Contribution of Arg288 of Escherichia Coli Elongation Factor Tu to ...

Eur. J . Biochem. 249, 408-414 (1997) 0 FEBS 1997

Contribution of Arg2SS of Escherichia coli elongation factor Tu to translational functionality Thomas RATTENBORG, Gitte NAUTRUP PEDERSEN, Brian F. C. CLARK and Charlotte R. KNUDSEN Institute of Molecular and Structural Biology, Aarhus University, Arhus, Deninark (Received 18 August 1997)

~

EJB 97 1186/3

The recently solved structure of the ternary complex formed between GTP-bound elongation factor Tu and aminoacylated tRNA reveals that the elements of aminoacyl-tRNA that interact with elongation factor Tu can he divided into three groups: the T stem: the 3‘-end CCA-Phe; and the 5’ end. The conserved residues Arg2X8, LysX9 and Asn90 are involved in the binding of the 5’ end. In the active, GTP-bound form of the elongation factor, Arg288 and A m 9 0 are involved in the forination of a network of hydrogen bonds connecting the switch regions I and I1 of domain 1 with the rest of the molecule. This network is disrupted upon formation of the ternary complex. Arg288 was replaced by alanine, isoleucine, lysine or glutamic acid, and the resulting mutants have been subjected to an in vitro characterisation with the aim of clarifying the function of Arg288. Unexpectedly, the mutants behaved like the wild-type factor with regard to thc association and dissociation o f guanine nucleotides, and the intrinsic GTPasc activities are unchanged. Furthermore, the mutants were as efficient as the wild-type factor i n carrying out protein synthesis in vitro in the presence of an excess of aminoacyl-tRNA. However. the mutants’ abilities to bind aminoacyl-tRNA and protect the labile arninoacyl bond were impaired, especially where the charge had been reversed. Kryct~orzls:elongation factor Tu ; aminoacyl-tRNA; protein-RNA interaction ; mutational study.

The main function of elongation factor Tu (EF-Tu) is to bring aminoacylated tRNA (Xaa-tRNA) to the A-site of the mRNA-programmed ribosome. This requires EF-Tu to be in its active conformation, i.e. bound to GTP. When codon-anticodon interaction is established, GTP hydrolysis is triggered and EFTu is converted into its inactive, GDP-bound form. EF-Tu . GDP, which has a low affinity for the ribosome, dissociates froin the ribosome, while Xaa-tRNA remains bound. EF-Tu is reactivated by a sccond clongation factor, EF-Ts, catalysing the exchange of GDP for GTP (Kjeldgaard et al., 1996). EF-Tu also plays a role in regulating the accuracy with which amino acids arc incorporated into the nascent polypeptide chain (Hopfield, 1974: Ninio, 1975; Thompson and Stone, 1977: Ruusala et al., 1982). The structure of EF-Tu on its way round the elongation cycle is well characterised. The following structures have been solved by means of X-ray crystallography: EF-Tu . GDP (Kjeldgaard and Nyborg, 1992; Polckhina et al., 1996; Abel and Jurnak. 1996), EF-Tu . GuoPPINH]P (Berchtold et al., 1993; Kjcldgaard et al., 1993), EF-Tu . EF-Ts (Kawashima ct 211.. 1996) and the ternary complex, EF-Tu . GTP Xaa-tRNA (Nissen et al., 1995). EF-Tu conaists of three structural domains, whose relntive orientations and surfaces change through the cyclc, thcrcby regulating EF-Tu‘s affinity for its different interaction partners. ~

~

~

~~~~~

C ’ o r - r - ~ ~ . s / ~ o r ~ ~ l ~to ~ / cC. . r l cR. . r Knudsen. liistitute or Molccular and Structural Biology, Aarhus University. G L I S ~Wieda ~ V Vc,i IOC, DK-8000 A r h w C. Deninark Fu.4.: +45 86 20 12 22. E-nrtr il : crk @ i insb.au .dh A h h v i t r t i o r t c . EF, elongation factor; Xna-tRNA. atminoacyl-tRNA ; GuoPP[NH]/’, guanosine S’-[/l,j~-inlino]-tripho~pI~~~t~; RNase, ribonuclense.

EH:JWIO.\.Mycrltinase

(tT2.7.4.3): pyruvate kinase (2.7.1.40).

The GDP-bound form of EF-Tu is rather ‘loose’, with the three domains positioned far apart and ii characteristic hole in the middle of the molecule, whereas the GTP-bound form is more cornpact and thus appears to be without the hole. The structure of the ternary complex, Phe-tRNA . EF-Tu . GuoPPl NHIP, formed between Phe-tRNA from yeast and EFTu from Tlirnnus nquuticus shows that all three domains of EFTu are involved in the intcractions with Phe-tRNA, which primarily takes place at three positions: thc T stem; the 3‘-end CCA-Phe; and the 5’ end. Thc 5’ cnd is bound at the threedomain interfxe of EF-Tu, where a cavity lined by helix A”. the C-terminal part of helix B. and two P-strand-connecting loops (rcsidues 300-303 and 346-348 connecting /J-strands e, with f, and b, with c l , respectively) is formed. The structure suggests that the conserved residue Arg300 (Arg288 in Esclwrichici c d i : hereafter the numbers in brackets refer to E. coli EF-Tu) can form a salt bridge to the 5’ phosphate of G I . Another conscrved residue. LysOCl (Lys89) is positioned between the phosphates of G1 and G2. and a third conscrvcd residue. Am91 (Asn90), is able to forin ii hydrogen bond to the ribose of G1 (Nissen et al., 1907). Apart froin playing this rather specialised role in the binding of the 5‘ end of Xaa-tRNA, Arg300 might be part of a signaltransduction system transmitting the ‘onhff’ signal determined by the nature of the bound nucleotide. throughout the entire molcculc. In the structure of EF-Tu . GuoPP[NH]P (Kjeldgaard et al., 1993). the: side chain of Arg300 is in direct contact with the switch-I1 region via a hydrogen bond to Asn91, and indirectly with the main chain of the switch-1 region, where Ile63 is hydrogen bonded to Asn91. These two switch regions are the inajor locations of intradornain rearrangements in all G-binding proteins for which the structures of both the GDP-bound and the GTP-bound forins arc known. They are very important in their

Rattenborg et al. (Eur: J. Biochem. 249)

effects on the change of the switch for the communication with other interaction partners (Kjeldgaard et al., 1996). This study aims at examining the role of the conserved Arg300 in more detail by mutation of the corresponding residue in E. coli EF-Tu, Arg288, to alanine, isoleucine, lysine or glutamic acid. The resulting four point mutants were named [Ala288]EF-Tu, [Ile288]EF-Tu, [Lys288J EF-Tu and [Glu288]EF-Tu, respectively. An array of in vitro assays have been carried out to characterise these mutants with regard to their binding of guanine nucleotides and Xaa-tRNA, their intrinsic GTPase activity, and their ability to translate a poly(U) message into poly(Phe).

METHODS Construction of the mutants. Site-directed mutagenesis of Arg288 was performed by the phosphorothioate method according to Taylor et al. (1985). The template used was a M13mpll vector containing the tufA gene encoding EF-Tu with a stretch of nucleotides encoding the recognition site for the protease factor X, at the 5' end (Knudsen et al., 1992). The four mutagenic primers were S'-GAAATCGAAGCTGGTCAGGT-3', 5'-GAAATCGAAATCGGTCAGGT-3', S-GAAATCGAAAAAGGTCAG-3' and S'-GAAATCGAAGAAGGTCAGGT-3', giving rise to the mutations to Ala, Ile, Lys and Glu, respectively. The mutated tufA genes were confirmed by ssDNA sequencing using the Sanger dideoxynucleotide procedure (Sanger et al., 1977), and subsequently subcloned into a pGEX expression vector. The mutations were confirmed by dsDNA sequencing (Sambrook et al., 1989) of the resulting pGEX-FX-tufA plasmid. Expression and purification. The mutated tufA genes were expressed as glutathione-S-tranferase-EF-Tu fusion proteins as described by Knudsen et al. (1995), in the host E. coli strain JM109 (Sambrook et al., 1989). By this method full-length EF-Tu without any additional amino acids is obtained. Wildtype EF-Tu protein was purified by the 5ame method to serve as a reference for the subsequent characterisation of the mutant proteins. Protein activity and stability. The concentration of active EF-Tu was determined by estimation of maximum GDP binding (Miller and Weissbach, 1977). EF-Tu was saturated with ['HJGDP (785 dpdpmol) and bound to celluloseacetate filters, washed and measured. Thermal stability was determined as described by Andersen and Wiborg (1994). The stabilities of the GTP and the GDP forms were determined. For ['HIGDP and ['4C]Phe assays cellulose-acetate filters from Gelman Sciences were used, and for [IHIGTP assays nitrocellulose filters (BA85) from Schleicher & Schull were used. Prior to use, filters were soaked in 10% trichloroacetic acid for ['"CIPhe assays, or in 10 mM Tris/HCl, pH 7.6, 10 mM MgCl,, 10 mM NH,Cl. All concentrations of EF-Tu used were determined by the GDP-binding assay. Formation of EF-Tu * GTP. EF-Tu . GDP was incubated with 100-fold excess GTP, 2000-fold excess phosphoenolpyruvate and pyruvate kinase (1 mg/mL) on ice. The time needed for formation of EF-Tu . GTP was individually determined for the wild-type protein and each mutant protein. The GTP solution was incubated with 13-fold excess of PEP and 0.1 mg/mL pyruvate kinase for 20 min at room temperature to convert any GDP to GTP. Charging of tRNAPhe.tRNAPhewas aminoacylated by incubating 40 pM yeast tRNAPhewith 200 pM [I4C]Phe (210 d p d pmol) and 25 pL aa-tRNAPh' synthetase in 0.1 M Tris/HCl, pH 7.6, 50 mM NH,CI, 12.5 mM MgCl,, 2 mM ATP, 0.24 mM

409

CTP and 2.8 mM 2-mercaptoethanol for 13 min at 37°C. The synthetase was purified crudely from Saccharomyes cerevisiae (von der Haar, 1979). Prior to the non-enzymatic hydrolysis assay, the synthetase was removed by phenol and phenol/chloroform extraction, and the charged ['"C]Phe-tRNAPh" was precipitated and dissolved in 60 mM Tris/HCI, pH 7.6, 6 mM MgCl,, 30 mM NH,Cl and 30 mM KC (buffer A).

Apparent dissociation rate constant, k-,, of the ternary complex. Determination of k - , for the ternary complex was carried out as described by Louie and Jurnak (1985). The ternary complex, EF-Tu . GTP . Phe-tRNAPhe,was formed by mixing in buffer 200 pmol EF-Tu . GTP with 70 pmol [L4C]Phe-tRNAPhe A (total volume 88 pL) for 30 min on ice. Ribonuclease A (RNaseA) was added to 10 pg/mL. Aliquots were withdrawn at different times, precipitated in 10% trichloroacetic acid and filtered. The dissociation follows first-order kinetics, thus k- , can be determined as the slope of In(c,/c,) plotted versus time (c, is the concentration of ternary complex at time t , and c,, is the initial concentration). Non-enzymatic hydrolysis of the aminoacyl bond. The assay for the protection of the aminoacyl bond by EF-Tu in the ternary complex was performed as described by Pingoud and Urbanke (1979). The ternary complex was formed by mixing 200 pmol EFTu . GTP with 60 pmol [14G]Phe-tRNAPhe (extracted) in 200 pL buffer A for 30 rnin on ice. The ternary complex was shifted to 20"C, and aliquots were withdrawn at various times, precipitated in 10% trichloroacetic acid and filtered. From a plot of ln(c,/c,) versus time the half-life of the ternary complex can be determined. In vitro translation. The poly(U)-directed poly(Phe)-synthesis assay was performed essentially as described by Ehrenberg et al. (1988). Two mixtures, factor mix and ribosome mix, were prepared separately on ice. Before mixing together they were incubated for 15 rnin at 30°C. After mixing, aliquots were withdrawn at different times, precipitated in 10% trichloroacetic acid, boiled at 85°C for 30 min, cooled on ice and filtered. Factor mix was 5 mM KPO,, pH 7.3, 1 mM dithiothreitol, 1 mM ATP, 10 mM phosphoenolpyruvate, 0.1 mM GTP, 0.1 mg/ mL pyruvate kinase, 2.1 U/mL myokinase, 7.3 pM [I4C]PhetRNAPh"(92 dpm/pmol), 1.1 pM EF-G, 8 pM EF-Ts and 40 nM EF-Tu in buffer B ( 5 mM magnesium acetate, 0.5 mM CaCI,, 95 mM KC1, 5 mM NH,Cl, 8 mM putrescine, 1 mM spermidine, pH 7.5). Ribosome mix was 5 mM KPO,, pH 7.3, 1 mM dithiothreitol, 0.8.5 pM [l4C]Phe-tRNAPh',4 pM ribosomes, 2.6 mgl mL poly(U) in buffer B. Ribosome purification. Ribosome purification was based on a protocol described by Rodnina and Wintermeyer (1995). E. coli MRE 600 cells were grown to an A,,,,, of 1.2- 1.5, harvested and frozen. All steps of the purification were carried out at 4°C. Cells were suspended in a minimal amount of 20 mM Tris/HCl, pH 7.6, 100 mM NH,Cl, 10.5 mM MgCl,, 0.5 mM EDTA, 1 mM dithiothreitol. Cells were opened by passage through a French press twice followed by treatment with DNase 1 (20 min, 5 pg/g cells). Cell debris was removed by centrifugation for 30 min at 9800 g, and the supernatant was ultracentrifuged for 1 h at 26000 g. The resulting supernatant was layered on top of a 9-mL sucrose cushion [ 1.1 M sucrose in 20 mM Tris/HCl, pH 7.6, 0.75 M NH,Cl, 10.5 mM MgCI,, 0.5 mM EDTA, 1 mM dithiothreitol (buffer C)] and ultracentrifuged for 16 h at 110000 g. The pellet was carefully suspended in buffer C, layered on a 5 mL sucrose cushion and spun for 6 h at 250000 g. The pellet was carefully suspended in buffer C layered on a 5-mL sucrose cushion and spun for 13 h at 110000 g. The pellet was dissolved in a 10 mM Tris/HCl, pH 7.6, 30 mM NH,Cl, 10 mM MgCl,, I mM dithiothreitol, 10% glycerol, and the con-

Rattenborg et al. ( E m J . Biochem. 249)

410

Table 1. Association and dissociation rate constants determined at 0 "C. Values are calculated from plots, all with correlation coefficients above 0.91. Standard deviations are given, and, Kd values are determined as the ratio k-,lk+,.

Protein

GTP

GDP

Wild-type EF-Tu [AIa288]EF-Tu [IleZX8]EF-Tu [Lys288]EF-T~ [Gl~288]EF-Tu

k,,XlO-"

k- I X 10'

Ki

k,,X10

M Is-'

s-

nM

M 's

28 ? 3.5

1.74 i0.1 1.63 i0.1 1.57 i0.2 1.67 t 0.1 1.52 i0.1

0.62 0.68 0.47 0.42 0.63

1.35 i0.41 3.08 2 0.85 1.79 2 0.43 2.04 i0.60

24 i- 3.9

33 i- 3.7 40 t 4.7 24 t 3.9

I

Table 2. Kinetic parameters of the GTPase activity measured at 30°C. The values are determined from Hanes plots with correlation

coefficients above 0.96.

Wild-type EF-Tu [Ala288]EF-Tu [Ile288]EF-Tu [Lys288]EF-T~ [Glu288]EF-T~

G m n/%(crw

K,,,

k,,, X 10'

k;,, X 10'lK,,,

PM

S-'

pM/s

6.2420.75 1.86 2 0.78 5.12t0.60 2.82t0.02 3.51 -t 1.70

855 ? 47 635 t 207 725 2204 442? 73 655 5216

137 341 141 157 187

'

0.80+0.15

k-, X lo4

K*

s '

nM

46.7 i21 55.7 t 21 56.2? 6 39.82 7 56.9 i23

346 181 314 195 71 1

558

226 668 464 1128

I Op=s:

--7

0.5

-0.5

-2.5 0

50

100

I

I

I

I

150

200

250

300

350

Time (s)

centration determined by measuring A,,, (24 pmol ribosomes give an A,,, of 1). Ribosomes were stored at -80°C. Native PAGE of the ternary complex, EF-Tu GTP * PhetRNAPhe.This method was described by Alexander et al. (1995). 100 pmol EF-Tu . GTP was complexed with increasing amounts (0-400 pmol) of Phe-tRNA""'. The gels were 5 % polyacrylamide (19:l), pH 6.8, and were run at 4°C. Gels were stained with Coomassie blue. An apparent K , was estimated based on the amount of Phe-tRNAPheneeded to convert 5 0 % EF-Tu . GTP to ternary complex under the conditions applied. Interactions with guanine nucleotides. The dissociation rate constants, k - , , for EF-Tu . GDP and EF-Tu . GTP were determined as described by Knudsen et al. (1995). The association rate constants, k , ,, were determined essentially as described by Knudsen and Clark (1995). 10-15 pmol nucleotide-free EF-TU was mixed with 40 pmol [3H]GDP or 150 pmol ['HIGTP to achieve pseudo-first-order conditions. Nucleotide-free EF-Tu was prepared with a 2% charcoal solution as described by Knudsen and Clark (1995). K,,, and k,,, determination for the intrinsic GTPase activity. The GTPase activity was examined by measuring the liberation of inorganic phosphate (Arai et al., 1974). The assay was performed as described by Knudsen et al. (1995). K,,, and k,,, was determined from Hanes plots (a plot of substrate concentration/hydrolysis rate versus substrate concentration), where V,,,,, equals l/slope and the intercept of the y-axis equals KJV,,,,,,.

RESULTS Primary characterisation. All of the mutants and the wild-type protein were purified from about l o g wet cells. The yield of each purified protein was about 5 nig. The activities measured by GDP binding were 50-90%. The active concentrations were in the range 20-25 pM. The purity of the proteins were higher than 95 c/o based on SDS/PACE.

Fig. 1. Dissociation rate of ternary complex measured by RNase-Aprotection assay. (0)Wild-type EF-Tu; ( 0 ) [.Ala288]EF-Tu; (m)

[Ile288]EF-Tu; (+) [Lys288]EF-Tu;(A)[Glu288]EF-Tu;(V)free PhetRNAP'". ci, and c, are the concentrations of ternary complex initially and at time t , respectively. The values of k are given in Table 3.

The time needed for reaching equilibrium in GDP/GDP exchange on ice was 40inin for wild-type EF-Tu and the four mutant proteins. For GDP/GTP exchange on ice the time required for establishment of equilibrium was about 75 min for all proteins. The irreversible heat-denaturation profiles for the GTP and GDP forms were determined (data not shown). The temperature at which 5 0 % of the active protein was inactivated was approximately 50°C and 42°C for EF-Tu . GDP and EF-Tu . GTP, respectively, for all proteins. All assays were carried out at a maximum temperature of 30°C.

Guanine-nucleotide interactions. The dissociation rate constants, k - , , and the association rate constants, k , were measured for the mutant EF-Tu proteins and wild-type EF-Tu in GDP and GTP complexes. The results are given in Table 1. None of mutants differed considerably from the wild-type protein in the GDP or GTP forms. Hence, the mutations have no effect on the nucleotide affinities. GTPase activity. K , and k,.,, of the intrinsic GTPase activity were determined from Hanes plots (Table 2). The system used was a one-round system, i.e. less than 1 p o l GTP hydrolysed/ pmol EF-Tu. As in the case of the nucleotide affinities no changes were observed when comparing the wild-type protein with the mutant proteins. Characteristics of the ternary complex. The apparent dissociation rate constants of the ternary complexes, EF-Tu . GTP . Phe-tRNAP"", were measured using the RNaseA-protection assay. The results are shown in Fig. 1, and the rate constants are

41 1

Rattenborg et al. (Eur: J . Biochem. 249) 0.5

-1.5

Table 3. Dissociation rate, k-l, half-life, f,,2, and apparent K , of the ternary complex. k- , was determined using the RNase-A-protection assay at 0°C. whereas t,,, was measured using the non-enzymatic hydrolysis assay at 20°C. The values are calculated from the plots shown in Figs 1 and 2, all with correlation coefficients above 0.91. The apparent constant for the dissociation of tRNA from the ternary complex, &, was estimated from native polyacrylamide gels (Fig. 3).

i

'V

EF-TU

k - , X 10' S-'

-2

1

0

Wild-type EF-Tu [Ala288]EF-Tu [Ife28 81EF-Tu [Lys288]EF-T~ [GIu~~~IEF-Tu

I

50

t1/2

Apparent K,,

min 1193 ? 532 3002 130 3732118 6422202 87? 16

PM 0.5 5 2.5 0.5 12.5

\

100

200

150

250

Time (min)

Fig. 2. Protection of amino acyl bond against non-enzymatic hydrolysis. (0)Wild-type EF-Tu;).( [Ala288]EF-Tu;).( [Ile288]EF-Tu; (+) [Lys288]EF-Tu; (A)[Glu288]EF-Tu; (V)free Phe-tRNAPhc.c,, and c, are the concentrations of ternary complex initially and at time t, respectively. The half-lives, tIiz.are listed in Table 3.

1.06 -I: 0.42 5.04 ?r 0.24 2.13 ir0.16 2.10 2 0.14 43.24r ! 8.13

protection by [Glu288]EF-Tu was close to that of the control. However, [Glu288]EF-Tu gave some protection. The half-life (t,J of the aminoacyl bonds are listed in Table 3. Using native PAGE, the apparent rC, values (dissociation constant of the equilibrium EF-Tu . GTP . Phe-tRNAPh'-EFTu . GTP+ Phe-tRNAPhe)were estimated (Table 3). The values agree very well with the results obtained by the RNase and nonenzymatic hydrolysis assays. Wild-type EF-Tu showed a 25fold higher affinity for Phe-tRNAPhe than [Glu288]EF-Tu. [Lys288]EF-Tu showed the same affinity for Phe-tRNAPh'as the wild type, whereas [Ala288]EF-Tu and [Ile288]EF-Tu had reduced Phe-tRNAPheaffinities. In Fig. 3, the gels of wild-type EF-Tu and [Glu288]EF-Tu are shown. The ternary complex migrates faster than EF-Tu . GTP on native gels due to the high negative charge on Phe-tRNAPh'.

listed in Table 3. Phe-tRNAPh"in complex with [Lys288]EF-Tu and [Ala288]EF-Tu were almost as resistant to RNase degradation as Phe-tRNAPhein the wild-type ternary complex. The ternary complex of [Ile288]EF-Tu had a slightly reduced resistance to RNase, whereas [Glu288]EF-Tu hardly protected the tRNA against digestion. k - , of the ternary complex formed by [Glu288]EF-Tu was about 40-fold higher than the apparent dissociation rate constant of the wild-type ternary complex, but differed from the control (no EF-Tu present). The ability of EF-Tu to protect the aminoacyl bond was determined by measuring the non-enzymatic hydrolysis. The overall picture was the same as in the RNase-protection assay (Fig. 2). All mutants, except [Glu288]EF-Tu, protected the amino acyl bond fairly well against hydrolysis. [Lys288]EF-T~ In-vitro translation assay. The efficiencies of the mutants in protected the bond as effectively as the wild type, whereas the achieving poly(Phe) synthesis on poly(U)-programmed ribo-

A

Wild-type EF-Tu 0

25

50

15

100

200

400

Fig.3. Native PAGE of ternary complex. (A) Wild-type EF-Tu, (B) [Glu288]EF-Tu. 100 pmol of the given EF-Tu was titrated with Phe-tRNAPh' (0, 25, 50, 75, 100, 200 and 400 pmol), and the band-shift was used to estimate an apparent Kd for the ternary complex. The upper band is EF-Tu . GTP, while the lower is the ternary complex, EF-Tu GTP . Phe-tRNAPhe.The gels of the other mutants are not shown, but the estimated K, values are given in Table 3.

Rattenborg et al. (Eur: J. Biochem. 249) 120

--

0

100

a0 60

40

20 0 0

1

2

3

4

5

6

Time (rnin)

Fig. 4. In vitro poly(U)-directed poly(Phe) synthesis. (0)Wild-type EF-Tu; (0)[Ala288]EF-Tu; (m) [Ile288]EF-Tu ; (+) [Lys288]EF-Tu; (A)[Glu288]EF-Tu; (V)control The results shown are for assays in the presence of EF-Ts. Fig. 6. Binding of the 5' end of Xaa-tRNA. The structures of EF-Tu . GTP and of the ternary complex, EF-Tu GTP . Phe-tRNAP'", are shown in gray and black, respectively. The structure solved is of I: aquaticus EF-Tu, and the corresponding conserved residues of E. coli EF-Tu are Arg288, Asp336, Am90 and Lys89. The binding pocket for the 5' end of the Xaa-tRNA is formed by residues from all three domains of EFTu (Lys90 and Am91 of domain 1, Arg300 of domain 2 and Asp348 of domain 3). As shown in the figure, the binding o f tRNA only changes the positions of the side chains, whereas the backbone is unaffected. The figure was produced with OPLOT (Jones et al., 1991).

-

Fig.5. The structure of the ternary complex EF-Tu GTP PhetRNA"h'. Thermus nqLtaricu.7 EF-Tu was used to solve the structure, and Arg300 corresponds to Arg288 of E. coli EF-Tu. Arg288 is situated in the three-domain-interface region, where it forms a salt bridge to the 5' phosphate of Xaa-tRNA. Before association with tRNA, in the active form, EF-Tu . GTP, Arg288 makes an interaction with the switch-I1 region of domain 1. The figure was produced with RasMol v2.6p by Roger Sayle.

somes were measured. The system was tested with (Fig. 4) and without EF-Ts (data not shown). The system was optimised to having EF-Tu as the rate-limiting factor, and under these conditions none of the mutants showed any significant variations from the wild type. The incorporation rate, with the background subtracted, was about 13-15 pmol Phe . pmol EF Tu-' . min-' in the presence of EF-Ts. EF-Ts stimulated all the mutants to the same degree as the wild type; thus the mutants interact in the same manner as the wild type with EF-Ts.

DISCUSSION The aim of this study was to elucidate biochemical details of the role of Arg288 of EF-Tu from E. coli (Fig. 5). Structural

studies had earlier indicated that this residue might be important in regulating the allosteric mechanism of EF-Tu by fine tuning the delicate balance of affinities for GDP and GTP and by playing a role in the interaction between EF-Tu and Xaa-tRNA by binding of the 5' phosphate of the tRNA. These ideas are based on the structures of EF-Tu . GuoPP[NH]P and EF-Tu . GuoP P [ N H ] P . Phe-tRNAPhe (Berchtold et al., 1993; Kjeldgaard et al., 1993 ; Nissen et al., 1995). These crystallographic structures are static pictures of molecules, which in their natural environment are highly mobile. Models based on structural data should therefore be confirmed by biochemical studies whenever possible. For this purpose, Arg288 was mutated to alanine, isoleucine, lysine and glutamic acid. The resulting four point mutants showed normal thermostability compared with the wild-type EF-Tu. Hence, we conclude that Arg288 does not play a crucial role in the formation and stabilisation of the overall three-dimensional structure of EF-Tu. However, we considered whether Arg288 would interfere with the overall structure, as indication of such interference was given by the study of the Arg300-Ile mutation (Zeidler et al., 1995) of Thermus thermophilus EF-Tu (Arg288 of E. coli EF-Tu). Their attempt to purify the mutated protein failed, probably due to proteolytic cleavage during overexpression of the protein. The crystal structure of EF-Tu in its active conformation with a GTP analogue (Kjeldgaard et al., 1993), reveals that Arg288 forms a hydrogen bond with Asn90 in the switch-I1 region of domain 1. Asn90 also forms a hydrogen bond with He62 of the effector loop (switch-I region). We therefore proposed that Arg288 could be an important residue in domain-domain interactions, thereby influencing the overall structure. Furthermore: the indirect interaction with the effector loop could lead to a series of Arg288 mutants with an altered nucleotide interaction. To our surprise, only small and insignificant variations in the guanine nucleotide binding (Table l), and in the GTPase activity (Table 2) were observed. In an earlier study, residues Gly94 and Gly 126, involved in the interaction between domains

Rattenborg et al. (Eul: J. Biochem. 249)

1 and 3, were mutated. As a result, the characteristic differences in affinities between GDP and GTP, with GDP being bound more than two-fold stronger than GTP, were strongly reduced (Knudsen et al., 1995). Recently, we have shown that also the interaction between domains 1 and 2 are important for the association of GTP (Mansilla et al., 1997). In the extreme case, that of the isolated domain 1, the difference in nucleotide affinities is completely eliminated (Jensen et al., 1989). Xaa-tRNA binding was very sensitive to the mutations. [Glu288]EF-Tu only gave very poor protection of Phe-tRNAPh' in the RNaseA-protection and non-enzymatic hydrolysis assays (Figs. 1 and 2, and Table 3), whereas [Ala288]EF-Tu and [Ile288]EF-Tu showed reduced protection of Phe-tRNAPhe. [Lys288]EF-Tu is as protective as the wild-type protein. These results confirm the crystal structure of the ternary complex (Nissen et al., 1995). According to this structure, the Send of the tRNA is tightly bound in a pocket formed by the switch-I1 region, the loops between p-strands e and f of domain 2 and between P-strands b and c of domain 3 (Fig. 6). Arg288, which is situated in the loop connecting @-strands e, and f2, forms a salt bridge with the 5' phosphate of the tRNA. Lys89 contributes with positive charge to the pocket, and Asp336 holds Arg288 in position by ionic interaction. Asn90 makes hydrogen bonds to the ribose of GI and Ile62. Thus, when the opposite charge is induced, as in [Glu288]EF-Tu, it is expected that the negatively charged phosphate is repulsed, thereby reducing the affinity for tRNA. Also, the removal of the charge ([Ala288]EF-Tu and [Ile288]EF-Tu) gave the expected decrease in affinity for tRNA. The size of the side chain seems also to have importance, since [Ile288]EF-Tu protects the tRNA to a greater extent than [Ala288]EF-Tu. Ile is bigger and more bulky than A h , and therefore induces a better fit for the 5' end into the pocket, Mutational studies of the two residues of the switch-I1 region involved i n 5'-phosphate binding, Lys89 and Asn90, showed decreased affinity for tRNA for all of the mutants (Wiborg et al., 1996). The activities of the EF-Tu mutants were measured by the poly(U)-directed poly(Phe)-synthesis in vitro assay. None of the mutants showed any variation in their ability to sustain protein synthesis compared with wild-type EF-Tu (Fig. 4). All-mutants were stimulated to the same extent by the guanine-nucleotideexchange factor EF-Ts, which indicates that their interactions with EF-Ts and the nucleotides are unaltered. That the mutations do not affect poly(Phe) synthesis can be explained by the relatively high concentration of Phe-tRNAPhcin the assay. Thus, the equilibrium is driven towards ternary-complex formation. The estimation of the apparent dissociation constant (Table 3), Kd, confirmed the previously obtained results. All mutants are capable of forming the ternary complex, though with very different affinities for Phe-tRNAPhe.[Glu288]EF-Tu has a very low affinity for Xaa-tRNA, but still enough to sustain protein synthesis in the presence of excess amounts of Xaa-tRNA. rIle2881EF-Tu showed higher affinity for Xaa-tRNA than [Ala288]EF-Tu which supports the results achieved in the RNaseA-protection and non-enzymatic hydrolysis assays. The importance of the 5' phosphate has been described previously (Sprinzl and Graeser, 1980). In this study, the 5' phosphate of tRNAPhr was removed, and the resulting tRNA was tested in various assays. It was found that this tRNA was capable of being aminoacylated with Phe, forming a ternary complex, and was protected to the same extent as normal Phe-tRNAPh" by EF-Tu. However, the tRNA was less efficient in poly(Phe) synthesis. The decreased rate of protein synthesis is probably due to a looser structural fit of the tRNA induced by EF-Tu and therefore a less accurate interaction with the ribosome. In addition, the 5' phosphate might interact directly with the ribosome

413

after GTP hydrolysis and dissociation of EF-Tu (Joseph and Noller, 1996). 5'-end binding of the tRNA is accomplished by the evolutionary conserved residues Arg288, Lys89 and Am90 as described earlier. A similar binding is seen for Asp-tRNA synthetase, a class-I1 Xaa-tRNA synthetase, where a lysine and an asparagine create a positive cave for the 5' phosphate (Cavarelli et al., 1993). Binding of the CCA end of the tRNA also appears to be an evolutionary-conserved phenomenon. Both EF-Tu and Asp-tRNA synthetase induce a helical curvature of the 3' singlestranded RNA upon binding of the CCA end. Thus, it seems that nature has evolved some general features in recognising tRNA. Apart from the 5' end and the CCA end, EF-Tu and XaatRNA synthetase do not recognise the same elements of the tRNA (Nissen et al., 1997) consistent with their different roles: EF-Tu must recognise a common feature of tRNA, whereas each Xaa-tRNA synthetase recognises a unique feature of its cognate tRNA. This study has provided information on the binding of tRNA to EF-Tu . GTP. When binding occurs, Arg288 is shifted away from Am90 to form a salt bridge with the 5' phosphate of the tRNA (Fig. 6). This shift concerns only the side chain of Arg288. The backbone is unaffected and so is the switch-I1 region. The salt bridge is important for the binding of tRNA, but is not crucial. EF-Tu can adjust to the phosphate without Arg288 but with decreased affinity for the tRNA. The correct fit is a combination of charge and size of the side chain. The interaction with Am90 in EF-Tu . GTP is of no significant effect. Why is this residue so well conserved? It is very likely that the role of this Arg is to ensure that the Xaa-tRNA enters into the initial binding site of the ribosome at the correct angle. In this way, an optimal balance of speed and accuracy is obtained. We would like to thank I>r Poul Nissen for providing Fig. 6 and for indispensable discussions, and Karen Margrethe Nielsen and Gitte Hartvigsen for their very skilled technical help. This work was funded by the Danish Biotechnology programme (Protein Engineering Research Center and Center for Interaction, Structure, Function and Engineering of Macromolecules), and the Human Capital and Mobility Programme. C. R. K. was funded by the Cxlsberg Foundation.

REFERENCES Abel, K. & Jurnak, F. (1996) A complex profile of protein elongation: translating chemical energy into molecular movement, Structure (Lond.) 4, 229-238. Alexander, C., Bilgin, N., Lindschau, C., Mesters, J. R., Kraal, B., Hilgenfeld, R., Erdmann, V. A . & Lippmann, C. (1995) Phosphorylation of elongation factor Tu prevents ternary complex formation, J. Bid. Chern. 270, 14541-14547. Andersen, C . & Wiborg, 0. ( 1994) Escherichia coli elongation-factorTu mutants with decreased affinity for arninoacyl-tRNA, Eur: J . Biochenz. 220, 139-744. Arai, K., Kawakita, M. & Kaziro, Y. (1974) Studies on the poly-peptide elongation factors from E. coli, J . Biochem. (Tokyo) 76, 293-306. Berchtold, H., Reshetnikova, L,., Reiser, C. O., Schirmer, N. K., Sprinzl, M. & Hilgenfeld, R. (1993) Crystal structure of active elongation factor Tu reveals major domain rearrangements [erratum appears in Nature 365, 3681, Nature 265, 126-132. Cavarelli, J., Rees, B., Ruff, M., Thierry, J. & Moras, D. (1993) Yeast tRNAA'precognition by its cognate class I1 aminoacyl-tRNA synthetase, Nature 362, 181- 184. Ehrenberg, M., Bilgin, N. & Kurland, C. G. (1988) Design and use of a fast and accurate in v i m translation system, in Ribosomes and protrin synthesis, a pratical approach (Spedding, G., ed.) pp. 101- 128, IRL press, Oxford. Hopfield, J. J. (1974) Kinetic proofreading: a new mechanism for reducing errors in biosynthetic processes requiring high specificity, Proc. Natl Acad. Sci. USA 71, 4L35-4139.

414

!I: J. Biochem. 249) Rattenborg et al. (EL(

Jones, T. A,, Cowan, S., Zou, J.-Y. & Kjeldgaard, M. (1991) Improved methods for building proteins models in electron density maps and the location of errors in these models, Acta Crystallogr: A46, 110119. Joseph, S. & Noller, H. F. (1996) Mapping the tRNA neighborhood of the acceptor end of tRNA in the ribosome, EMBO J. 15, 910-916. Kawashima, T., Berthet Colominas, C., Wulff, M., Cusack, S. & Leberman, R. (1996) The structure of the Escherichia coli EF-Tu . EF-Ts complex at 2.5 A resolution, Nature 379, 511-518. Kjeldgaard, M. & Nyborg, J. (1992) Refined structure of elongation factor EF-Tu from Escherichia co/i, J. Mol. Biol. 223, 721 -742. Kjeldgaard, M., Nissen, P., Thirup, S. & Nyborg, J. (1993) The crystal structure of elongation factor EF-Tu from Thermus aquaticus in the GTP conformation, Structure (Lond.) I , 35-50. Kjeldgaard, M., Nyborg, J. & Clark, B. F. C. (1996) The GTP binding motif: variations on a theme, FASEB J. 10, 1347-1368. Knudsen, C. R., Clark, B. F., Degn, B. & Wiborg, 0. (1992) One-step purification of E. coli elongation factor Tu, Biochem. Znt. 28, 353362. Knudsen, C. R. & Clark, B. F. C. (1995) Site-directed mutagenesis of Arg58 and Asp86 of elongation factor Tu from Escherichia coli: effects on the GTPase reaction and aminoacyl-tRNA binding, Protein Eng. 8, 1267-1273. Knudsen, C. R., Kjaersgard, I. V., Wiborg, 0. & Clark, B. F. C. (1995) Mutation of the conserved Gly94 and Gly126 in elongation factor Tu from Escherichia coli. Elucidation of their structural and functional roles, Eur: J. Biochem. 228, 176-183. Louie, A. & Jumak, F. (1985) Kinetic studies of Escherichia coli elongation factor Tu-guanosine 5’-triphosphate-aminoacyl-tRNAcomplexes, Biochemistry 24, 6433 -6439. Mansilla, F., Knudsen, C. R., Laurberg, M. & Clark, B. F. C. (1997) Mutational analysis of Escherichia coli elongation factor Tu in search of a role for the N-terminus, Protein Eng. 10, 927-934. Miller, D. L. & Weissbach, H. (1977) Factors involved in the transfer of aminoacyl-tRNA to the ribosome, in Molecular mechanisms of protein biosynthesis (Weissbach, H. & Petska, s., eds) pp. 323-373, Academic Press, New York. Ninio, J. (1975) Kinetic amplification of enzyme discrimination, Biochimie (Paris) 57, 587-595. Nissen, P., Kjeldgaard, M., Thirup, S., Polekhina, G., Reshetnikova, L., Clark, B. F. C. & Nyborg, J. (1995) Crystal structure of the ternary complex of Phe-tRNAPhe, EF-Tu, and a GTP analog, Science 270, 1464- 1472.

Nissen, P., Kjeldgaard, M., Thirup, S., Clark, B. F. C. & Nyborg, J. (1997) The ternary complex of aminoacylated tRNA and EF-TuGTP. Recognition of a bond and a fold, Biochimie (Paris) 78, 921 933. Pingoud, A. & Urbanke, C. (1979) A quantitative assay for ternary complex formation of elongation factor Tu, GTP, and aminoacyl-tRNA, Anal. Biochem. 92, 123-127. Polekhina, G., Thirup, S., Kjeldgaard, M., Nissen, P., Lippmann, C. & Nyborg, J. (1996) Helix unwinding in the effector region of elongation factor EF-Tu-GDP, Structure (Lond.) 4, 1141- 115 1. Rodnina, M. V. & Wintermeyer, W. (1995) GTP consumption of elongation factor Tu during translation of heteropolymeric mRNAs, Proc. Natl Acad. Sci. USA 92, 1945-1949. Ruusala, T., Ehrenberg, M. & Kurland, C. G. (1982) Is there proofreading during polypeptide synthesis? EMBO J . 1, 741-745. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular cloning: a laboratov nzanual, 2nd edn, Cold Spring Harbor Laboratory, Cold Spring Harbor NY. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) DNA sequencing with chain-terminating inhibitors, Proc. Natl Acad. Sci. USA 74, 5463 5461. Sprinzl, M. & Oraeser, E. (1980) Role of the 5’-terminal phosphate of tRNA for its function during protein biosynthesis elongation cycle, Nucleic Acids Res. 8, 4737-4744. Taylor, J. W., Ott, J. & Eckstein, F. (1985) The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA, Nucleic Acids Res. 13, 8765- 8785. Thompson, R. C. & Stone, P. J. (1977) Proofreading of the codon-anticodon interaction on ribosomes, Proc. Nut/ Acad. Sci. USA 74, 198202. von der Haar, F. (1979) Purification of aminoacyl-tRNA synthetases, Methods Enzymol. 59, 257-267. Wiborg, O., Andersen, C., Knudsen, C. R., Clark, B. F. C. & Nyborg, J. (1996) Mapping Escherichia coli elongation factor Tu residues involved in binding of aminoacyl tRNA, J. B i d . Chem. 271, 204062041 1. Zeidler, W., Egle, C., Ribeiro, S., Wagner, A., Katunin, V., Kreutzer, R., Rodnina, M., Wintermeyer, W. & Sprinzl, M. (1995) Site-directed mutagenesis of Thermus thermophilus, Replacement of His85, Asp81 and Arg300, Eul: J. Biochem. 229, 596-604.