©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Studies on the Polypeptide Elongation Factor 2 from Sulfolobus solfataricus
INTERACTION WITH GUANOSINE NUCLEOTIDES AND GTPase ACTIVITY STIMULATED BY RIBOSOMES (*)

(Received for publication, May 10, 1995; and in revised form, June 28, 1995)

Gennaro Raimo Mariorosario Masullo Vincenzo Bocchini (§)

From the Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, Via S. Pansini 5, I-80131 Napoli, Italia

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The elongation factor 2 from the thermoacidophilic archaeon Sulfolobus solfataricus (SsEF-2) binds [^3H]GDP at 1:1 molar ratio. The bound [^3H]GDP is displaced by GTP or its nonhydrolyzable analogue guanyl-5`-yl imidodiphosphate (Gpp(NH)p) but not by ATP, thus indicating that only the two guanosine nucleotides compete for the same binding site. The affinity of SsEF-2 for [^3H]GDP is higher than that for GTP and Gpp(NH)p. On the contrary, in the presence of ribosomes the affinity of SsEF-2 for GDP is lower than that for Gpp(NH)p. SsEF-2 is endowed with an intrinsic hardly detectable GTPase activity that is stimulated by ribosomes up to 2000-fold. The ribosome-stimulated SsEF-2 GTPase (GTPase^r) reaches a maximum at pH 7.8 and is not affected by ATP but is competitively inhibited by either GDP or Gpp(NH)p. Both K for [-P]GTP and k of GTPase^r increase with increasing temperature, and the highest catalytic efficiency is reached at 80 °C. The ADP-ribosylation of SsEF-2 does not significantly affect either the binding of GDP and GTP or the kinetics of the GTPase^r. A hypothesis on the stimulation by ribosome of SsEF-2 GTPase is proposed.


INTRODUCTION

In the course of the protein synthesis the translocation of the peptidyl-tRNA from the A-site to the P-site of the ribosome is catalyzed in eubacteria by the elongation factor G and in eukarya and archaea by the elongation factor 2, both in the GTP-bound form. Following the interaction of the elongation factor-GTP complex with the ribosome, the GTP is hydrolyzed to GDP and P(i), and the inactive GDP bound form of EF-2/EF-G (^1)leaves the ribosome. Therefore, the biological activity of EF-2/EF-G is regulated by the alternate binding of GDP and GTP(1, 2, 3) .

In a previous study we have reported that the primary structure of the elongation factor 2 in the hyperthermophilic archaeon Sulfolobus solfataricus (SsEF-2, formerly aEF-2) possesses the consensus sequences typical of the GTP-binding proteins(4) . In the present report we show that SsEF-2 is able to bind guanosine nucleotides and to promote the hydrolysis of GTP in the presence of ribosomes as well. These findings support the hypothesis that the translocation step in the elongation of the polypeptide chain in archaeal systems involves similar mechanisms as those described for eukarya and eubacteria(1, 5, 6, 7) .


EXPERIMENTAL PROCEDURES

Materials

[^3H]GDP and [-P]GTP were purchased from Amersham Corp.; unlabeled nucleotides and Gpp(NH)p were from Boehringer Mannheim. Diphtheria toxin was obtained from Calbiochem. All of the reagents were of analytical grade.

The following buffers were used: buffer A, 25 mM Tris/HCl, pH 7.8, 10 mM NH(4)Cl, 10 mM Mg(CH(3)COO)(2); buffer B, 50 mM Tris/HCl, pH 7.8, 2 mM Mg(CH(3)COO)(2), 50 mM KCl, 1 mM dithiothreitol; buffer C, 20 mM Tris/HCl, pH 7.8, 0.4 mM NH(4)Cl, 10 mM Mg(CH(3)COO)(2), 0.05 mg/ml poly(U), 1 mM spermine, 1 mM ATP.

Methods

SsEF-2 was purified from S. solfataricus (ATCC 49255) as previously described (8) and stored at -20 °C in 20 mM Tris/HCl, pH 7.8, 10 mM MgCl(2), 50% (v/v) glycerol. The purity of isolated SsEF-2 was assessed by SDS-polyacrylamide gel electrophoresis (9) or by reverse phase chromatography on a C(4) column (Vydac) connected to a high performance liquid chromatography apparatus (Kontron).

Ribosomes were prepared according to the procedure described elsewhere (10) with an additional ultracentrifugation step at 100,000 g at 20 °C in buffer A supplemented with 18% sucrose and 500 mM NH(4)Cl. The pellet was then suspended in buffer A containing 50% glycerol and stored at -20 °C. One A unit was taken to correspond to 25 pmol of ribosomes.

Poly(U)-dependent poly(Phe) synthesis was performed as described previously(6) .

The amount of the SsEF-2bullet[^3H]GDP complex, formed upon incubation of [^3H]GDP with SsEF-2 in buffer B, was estimated by counting the radioactivity sticking on nitrocellulose filters(11) , using a Packard Tri-Carb 1500 liquid scintillation analyzer. The apparent dissociation equilibrium constant, K`, of the SsEF-2bulletGDP complex was determined by titrating SsEF-2 with [^3H]GDP; the results were plotted according to the Scatchard equation(12) , and both the K` of the complex and the number of binding sites for [^3H]GDP on SsEF-2 were derived. The values of K` of the SsEF-2bulletGTP and SsEF-2bulletGpp(NH)p complexes were determined by competitive binding experiments between [^3H]GDP and GTP or Gpp(NH)p following the procedure described previously for SsEF-1alpha(6) ; other experimental details are given in the legend to Fig. 1.


Figure 1: Binding of [^3H]GDP, GTP, and Gpp(NH)p to SsEF-2. A, kinetics of the [^3H]GDP binding to SsEF-2. 600 µl of buffer B containing 50 µM [^3H]GDP (specific activity, 430 cpm/pmol), and 0.38 µMSsEF-2 were incubated at 0 °C; at the times indicated 90-µl aliquots were withdrawn, and the amount of SsEF-2bullet[^3H]GDP formed was determined as described under ``Methods.'' B, competitive binding on SsEF-2 between [^3H]GDP and ATP, GTP, or Gpp(NH)p. 160 µl buffer B containing 6.5 µM [^3H]GDP (specific activity, 900 cpm/pmol) and 0.2 µMSsEF-2 were incubated in the presence of increasing concentrations of GTP (bullet), Gpp(NH)p (), or ATP (up triangle, filled). After 30 min of incubation at 60 °C, a time chosen to be sure that the equilibrium was attained, the amount of the SsEF-2bullet[^3H]GDP complex formed was determined in duplicate on 70-µl aliquots as described under ``Methods.'' Under these conditions the amount of GTP hydrolyzed by SsEF-2 was negligible (from 0.04 to 1.7%). C, Scatchard plot for the dissociation of the SsEF-2bullet[^3H]GDP complex. 300 µl of buffer B containing 1 µMSsEF-2 and 0.5-40 µM [^3H]GDP (specific activity, 250-7000 cpm/pmol) were incubated for 30 min at 60 °C; at each concentration of [^3H]GDP the amount of SsEF-2bullet[^3H]GDP formed was determined in triplicate on 90-µl aliquots. The data were analyzed according to the Scatchard equation; r, [^3H]GDP bound/SsEF-2 (by moles) at equilibrium.



The ribosome-stimulated GTPase activity of SsEF-2 was measured in buffer C; ATP was maintained throughout even though it was essential only when the GTPase was tested in the presence of partially purified ribosomes. The [-P]GTP hydrolyzed was estimated from the amount of P(i) released, measured by the charcoal method(13) ; the reaction was followed kinetically at 60 °C. Blanks run in the absence of SsEF-2 were subtracted. The rate of [-P]GTP breakdown was calculated from the slope of the linear kinetics of the hydrolytic reaction. The values of K and K` for [-P]GTP, k and K` of GDP and Gpp(NH)p for GTPase^r were determined as reported previously (13) .

The ADP-ribosylation of SsEF-2 was performed as previously described (8) using 12 µMSsEF-2, 100 µM NAD, and 1 µM diphtheria toxin. The ADP-R-F2 was freed of diphtheria toxin, nonmodified SsEF-2, and NAD excess by chromatography on a Mono Q column equilibrated with 20 mM Tris/HCl, pH 7.8, connected to a fast protein liquid chromatography system (Pharmacia Biotech Inc.) and eluted by a linear 0-150 mM KCl gradient in the same buffer. ADP-R-F2 was collected at about 60 mM KCl, whereas the residual SsEF-2 was eluted at about 100 mM KCl.

Protein concentration was determined by the method of Bradford (14) using bovine serum albumin as standard.


RESULTS AND DISCUSSION

Binding of Guanosine Nucleotides to SsEF-2

The rate of [^3H]GDP binding to SsEF-2 was very high, even at 0 °C (Fig. 1A), and it did not change appreciably in the pH range 5-9. Since SsEF-2bulletGTP is not retained on nitrocellulose filters, the binding of GTP to SsEF-2 was measured by competitive binding experiments in which the amount of the SsEF-2bullet[^3H]GDP complex formed decreased at increasing concentrations of GTP (Fig. 1B). The nonhydrolyzable GTP analogue Gpp(NH)p was less efficient, whereas ATP added up to 1000-fold molar excess over [^3H]GDP was ineffective. The Scatchard plot shows that 1 mol of SsEF-2 binds 1 mol of [^3H]GDP (Fig. 1C). At 60 °C the affinity of SsEF-2 for [^3H]GDP (K` = 1 µM) is 10-fold higher than that for GTP and 51-fold higher than that for Gpp(NH)p, thus resembling the behavior of EF-G/EF-2 from other sources(3, 15, 16) .

SsEF-2 GTPase Activity in the Presence of Ribosome

Ribosomes isolated from S. solfataricus possess a tightly associated GTPase activity(17) . An additional ultracentrifugation step (see ``Methods'') reduced the ribosomal GTPase activity by 5-10-fold without affecting the ability of the ribosomes to support the in vitro poly(Phe) synthesis. When assayed in buffer C, purified SsEF-2 showed an intrinsic hardly detectable GTPase activity (less than 0.5 mol of [-P]GTP hydrolyzed by 1 mol of SsEF-2 in 30 min at 60 °C). The addition of ribosomes stimulated a turnover SsEF-2 GTPase (Fig. 2A); its extent was maximum at 0.5 µM ribosome, which at higher concentrations produced inhibition (Fig. 2B), as already reported for the E. coli EF-G GTPase^r(18) . The enzymatic [-P]GTP breakdown followed linear kinetics and depended on the amount of SsEF-2 added. The possibility that SsEF-2 GTPase^r was due to a triphosphatase contaminant was ruled out by the following observations: (i) the sample of SsEF-2 used appeared homogenous when analyzed by either SDS-polyacrylamide gel electrophoresis or by reverse phase chromatography on a C(4) column; (ii) the typical properties of SsEF-2 (ability to be ADP-ribosylated by diphtheria toxin, GDP binding, and GTPase^r) co-purified with SsEF-2; (iii) the presence of a 10-fold molar excess of ATP over [-P]GTP did not have any effect on the amount of P(i) released.


Figure 2: Hydrolysis of [-P]GTP promoted by SsEF-2 in the presence of ribosomes. 250 µl of buffer C containing 100 µM [-P]GTP (specific activity, 180 cpm/pmol) were incubated at 60 °C, and the amount of P(i) released was determined on 40-µl aliquots as described under ``Methods.'' A, time course of the GTPase activity in the presence of 0.1 µMSsEF-2 (up triangle, filled), 0.5 µM ribosomes (), or 0.1 µMSsEF-2 plus 0.5 µM ribosomes (bullet). B, GTPase activity of ribosomes at increasing concentrations in the absence () or in the presence (bullet) of 0.1 µMSsEF-2. Blanks were subtracted.



pH and Temperature Dependence, Catalytic Efficiency, and Energetic Aspects of GTPase^r

At 60 °C the pH optimum for the GTPase^r was in a narrow range, centered around pH 7.8, that was not substantially affected by the buffer used (Fig. 3).


Figure 3: Effect of pH on SsEF-2 GTPase^r. The reaction mixture contained 0.5 µM ribosomes, 100 µM [-P]GTP (specific activity, 80 cpm/pmol), and 0.1 µMSsEF-2 in 50 µl of buffer C in which 20 mM Tris/HCl was substituted for 50 mM of each of the following buffers: sodium citrate (bullet), glycine/NaOH (), imidazole/acetate (black square), Tris/HCl (). The reaction was carried out for 3 min at 60 °C, and the P(i) released was determined; blanks were subtracted.



In order to evaluate the effect of temperature on the rate and on the affinity of GTPase^r for [-P]GTP, K and k were determined by Lineweaver-Burk plots that in the temperature range 50-91 °C were all linear. The data reported in Table 1show that at increasing temperatures both k and K increased; the catalytic efficiency, expressed by the ratio k/K, reached a maximum at 80 °C. The possibility that the increase with temperature of K of SsEF-2 GTPase^r for [-P]GTP could also be due to a competitive inhibition by the GDP produced upon hydrolysis of [-P]GTP was ruled out since such an amount was negligible compared to that required to get a significant increase of K (see below). A linear Arrhenius plot gave a value for the energy of activation equal to 85 kJ/mol; at 87 °C the calculated values for DeltaH*, DeltaS*, and DeltaG* were 82 kJbulletmol, -3 JbulletmolbulletK, and 83 kJbulletmol, respectively. These energetic data were quite close to those reported for Escherichia coli EF-G GTPase^r at 30 °C(19) , thus indicating that the two enzymatic systems have similar energetic requirements.



It is remarkable that at the growth temperature of the respective source organisms, the affinity for GTP of GTPase^r is very similar between S. solfataricus SsEF-2 (K = 32 µM) and E. coli EF-G (K = 41 µM) (20) .

Affinity of SsEF-2 for Guanosine Nucleotides in the Presence of Ribosomes, Evaluated by the Inhibition of GTPase^r by GDP and Gpp(NH)p

GTPase^r was competitively inhibited by both GDP and Gpp(NH)p, the latter being a stronger inhibitor (Fig. 4). At increasing concentrations of both inhibitors the value of v remained unchanged, whereas Kfor [-P]GTP, that was 9 µM in the absence of inhibitors, increased to 18, 29, 47, and 93 µM in the presence of 5, 10, 30, and 60 µM GDP, and to 48 µM and 312 µM in the presence of 2 µM and 32 µM Gpp(NH)p. Values of 7 and 1 µM were calculated for the inhibition constant, K`, in the presence of GDP and Gpp(NH)p, respectively; they represent also the values of K` of SsEF-2bulletGDP and SsEF-2bulletGpp(NH)p in the presence of ribosomes. In fact, the instability during filtration on nitrocellulose of the SsEF-2bulletGDP and SsEF-2bulletGpp(NH)p complexes with ribosomes did not allow the directed determination of the respective K` values. The data reported in Table 2show that ribosomes reverse the relative affinity of SsEF-2 for GDP and Gpp(NH)p; in fact, the affinity for GDP, that in the absence of ribosomes was 51-fold higher compared to that for Gpp(NH)p, in the presence of ribosomes became 7-fold lower. A similar behavior has been described for E. coli EF-G(3, 15) and rat liver EF-2(16) . The increased affinity of SsEF-2 for GTP might explain the stimulatory effect by the ribosome on the SsEF-2 GTPase; in addition, the decreased affinity for GDP should reduce the inhibitory effect by the GDP formed upon the hydrolysis of GTP.


Figure 4: Competitive inhibition of GTPase^r by GDP and Gpp(NH)p. 200 µl of buffer C contained 0.07 µMSsEF-2, 2-200 µM [-P]GTP (specific activity, 84-7600 cpm/pmol), in the absence () or in the presence of 30 µM GDP (black square) or 32 µM Gpp(NH)p (); the amount of [-P]GTP hydrolyzed was followed kinetically at 60 °C on 40-µl aliquots. The reaction rate (v) was expressed as picomoles of [-P]GTP hydrolyzed per min.





Effect of ADP-ribosylation of SsEF-2 on the GTPase^r

Since the ADP-ribosylation of SsEF-2 inhibits poly(Phe) synthesis(7, 21) , its effect on the SsEF-2 GTPase^r was investigated. The ADP-R-F2 was still able to sustain the GTPase^r whose kinetics was not greatly affected. In fact, at 60 °C the values of k and K for [-P]GTP are 0.6 s and 12 µM, respectively (for a comparison with nonmodified SsEF-2, see Table 1). ADP-R-F2 GTPase^r was competitively inhibited by GDP, and the value of K` was identical to that determined with not ADP-ribosylated SsEF-2. Therefore, the ADP-ribosylation of SsEF-2 does not affect the binding of GDP and GTP to SsEF-2; a similar finding has been reported for rat liver EF-2(22) .

To our knowledge, this is the first report on the characterization of an archaeal hyperthermophilic EF-2 regarding either its affinity for GDP and GTP or its ribosome-stimulated GTPase activity. Our recent report that the elongation factor SsEF-1alpha binds GDP and GTP (6) and elicits an intrinsic GTPase (13) and the data reported in the present work prove that the S. solfataricus elongation factors are functionally similar to the corresponding bacterial and eukaryal counterparts (1, 3, 5, 7) despite that the temperature for optimum growth of S. solfataricus is about 50 °C higher.


FOOTNOTES

*
This investigation was supported by Ministero dell'Università e della Ricerca Scientifica e Tecnologica, by Consiglio Nazionale delle Ricerche, Target Project on Biotechnology and Bioinstrumentation (Roma), and partially by the European Community Human Capital and Mobility Programme, contract no. ERBCHRXCT940510. 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.: 39.81.7463120; Fax: 39.81.7463653.

(^1)
The abbreviations used are: EF, elongation factor; SsEF, Sulfolobus solfataricus EF; EF-1alpha and EF-2, elongation factors 1alpha and 2 from other archaea and eukarya, respectively; EF-G, eubacterial elongation factor G; Gpp(NH)p, guanyl-5`-yl imidodiphosphate; GTPase^r, ribosome-stimulated GTPase activity; k, rate constant of the hydrolytic cleavage of GTP in GDP and P(i); K`, Michaelis constant in the presence of inhibitor; K`, apparent dissociation equilibrium constant; K`, apparent inhibition constant; ADP-R-F2, ADP-ribosylated SsEF-2.


ACKNOWLEDGEMENTS

We thank A. Fiengo for skillful technical assistance.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.