onto a 12% polyacrylamide gel. The gel was stained with Coomassie Brilliant Blue R (Sigma) after electronhoresis and an autoradiogram was taken of the dried gel.

Autophosphorylating activity of the purified protein was deter- mined by incubating the protein with ~T-~‘P~GTP at 30 “C. Aliquots were taken at increasing incubation periods and pipetted into 2 ml of ice-cold 10% trichloroacetic acid. After lo-20 s, this was filtered onto nitrocellulose (Sartorius SM 11306, 0.45 rrn); followed by a B-fold washing with 2 ml of 10% trichloroacetic acid (0 “C), as described (29). The filters were dried and counted in organic countingscintillant (Amersham Corp.). Alternatively, the aliquots were mixed with an excess of GTP and EDTA. and incubated for 15 min at 30 ‘C to replace the noncovalently bound radioactivity with nonlabeled nu- cleotide (30). Filtration on nitrocellulose, followed by two washes with 3 ml of 50 mM Tris-HCl, pH 7.7, gave similar results as the previous method.

Identification of the Phosphor&ted Amino Acid Residue-Analysis of the labeled amino acid in phosphorylated G domainPT82 was carried out after total hydrolysis of i8 pg.of 32P-labeled protein (1700 cnm/nmol of labeled G domain) after a 3-h incubation at 110 “C in 6 & HCl. The hydrolysate was spotted on thin-layer cellulose (MN300) and separated in two dimensions in the presence of 2 pg of phospho- threonine (Sigma) as described by Shih et al. (22). The first dimension was an electrophoresis for 85 min at 500 V and 20 mA in acetic acidformic acidwater (78:25:897; pH 1.9), the second a chromatog- raphy in 0.5 M NHIOH:isobutyric acid (3:5). After drying the cellulose an autoradiogram was made and finally the plate was sprayed with a ninhydrin solution.

Tryptic digestion and analysis of the labeled peptide of G domainPT82 was carried out essentially as described by Jonik et al. (31). Of unlabeled and labeled G domainPT82, 60-80 pg were dis- solved in 7 M guanidine HCl, 1 M Tris-HOAc, pH 8.5, 0.1 M EDTA, treated with iodoacetamide in order to protect the cysteine residues, and digested with 1.6 pg of N-tosyl-L-phenylalanylchloromethane- treated trypsin (Worthington) for 5 h at 37 “C. Part of the mixture

of peptides was separated by high performance liquid chromatography (Knauer) using a O-90% gradient of acetonitrile in 0.05% trifluoroa- cetic acid over a Vydac Cla column (4 X 280 mm). The peaks were collected in fractions of which part was counted in a scintillator. The labeled peptide was sequenced by Edman degradation in a gas-phase sequenator with subsequent identification of the liberated modified amino acids by reversed-phase chromatography on high performance liquid chromatography.

RESULTS

Purification and General Properties of G DomainPTBB- The mutated product was expressed in the E. coli cell as effectively as the wild-type G domain. It was overproduced up to 20% of the total protein of the cell; no anomalies in the growth rate of the cells containing overproduced G domainPT82 were observed. Solubility of the G domain after centrifugation at 20,000 x g for 40 min was about 60%. After the three purification steps (see “Materials and Methods”) the mutant protein was at least 99% pure. The yield of the purified product was approximately 1 mg of protein/g of cell. Neither proteolytic or aggregation phenomena were observed during and after purification as determined by SDS-PAGE. The mutant protein was stored in 25 mM Tris-HCl, pH 7.7, 50 IXIM NH&l, 10 mM MgCl*, 20 pM GDP, 7 mM 2-mercap- toethanol, and 50% glycerol. It retained the same level of guanine nucleotide binding for at least 6 months when kept at -30 “C.

GTPase Activity and Autophosphorylation of G Domain- PTBZ-The mutant protein displays no intrinsic GTPase activity, at least when measured by the amount of liberated organic phosphate. Whereas the GTPase activity of both EF- Tu and G domain wild-type can be stimulated by monovalent salt (18), there is no effect whatsoever on G domainPT82. Fig. 1 shows the consequence of substitution of Pros2 on GTPase activity at 1 M KCl, as measured by the liberation of Pi.

A striking property of rus protein p21 is represented by the occurrence of autophosphorylation of position 59 when the wild-type residue alanine is substituted by threonine (21-23). It was therefore important to see whether G domainPT82 also could express this activity.

We first determined the autokinase activity in the cell extract. Comparison of the Coomassie Blue-stained patterns (Fig. 2.4) with the autoradiogram of the same gel (Fig. 2B), shows that there is radioactivity co-migrating with G domainPT82 and that the labeling of the protein increases by increasing the time of incubation with [T-~‘P]GTP. No radio- activity could be detected co-migrating with wild-type G do-

0 20 40 60 80 time (min)

FIG. 1. GTPase activities of G domainPT82 as compared to wild-type G domain. G domain (0) or G domainPT82 (B) at a concentration of 1 pM was incubated at 30 “C with 18 pM [Y-~‘P]GTP (3800 cpm/pmol) in 580 pl of standard buffer containing 1 M KC1 as monovalent ions. Samples were taken after the indicated time inter- vals and treated by the isopropylacetate method (see “Materials and Methods”).

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6746 Autophosphorylation of Thrs’ in EF-Tu

EF-Tu G domain G domainPTX2 EF-TU G domain G domainPI‘ I 2 5 IS 45 I 2 5 IS 45 5 IS 45 90 180 min I 2 5 I5 45 I 2 S 15 45 5 IS 45 90 180 mill

A B

EF- ru G domam G domamFT82 I 2 5 I5 45 I 2 5 15 45 5 15 45 90 ,*,I ml,”

C

EF-Tu G domain G domainPTX2 / 2 5 15 4s I 2 J IS 45 5 IS 45 90 IX0 ml”

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;&$ 4’ : t . . ,

$f$ ‘T -3 I*r +I” 4 EF-Tu ,

“ ,aa;d,Gdomom, *Q**++

-- -A- - -

FIG. 2. Phosphorylation of G domainPTS2 in cell extract. Cultures of 7 ml of E. coli N4830 containing pCP40 with the inserted gene coding for EF-Tu, G domain, or G domainPT82, were grown at 28 “C. Induction was started at Ah,H, 0.4-0.7 by raising the temperature to 42 “C. After incubation for 1.5 h, cells were harvested, resuspended in 1 ml of sonication buffer, sonicated, and centrifugated for 40 min at 20,000 X g. Of the supernatant, 150 ~1 were taken and diluted to 600 ~1 of 50 mM imidazole acetate, pH 7.7, 100 mM KCl, 10 mM MgC12, 1 mM dithiothreitol, and 100 pM [-y-“*P]GTP (100 cpm/pmol). During incubation at 30 “C, samples of 60 ~1 were either directly precipitated by adding trichloroacetic acid to 10% in ice (panels A and B), or first EDTA and non-labeled GTP were added to 6 and 4 mM, respectively, with an additional incubation of 15 min at 30 “C, followed by the trichloroacetic acid precipitation (panels C and D). After electrophoresis the gel was stained (panels A and C) and dried, whereafter an autoradiogram was made of the gel (panels B and D).

main or EF-Tu. Another, still unidentified protein, which migrates somewhat slower than EF-Tu, was labeled with [y- ‘“P]GTP within a short interval of time (about l-2 min, see Fig. 2, A and B). However, treatment of the samples with an excess of cold GTP and EDTA removed nearly all the radio- activity of this component (Fig. 2, C and D). This treatment had no detectable effect on the radioactivity co-migrating with the G domainPT82, strongly suggesting that T82 can bind covalently the y-phosphate of GTP. In this particular experiment EF-Tu and G domain wild-type were not overpro- duced well. However, other experiments with better induction confirmed our observation that these proteins are not labeled as determined by this method (not shown).

To examine further whether the labeling of G domainPT82 was a consequence of an autokinase activity, we incubated highly purified G domainPT82 with [-Y-~‘P]GPT at 30 “C and quantified the labeled protein by filtration on nitrocellulose after precipitation with 10% trichloroacetic acid. As shown in Fig. 3, the bound radioactivity increased in time, pointing out that the autophosphorylating activity of G domainPT82 is independent of the presence of other proteins. Moreover, the reaction is independent of the protein concentration at least up to 10 PM when incubated with a large concentration of GTP (Fig. 4). This indicates that the autokinase activity is an intermolecular reaction, similar to the autophosphorylat- ing activity of p21 (30) and protein kinase C (32).

To compare the autophosphorylation reaction with the GTPase of wild-type G domain we tested the effect of different experimental conditions on the phosphorylation reaction (Fig. 5). The results show that KC1 and pH have essentially iden- tical influence on both activities, while the Mg2+ optimum appears to be slightly shifted to lower concentrations: from

OY I I 0 I* 2csl ml 41x)

time (min)

FIG. 3. Kinetics of the autophosphorylation activity of G domainPTS2. G domainPT82 (10 pM) was incubated with 17 pM [r-“*P]GTP (2650 cpm/pmol) at 30 “C in 720 ~1 of 50 mM imidazole acetate, pH 7.7, 1 M KCl, 0.5 mM MgCl,, and 1 mM dithiothreitol. Aliquots of 4 ~1 were taken at different time intervals, precipitated in 10% trichloroacetic acid (0 “C), and filtrated as described under “Materials and Methods.”

2-3 mM2 to 0.1-l mM. However, presence of some Mg2+ is a requisite, since in both cases 3 mM EDTA abolishes any activity (Ref. 18, and Fig. 5B).

Identification of the Autophosphorylated Residue in G DonainP7’82-In order to verify that residue Thrs2 is phos- phorylated by autokinase activity of G domainPT82, we la- beled in uitro a preparative amount of G domainPT82 by incubating with [T-“~P]GTP, as reported in Fig. 3. The final percentage of labeled protein was about 20%.

The acidic hydrolysis of labeled G domainPT82 and sepa- ration into two dimensions in the presence of commercial phosphothreonine, as described under “Materials and Meth- ods,” first of all shows that labeling of the amino acid can

2 Ref. 18, and K. Harmark and R. H. Cool, unpublished observa- tions.

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Autophosphorylation of Thr a’ in EF-Tu 6747

protein (pmol)

FIG. 4. The autophosphorylation reaction is a first order rate reaction. Different quantities of G domainPT82 were incubated with 59 pM [-y-32P]GTP (2300 cpm/pmol) in 50 ~1 of 50 mM imidazole acetate, pH 7.4, 500 mM KC1 and MgCL, varying between 0.06 and 0.57 mM (brought along with the storage buffer of the protein), for 3.5 b at 30 “C. Thereafter. an excess of EDTA and GTP was added in 20 ~1 to a final concentration of, respectively, 12 and 20 mM, and after an additional incubation of 15 min at 30 “C an aliquot of 60 ~1 was filtered on nitrocellulose and washed twice with 3 ml of 50 mM imidazole acetate, pH 7.4 (0 “C).

survive a severe treatment such as 3-h incubation in 6 M HCl at 110 “C, further confirming the existence of a covalent bond. Second, the fact that the ninhydrin colored spot coincides exactly with the labeled amino acid disclosed by the autora- diogram (not shown), proves that a threonine residue is phos- phorylated in labeled G domainPT82.

The patterns of peptides of the nonlabeled and labeled G domainPT82 obtained by trypsin digestion and separation on high performance liquid chromatography were essentially identical, as can be seen in Fig. 6. Scintillation counting revealed only one radioactive fraction (Fig. 6C). Analysis of the amino acid sequence of the radioactive peptide by sequen- tial Edman degradation and identification of the modified amino acids on high performance liquid chromatography showed that this peptide is indeed the peptide consisting of amino acids His75-Lys8g, and that Pro’* has been replaced by a Thr. Although because of technical reasons no peak of radioactivity could be measured (due to the treatment of the peptide and sticking of the 32P to the filter), this confirms that Thr” is phosphorylated in G domainPT82.

Interactions with GDP and GTP-In Table I we have re- ported the apparent dissociation constants and the dissocia- tion rate constants of the complexes formed by G domain-

PT82 with GDP or GTP, as compared to wild-type G domain. Apparently, the mutation has no large effect on the affinities for GDP or GTP, as shown by the nearly identical dissociation constants of the two G domain species. One can, however, observe a marked retardation of the dissociation rates of the complexes: the GTP complex three times and the GDP com- plex about eight times. The association rate constants of these complexes of G domainPT82 are decreased to a similar extent.

DISCUSSION

Of the many biological activities of bacterial elongation factor Tu the only function that has been elucidated in three- dimensional detail is the binding of GDP. The two first elements (G18HVDHGK24 and DBOCPG83) of the consensus sequence, constituting the guanine nucleotide pocket, concern the interaction with the phosphoryl groups, as indicated by functional, chemical, and crystallographic studies (6, 11, 12, and references within). Pro” is a residue which is conserved in elongation and initiation factors from different organisms but not in other families of GTP-binding proteins. The three- dimensional model derived from EF-Tu. GDP (13, and Fig. 7A) shows that Pros2 is situated close to the D-phosphate, but not so close as to form Pydrogen bonds. The C, atom of Pro” is approximately 6.1 A from the nearest oxygen of the /3- phosphate. The functional and structural similarities and diversities found so far between EF-Tu and p21 make the substitution of Pro” particularly interesting, considering that the corresponding mutation in p21 (AT59) induces biochem- ically well-defined effects, like autophosphorylation and in- hibition of the catalytic activity. Moreover, in p21 the transfer of the terminal phosphate of GTP to the protein is highly specific for Thr5’. Its neighbor Thr58 is not phosphorylated in either the protooncogenic or the oncogenic form (22), confirm- ing the strict correlation of activity with structure.

Our results show that the catalytic activity of the G domain is strongly decreased by the substitution. At the same time, mutation of Pro’* into Thr induces an autophosphorylation activity in the G domain of EF-Tu. Analysis by two-dimen- sional separation of the hydrolysate of labeled G domainPT82 together with the Edman degradation of the labeled peptide shows that the y-phosphate of GTP is transferred to Thr’*. The autophosphorylation reaction is under the same con- straints as the GTPase of the wild-type G domain, as shown by essentially the same behavior when tested as a function of

d 0-l I 0-I 1 I ,’ 0 2 0 xxi lcal 15co 2030 0 5 10 15 5 6 7 8 9 IO

WC11 (mM) lMgCI21 (mM) PH

FIG. 5. The autophosphorylating reaction is dependent on the buffer composition. A and B, in 45 ~1 of buffer containing 50 mM imidazole acetate, pH 7.7, 1 mM dithiothreiol, and MgClz (10 mM in A) and KC1 (50 mM in B) as indicated, 2 pM of G domainPT82 was incubated with 15 pM [Y-~‘P]GTP (1800 cpm/pmol) for 150 min at 30 “C. After an additional incubation in the presence of 6 mM EDTA and 2.6 mM GTP for 15 min at 30 “C, the mixture was filtrated on nitrocellulose and washed twice with 3 ml of 50 mM imidazole acetate, pH 7.7. In B, the first two incubations were carried out in the presence of, respectively, 3 mM EDTA (= 0 mM Mg2+ in the graph) and 0.14 mM Mg?. C, in 40 ~1 of buffer containing 1 M KCl, 1 mM MgCh, 1 mM dithiothreitol, 12 @M [T-~~P]GTP (4700 cpm/pmol), and 50 mM imidazole acetate of the indicated pH, 1.6 +M of G domainPT82 was incubated for 150 min at 30 “C. After a second incubation of 15 min in the presence of 5.9 mM EDTA and 3.3 mM GTP, the mixture was filtrated on nitrocellulose and washed twice with 50 mM imidazole acetate, pH 7.7 (0 “C).

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6748 Autophosphorylation of ThP in EF-Tu

non-labeled G domainPT82

-L

labeled C domainPT82

LJ

B Ll

FIG. 6. Peptide pattern of a tryptic digest of labeled and nonlabeled G domainPTS2, separated on high performance liquid chromatography. After tryptic digestion (see “Materials and Methods”), 60-80 pg of nonlabeled (panel A) or labeled G domain- PT82 (panel B) were loaded on a HPLC column. The mixtures of peptides were separated on an acetonitrile gradient (panel D). Part of the fractions was used for determination of radioactivity, as shown in panel C.

mono- and divalent cations and pH (Fig. 5). This indicates that both G domain wild-type and G domainPT82 have the same general conformation, as already pointed out by their near identical affinities for the guanine nucleotides (Table I). Moreover, it suggests that the mechanisms underlying the GTPase and autokinase activities are identical. As shown in Fig. 4, the autophosphorylation activity is independent of the protein concentration and therefore should result from an intermolecular reaction. This implies that the hydroxyl group of Thr8’ acts as a nucleophil attacking the y-phosphate of GTP. Remarkably, the same first order mechanism has been found for p21AT59 (30).

The observation that in EF-Tu and p21 a substitution with

TR? 7

FIG. 7. Graphic modelling of the guanine nucleotide binding pocket of wild-type EF-Tu*GDP (A) and G domainPT82sGTP (B). In this partial representation of the binding pocket of the EF- Tu.GDP complex (A), derived from a three-dimensional model at 2.6-A resolution, Pros* has been substituted by Thr and the y- phosphate of GTP has been introduced following simple energy minimization (B). In A, the C, atoms of the amino acids are indicated with their one-letter symbol and number.

threonine of the same residue in the second consensus element gives rise to an autophosphorylating activity points to a similar local three-dimensional structure of these two pro- teins. Recent x-ray diffraction studies of the ~21. GTP com- plex (34-37) emphasize the great similarities with the three- dimensional model of the EF-Tu. GDP complex. The side chain of residue Ala5’ is very close to the y-phosphate of GTP, in agreement with the phosphotransfer reaction of p21AT59. Hence, the appearance of autokinase activity in the G domainPT82, as described in this article, is in line with the corrected model of ~21 (34).

Graphic modeling of the three-dimensional structure of the EF-Tu. GDP complex (Fig. 7A), with Thr substituting Pro in position 82 and GTP at the place of GDP (Fig. 7B), shows that the hydroxyl group of the Thr** side chain would be located approximately 5A from the y-phosphate of GTP,3 too distant for a direct interaction, and thus implying a local conformation difference between the GDP complex and the transition state of the hydrolysis of GTP.

3 J. Nyborg, personal communication.

Complexes

TABLE I Effect of substitution of Pro” by Thr on the GDP/GTP interactions of G domain

Dissociation constant, K; Association rate Dissociation rate

constant,” ki1 0 “C constant, kl, 0 "C 0 “C 30 “C

Dissociation half-time, 0 “C

PM M.C’ s-1 s

G domain. GDP* 2.1 3 1.3. lo3 2.8. 1O-3 248 G domainPT82. GDP 1.6 2.9 2.1.102 3.3. 1o-4 2100 G domain. GTPb 6.2 :D 1.0. lo3 5.9. 1o-3 118 G domainPT82. GTP 6.5 3.2.10’ 2.1.10+ 330

a Calculated with the equation: k,, = k-,/K+ ’ Taken from Jensen et al. (18). ’ ND, not determined.

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Autophosphorylation of Thr 82 in EF-Tu

It should be stressed that the N-terminal domain of EF-Tu and p21 only share 23% identical sequence similarity (4). Therefore, it is not surprising that although autophosphory- lation and low GTPase activity are features also found in the corresponding p21 protein (22, 23, 30, 38), the effects on the interactions with GDP and GTP complexes are different for the ~21 and G domain, as a consequence of subtle local structural differences arising from sequence diversity. A marked increase in the dissociation rate of the p21AT59. GDP complex has been observed (30), in contrast to the 8 fold lower dissociation rate of the complex of G domainPT82 and GDP (Table I). Furthermore, Clanton et al. (39) found a greatly decreased affinity of p21AT.59 for GDP, opposite to mutation PT82 in the G domain, which induces a slightly lower dissociation constant (Table I). Our results show that G domainPT82 is capable of hydrolyzing GTP in spite of the absence of liberation of inorganic phosphate (Fig. 1) due to the stoichiometric transfer of the y-phosphate to Thr’*. This differs from the results obtained with p21AT59, whose GTPase activity exceeds the phosphorylating activity (30). These disparities reflect the specific structure-function rela- tionships of EF-Tu and ~21. Similar conclusions were previ- ously reached by comparing the functional effects induced by Val and Gly in position 20 of EF-Tu with the homologous species of protein p21 (19, 20).

It is germane to mention that due to the autokinase activity part of the purified G domainPT82 may have already been phosphorylated in uiuo, and possibly be no longer able to hydrolyze GTP. In the mammalian cell, the phosphorylated fraction of p21AT59 corresponded to 14-50%, depending on the nature of residue 12 (23). Phosphorylation of Thr” does not change the mobility of G domainPT82 in 12% SDS- PAGE (Fig. 2), opposite to the effect on p21AT59 (23). However, we have recently been able to separate analytically phosphorylated from nonphosphorylated G domainPT82 by electrofocusing. In our preparations the phosphorylated frac- tion represented about 40-50%.4 Taking into account the autokinase activity (Figs. 3-5), we have estimated that non- phosphorylated G domainPT82 sustains about 5% of the GTPase activity of wild-type G domain. This reduction is consistent with that found for yeast protein RAS2AT66 (41), but larger as compared to the reduction found for p21AT59 (30,38,40,41) and yeast YPTlAT65 (29).

Preliminary results have indicated that mutation PT82 also induces an autophosphorylating activity in the intact mole- cule of EF-Tu.~ This construction will allow us to study the effect of the phosphorylation on the interaction between EF- Tu and the various ligands.

Acknowledgments-We would like to express our gratitude to Drs. J. Nyborg, T. F. M. la Cour, and M. Kjeldgaard for fruitful discussions, release of unpublished observations, and the structural modeling in Fig. 7.

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