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Involvement of histidine residues in the substrate binding of elongation factor Tu from Thermus thermophilus: Proton nuclear magnetic resonance and photooxidation study
ARCHIVES
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 196, No. 1, August, pp. 233-238, 1979
Involvement of Histidine Residues in the Substrate Elongation Factor Tu from Thermus thermophilus: Nuclear Magnetic Resonance and Photooxidation AKIHIKO
NAKANO,*
Binding of Proton Study
TATSUO MIYAZAWA,* SHUN NAKAMURA,? YOSHITO KAZIROt
AND
*Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan, and Wnstitute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108, Japan
Received March 2, 1979 Proton nuclear magnetic resonance (nmr) spectra (270 MHz) were measured of polypeptide chain elongation factor Tu (EF-Tu) from an extreme thermophile, Thewnus thermophilus. This protein was stable enough for a series of nmr measurements at temperature as high as 50°C. For histidine C, protons, pH dependences of nmr chemical shifts were measured in the pH range from 5.5 to 8.0. The nmr titration curve of one histidine residue of free EF-Tu was markedly affected by the binding with GDP. This titration curve was further affected by the ligand substitution from GDP to GTP, indicating that this histidine is involved in the binding of EF-Tu with guanine nucleotides. The nmr titration curve of another histidine was also affected by the ligand substitution from GDP to GTP. The results of photooxidation experiments suggest that histidine residues are involved in the binding of EF-Tu with guanine nucleotides as well as with aminoacyl-tRNA and/or ribosomes.
free EF-Tu and EF-Tu *GDP from E. coli have also been detected spectrophotometrically (8, 9). Recently, the tertiary structure of the modified form of E. coli EF-Tu . GDP has been reported by X-ray crystallographic analyses at 6.0 (10) and 2.6 A (11) resolution. Nuclear magnetic resonance (nmr) spectroscopy should provide important information for studying dynamic structure-function relationships of EF-Tu. However, EF-Tu from E. coli is rather unstable and it has been difficult to carry out a series of nmr measurements on this protein even at room temperature. On the other hand, EF-Tu from an extreme thermophile, Thermus thermophilus (12-14) is much more stable at high temperature and in wide pH range than E. coli EF-Tu (15) and is far more suitable for intensive nmr studies. how* Abbreviations used: EF-Tu and EF-Ts, poly- This EF-Tu from T. thermophilus, ever, does not have cysteine residues (14) peptide chain elongation factors Tu and Ts, respecand some other residues may be essential tively; buffer TMM, a buffer containing 20 mM Tris-HCl (pH 7.Q 10 mM magnesium acetate, and 5 mM 2-mer- for the binding with aminoacyl-tRNA. In view of the fact that the tryptic peptide captoethanol.
The polypeptide chain elongation factor Tu (EF-Tu)’ promotes the GTP-dependent binding of aminoacyl-tRNA to ribosomes through intermediate formation of a ternary aminoacyl-tRNA *EF-Tu *GTP complex [for recent reviews, see Refs. (1, Z)]. Since only EF-TueGTP, but not EF-TuaGDP, can interact with aminoacyl-tRNA to form the ternary complex, it is expected that the conformation of EF-Tu around the aminoacyl-tRNA site is altered upon ligand substitution from GDP to GTP [see Ref. (Z)]. For EF-Tu from Escherichia coli, the conformational studies with spin label (3-5) and fluorescence (6, 7) probes have demonstrated that the conformational transitions occur near the cysteine residue essential for aminoacyl-tRNA binding. Differences between the conformations of nucleotide-
233
0003-9861/79/090233-06$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
234
NAKANO ET AL.
of E. coli EF-Tu containing the cysteine residue essential for aminoacyl-tRNA binding is rich in histidine residues (16), the proton nmr analyses on histidine residues of T. thermophilus EF-Tu were attempted in the present study. From the amino acid analysis, this EF-Tu has been found to contain about 13 histidine residues (14). Photooxidation experiments were also carried out to elucidate the functional roles of histidine EF-Tu. residues in T. themophilus
No. CE-103). The direct meter readings are quoted in this paper. The 2H,0 solution of EF-Tu was prepared as follows. The solution containing EF-Tu was extensively dialyzed against 0.01 M NaCl and 0.01 mM dithiothreitol. The dialyzed solution was lyophilized, dissolved in *H,O, incubated at 50°C for 30 min for substituting exchangeable protons by deuterons, and lyophilized again. Then the residue was dissolved in a small volume of 2H,0, and the composition of the solution was adjusted to 0.1 M NaCl and 0.1 mM dithiothreitol by adding *H20 solution of 1 M NaCl and 1 mM dithiothreitol. The pH values were varied with 0.1 M NaOZH and 0.1 M *HCl in the range from 5.5 to 8.0. The nmr measurements at pH below 5.5 EXPERIMENTAL PROCEDURES were impossible because of the precipitation of the enzyme, while the measurements of CZproton signals Materials. EF-Tu.GDP and EF-Ts were purified from T. thewnophilus HB8 as previously described of histidine residues at pH above 8.0 were difficult because of hydrogen-deuterium exchange (20) and (12). EF-Tu.GTP was prepared from EF-Tu.GDP of significant signal overlapping. Protein concenby incubation with phosphoenolpyruvate and pyruvate kinase (17). Nueleotide-free EF-Tu was pre- tration was estimated from the absorbance at 280 nm pared as follows. Purified EF-Tu.GDP (-100 mg) using Agum = 0.8 for EF-Tu.GDP and EF-Tu. was extensively dialyzed against 10 mM imidazole- GTF, and A.jS~g’ml = 0.6 for free EF-Tu (14, 21). Photoo&&ion. EF-Tu was photooxidized using HCl (pH 5.0), 1 mM EDTA, and 5 mM 2-mercaptoethanol. It was then applied on an SP-Sephadex C-50 rose bengal as a photosensitizer (22). In a typical experiment, 0.5 ml of the reaction mixture containing column (2 x 40 cm) equilibrated with the above buffer, and the column was further washed with 1 liter of the 20 pM free EF-Tu or EF-Tu.GDP and 10 pM rose same buffer. The fraction containing free EF-Tu was bengal in 20 mM Tris-HCl (pH 7.5), 10 mM magnesium obtained by stepwise elution with 100 mM imidazole- acetate, and 6 rn~ &mercaptoethanol (buffer TMM’) was ice-cooled, aerated by continuous stirring, and HCl (pH 8.0), 1 mM EDTA, 5 mM 2-mercaptoethanol, was illuminated by a 375-W tungsten lamp from a and 200 mM NH&l. Pyruvate kinase and phosphoenolpyruvate were distance of 15 cm. Aliquots of 10 ~1 were withdrawn purchased from Boehringer. GDP and GTP were at appropriate intervals and diluted 10 times in buffer TMM in the dark. Then the activities of EF-Tu, i.e., obtained from Yamasa Shoyu Company, and [VH]GDP was purchased from the Amersham Radiochemi- GDP binding activity and the activity to promote cal Centre. All nucleotides were purified by Dowex 1 Phe-tRNA binding to ribosomes, were analyzed. For the photooxidation of the ternary aminoacylcolumn chromatography (18). L-[UJ4C]Phe-tRNA complex, the reaction mixture was also prepared as previously described (17). Rose tRNA.EF-Tu’GTP bengal was purchased from Wako Pure Chemical in the final volume of 0.1 ml contained 2 pM EF-Tu, Industries. Deuterated chemicals, Na02H, *HCl, 1 pM rose bengal, 6 pM [14C]Phe-tRNA, 0.1 PM EF-Ts, 30 pM GTP and its regenerating system (0.6 mM and 99.75% 2H,0 were obtained from E. Merck. phosphoenolpyruvate and 1.5 pg/ml pyruvate kinase) Assays of EF-Tu. The [3H]GDP binding activities of EF-Tu’GDP and free EF-Tu were measured by in 50 mM Tris-HCl (pH 7.5), 8 mM magnesium acetate, the nitrocellulose membrane filter procedure (15). 5 mM 2-mereaptoethanol, and 150 mM NH&l. PhotoThe activity for promoting the binding of [14C]Phe- oxidation reaction was then started as described above and the binding activity of the ternary comtRNA to ribosomes was assayed as described before (3). Nuclear magnetic resonance measurements. Proton plex to ribosomes were measured for the aliquots nmr spectra of T. thermophilus EF-Tu were recorded withdrawn at appropriate intervals. For determination of the amino acid composition, on Bruker WH270 pulse FT spectrometer at 50°C. photooxidized EF-Tu was precipitated with 5% (v/v) Accumulation of as many as 1000-2000 free induction decays over 8192 data points was necessary for trichloroacetic acid and was hydrolyzed in constant l-l.5 mM EF-Tu solution. Convolution difference boiling HCl in a sealed tube at 110°C for 24 h. The technique (19) was used to obtain well-resolved spec- amino acid composition of the hydrolyzate was detertra, where accumulated free induction decays were mined with a Hitachi Model 835-50amino acid analyzer. multiplied by a function, e-*lif - 0.9t-‘O”‘. The solvent peak was suppressed by homo-gated decoupling. RESULTS AND DISCUSSION The pH values were measured at 50°C using Radiometer PHM26 pH meter equipped with a long, thin Well-resolved proton nmr spectra were combination electrode from Nisshin Rika (Catalog observed for T. themophilus EF-Tu, with
HISTIDINE
RESIDUES IN T. thewnophilus EF-Tu
the molecular weight of 49,000. Figure 1 shows the convolution difference spectra of EF-Tu. GDP in the low field region at several pHs. Especially in the region of CZ proton resonances of histidine residues (from 8.6 to 7.5 ppm), as many as 10 signals are resolved. It must be noted that relative intensities of signals do not correspond to the number of protons because of the convolution difference method and of the selective spin diffusion (23). Nontitratable signals at 7.9 and 6.0 ppm are assigned to C8 and Cl,, protons of free GDP, respectively. The pH titration curves were obtained for nucleotide-free EF-Tu, EF-Tu *GDP, and EF-Tu .GTP by plotting the chemical shifts of low field signals versus pH (Fig. 2). Here, filled symbols represent relatively well-defined titration curves for C, proton resonances of histidine residues. First, the effect of the interaction of GDP with EF-Tu was studied by comparing the nmr titration curves of free EF-Tu and EF-Tu*GDP. As shown in Figs. 2A and B, the titration curves with filled circles and triangles are affected little by the GDP binding. However, the titration curve with filled squares shows a drastic shift to lower field on interaction of EF-Tu with GDP. Since either protonation or deprotonation of imidazole rings cannot be completed in this pH range from 5.5 to 8.0, it is difficult to determine the accurate pK, values of histidine residues. Nevertheless, the apparent pK, of the titration curve with filled squares in Fig. 2A appears to be lower than 6.0 and that in Fig. 2B is higher than 7.0. This large change in pK, value is quite reasonable if the histidine residue is directly involved in the binding with phosphate group of nucleotides; the electrostatic interaction between the cationic imidazolium group and the anionic phosphate group increases the p K, of the imidazolium group. Similar changes in pK, values have been definitely observed for His 12 and His 119 of ribonuclease A (20, 24); the pK, values of these histidine residues are increased by l-2 pH unit on the binding with 3’-CMP inhibitor. It is, however, difficult to ascertain that the signals with filled squares in Figs. 2A and B are due to the same histidine residue of free EF-Tu and EF-Tu *GDP,
235
Cherrkal Shii (ppm) FIG. 1. Lowfield region of the convolution difference proton nmr spectra of T. therrnophilus EF-Tu.GDP at various pHs. Chemical shifts are given in parts per million (ppm) from sodium 2,2-dimethyl-2-silapentane5-sulfonate.
because the dissociation rate of E. eoli EF-Tu *GDP is as low as 3.4 x lop4 s-l (25) and accordingly the exchange of EF-Tu between the nucleotide-free state and the GDP-binding state is supposed to be extremely slow. Nevertheless, the relative intensities of C, proton signals of the histidine residue (tilled squares in Figs. 2A and B) were observed as a function of the added amount of GDP. As shown in Fig. 3, the signal intensity of the histidine proton of free EF-Tu (open circle) is lowered while the signal intensity of EF-TunGDP (filled circle) is raised by the addition of GDP, but there are no other prominent signals which are appreciably affected by the addition of GDP. This observation suggests that at least one histidine residue of EF-Tu is directly involved in the binding with GDP. Furthermore the effect of the ligand change from GDP to GTP was studied by
236
NAKANO ET AL.
comparing the pH titration curves in Figs. 2B and C. The titration curves with filled circles are little affected by the ligand substitution. However, the titration curve with filled squares is most significantly affected
8.6
and its apparent pK, is decreased by 0.3-0.4 pH unit upon the ligand substitution from GDP to GTP, again suggesting that this resonance is due to the histidine residue in the nucleotide binding site of EF-Tu. B 8.6
I
28.4. a
78.4. E
E 8.2. 5 $.E 8.0. Q, 6 7.8. 7.6
7.6 t
6
I
eI
7
6
PH
PH
;; 8.4. B
7.6. I
7
6 PH
FIG. 2. Proton nmr titration curves of EF-Tu in *H,O solution at 50°C. Samples in a volume of 0.2 ml commonly contained 0.1 M NaCl and 0.1 mM dithiothreitol. Filled symbols represent relatively well-defined titration curves. (A) Titration curves of nucleotide-free EF-Tu. The solution contained 1.5 mM EF-Tu. (B) Titration curves of EF-Tu.GDP. The solution contained 1.5 mM EF-Tu, 2.5 mM GDP, and 10 mM MgCl,. (C) Titration curves of EF-Tu.GTP. The sample solution was prepared from that of EF-Tu.GDP by converting GDP to GTP enzymatically. Phosphoenolpyruvate and pyruvate kinase were added to the solution of EF-Tu.GDP to final concentrations of 10 mM and 0.5 mg/ml, respectively, and then the solution was incubated at 50°C for 30 min.
HISTIDINE
RESIDUES IN 2’. thermophilus EF-Tu
In addition, the titration curve with filled triangle is also affected; its p K, is increased by about 0.2 pH unit upon the ligand change from GDP to GTP. This resonance may be due to another histidine residue in the nucleotide binding site or to a histidine residue in another site which is affected indirectly by the ligand substitution. As described above, several histidine residues are found near the cysteine residue essential for aminoacyl-tRNA binding of E. coli EF-Tu (16), so that the latter histidine residue of T. thermophilus EF-Tu is possibly located in the neighborhood of aminoacyl-tRNA binding site, where the microenvironment is affected by the ligand substitution from GDP to GTP. Photooxidation experiments on T. thermophilus EF-Tu were useful for elucidating the role of histidine residues in the activity of EF-Tu. The amino acid analyses on the photooxidized EF-Tu *GDP showed that only histidine residues were modified by photooxidation. As shown in Fig. 4, the GDP binding activity of EF-Tu was appreciably reduced upon photooxidation of nucleotide-free EF-Tu for 60 min (filled circle). However, if GDP was present during photooxidation of EF-Tu, the GDP binding activity of EF-Tu was much less affected (open circle). These observations
237
Time( min)
FIG. 4. Time courses of photooxidative inactivation of EF-Tu. EF-Tu was photooxidized for the times indicated in the form of either free EF-Tu (0, n ), EF-Tu. GDP (0, q ), or the ternary Phe-tRNA.EF-Tu.GTP (A), and the activities to bind GDP (0,O) or to promote Pbe-tRNA binding to ribosomes (Cl, W, A) were measured. For details, see under Experimental Procedures.
indicate that the GDP binding site is protected by the nucleotide from photooxidation, supporting the suggestion from nmr analyses that histidine residue(s) is possibly essential for the binding with guanine nucleotides. Furthermore, the possible role of histidine residues for the aminoacyl-tRNA binding was also studied. If free EF-Tu (filled square in Fig. 4) or EF-Tu . GDP (open square) was photooxidized in the absence of aminoacyl-tRNA, the activity of EF-Tu to promote the binding of aminoacyl-tRNA to ribosomes was lost much faster than the GDP binding activity. However, the former activity was little affected if photooxidation experiments were carried out in the presence of GTP and aminoacyltRNA (open triangle in Fig. 4), indicating that the binding site for aminoacyl-tRNA and/or ribosomes was protected from photoI I oxidation by the binding with aminoacyl0 0.5 1.5 1.0 tRNA. These observations suggest that GOP (mM) histidine residue(s) is also essential for FIG. 3. Relative intensities of the signals tentatively the binding with aminoacyl-tRNA and/or assigned to C, proton of the histidine residue in the ribosomes. GDP binding site of EF-Tu as a function of added In summary, the involvement of histidine GDP. The solution contained 1.2 mM EF-Tu in 0.1 M residue(s) in the binding of T. thermophilus NaCl and 0.1 mM dithiothreitol at pH 6.7, 50°C. RelaEF-Tu with guanine nucleotides was revealed tive intensities of the two signals at 7.6 (0) and 8.4 ppm (0) are represented as peak height ratios to the by nmr studies on histidine C, protons and invariant signal at 7.3 ppm. Arrows indicate the point was confirmed by photooxidation experiments. Furthermore, the presence of histiwhere EF-Tu:GDP = 1:l.
238
NAKANO
dine residue(s) in the binding site for aminoacyl-tRNA and/or ribosomes was suggested by photooxidation experiments. These histidine residues may play essential roles in the biochemical functions of T. thermophiZus EF-Tu. ACKNOWLEDGMENT We express onr gratitudes to Dr. T. Oshima, Mitsubishi Kasei Institute of Life Sciences, for his valuable advice and kind supply of Thermus thermophilus HB8, end to Dr. S. Kawashima, Faculty of Medicine, University of Tokyo, for the amino acid analysis of photooxidized EF-Tn. REFERENCES 1. MILLER, D. L., AND WEISSBACH, H. (1977) in Molecular Mechanism of Protein Biosynthesis (Weissbach, H., and Pestka, S., eds.), pp. 323-373, Academic Press, New York. 2. KAZIRO, Y. (1978) Biochim. Biophys. Acta 505, 95- 127. 3. ARAI, K., KAWAKITA, M., KAZIRO, Y., MAEDA, T., AND OHNISHI, S. (1974) J. Biol. Chem. 249, 3311-3313. 4. ARAI, K., MAEDA, T., KAWAKITA, M., OHNISHI, S., AND KAZIRO, Y. (1976)J. Biochem. 80,1047-1055. 5. WILSON, G. E., COHN, M., AND MILLER, D. (1978) J. Biol. Chem. 253, 5764-5768. 6. CRANE, L. J., AND MILLER, D. L. (1974) Biochemistry 13, 933-939. 7. &AI, K., AELAI, T., GWAKITA, M., AND KAZIRO, Y. (1975) J. Biochem. 77, 1095-1106. 8. ARAI, K., ARAI, T., KAWAKITA, M., AND KAZlRO, Y. (1977) J. Biochem. 81, 1335-1346. 9. OHTA, S., NAKANISHI, M., TSUBOI, M., ARAI, K., AND KAZIRO, Y. (1977) Eur. J. Biochem. 78, 599-608.
ET AL.
10. K=~BSCH, W., GAS?W. H., S~.JLTZ,G. E., AND 11.
12.
13.
14.
15. 16.
17. 18. 19.
20. 21. 22. 23. 24.
25.
LEBERMAN,I, R. (1977) J. Mol. Biol. 117, 9991012. MORIKAWA, K., L, LA COUR, T. F. M., NYBORG, J., RASMUSSEN, IV, K. M., MILLER, D. L., AND CLARK, B. F. C. (1978) J. Mol. Biol. 125, 325-338. ARAI, K., OTA, Y., ARAI, N., NAKAMURA, S., HENNEKE, C., OSHIMA, T., AND KAZIRO, Y. (1978) Eur. J. Biochem. 92, 509-520. ARAI, K., ARAI, N., NAKAMURA, S., OSHIMA, T., AND KAZIRO, Y. (1978) Eur. J. Biochem. 92, 521-532. NAKAMURA, S., OHTA, S., ARAI, K., ARAI, N., CSHIMA, T., KAZIRO, Y. (1978) Eur. J. Biochem. 92, 533-543. ARAI, K., KAWAKITA, M., AND KAZIRO, Y. (1972) J. Biol. Chem. 247, 7029-7037. NAKAMURA, S., ARAI, K., TAKAHASHI, K., AND KAZIRO, Y. (1975) Biochem. Biophys. Res. Commun. 66, 1069-1077. ARAI, K., KAWAKITA, M., AND KAZIRO, Y. (1974) J. Biochem. 76, 283-292. KAZIRO, Y., INOUE-YOKOSAWA, N., AND KAWAKITA, M. (1972) J. Biochem. 72, 853-863. CAMPBELL, I. D., DoB~~N, C. M., WILLIAMS, R. J. P., AND XAVIER, A. V. (1973) J. Mugn. Resonance 11, 172-181. MARKLEY, J. L. (1975) Biochemistry 14, 35463554. ARAI, K., KAWAKITA, M., KAZIRO, Y., KONW, T., AND UI, N. (1973) J. Biochem. 73, 1095-1105. TAKAHASHI, K. (1970) J. Biochem. 67, 833-839. AKASAKA, K., KONRAD, M., AND GOODY, R. S. (1978) FEBS Lett. 96, 287-290. GORENSTEIN, D. G., WYRWICZ, A. M., AND BODE, J. (1976) J. Amer. Chem. Sot. 98,23082314. ARAI, K., KAWAKITA, M., AND KAZIRO, Y. (1974) J. Biochem. 76, 293-306.
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 196, No. 1, August, pp. 233-238, 1979
Involvement of Histidine Residues in the Substrate Elongation Factor Tu from Thermus thermophilus: Nuclear Magnetic Resonance and Photooxidation AKIHIKO
NAKANO,*
Binding of Proton Study
TATSUO MIYAZAWA,* SHUN NAKAMURA,? YOSHITO KAZIROt
AND
*Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan, and Wnstitute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108, Japan
Received March 2, 1979 Proton nuclear magnetic resonance (nmr) spectra (270 MHz) were measured of polypeptide chain elongation factor Tu (EF-Tu) from an extreme thermophile, Thewnus thermophilus. This protein was stable enough for a series of nmr measurements at temperature as high as 50°C. For histidine C, protons, pH dependences of nmr chemical shifts were measured in the pH range from 5.5 to 8.0. The nmr titration curve of one histidine residue of free EF-Tu was markedly affected by the binding with GDP. This titration curve was further affected by the ligand substitution from GDP to GTP, indicating that this histidine is involved in the binding of EF-Tu with guanine nucleotides. The nmr titration curve of another histidine was also affected by the ligand substitution from GDP to GTP. The results of photooxidation experiments suggest that histidine residues are involved in the binding of EF-Tu with guanine nucleotides as well as with aminoacyl-tRNA and/or ribosomes.
free EF-Tu and EF-Tu *GDP from E. coli have also been detected spectrophotometrically (8, 9). Recently, the tertiary structure of the modified form of E. coli EF-Tu . GDP has been reported by X-ray crystallographic analyses at 6.0 (10) and 2.6 A (11) resolution. Nuclear magnetic resonance (nmr) spectroscopy should provide important information for studying dynamic structure-function relationships of EF-Tu. However, EF-Tu from E. coli is rather unstable and it has been difficult to carry out a series of nmr measurements on this protein even at room temperature. On the other hand, EF-Tu from an extreme thermophile, Thermus thermophilus (12-14) is much more stable at high temperature and in wide pH range than E. coli EF-Tu (15) and is far more suitable for intensive nmr studies. how* Abbreviations used: EF-Tu and EF-Ts, poly- This EF-Tu from T. thermophilus, ever, does not have cysteine residues (14) peptide chain elongation factors Tu and Ts, respecand some other residues may be essential tively; buffer TMM, a buffer containing 20 mM Tris-HCl (pH 7.Q 10 mM magnesium acetate, and 5 mM 2-mer- for the binding with aminoacyl-tRNA. In view of the fact that the tryptic peptide captoethanol.
The polypeptide chain elongation factor Tu (EF-Tu)’ promotes the GTP-dependent binding of aminoacyl-tRNA to ribosomes through intermediate formation of a ternary aminoacyl-tRNA *EF-Tu *GTP complex [for recent reviews, see Refs. (1, Z)]. Since only EF-TueGTP, but not EF-TuaGDP, can interact with aminoacyl-tRNA to form the ternary complex, it is expected that the conformation of EF-Tu around the aminoacyl-tRNA site is altered upon ligand substitution from GDP to GTP [see Ref. (Z)]. For EF-Tu from Escherichia coli, the conformational studies with spin label (3-5) and fluorescence (6, 7) probes have demonstrated that the conformational transitions occur near the cysteine residue essential for aminoacyl-tRNA binding. Differences between the conformations of nucleotide-
233
0003-9861/79/090233-06$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
234
NAKANO ET AL.
of E. coli EF-Tu containing the cysteine residue essential for aminoacyl-tRNA binding is rich in histidine residues (16), the proton nmr analyses on histidine residues of T. thermophilus EF-Tu were attempted in the present study. From the amino acid analysis, this EF-Tu has been found to contain about 13 histidine residues (14). Photooxidation experiments were also carried out to elucidate the functional roles of histidine EF-Tu. residues in T. themophilus
No. CE-103). The direct meter readings are quoted in this paper. The 2H,0 solution of EF-Tu was prepared as follows. The solution containing EF-Tu was extensively dialyzed against 0.01 M NaCl and 0.01 mM dithiothreitol. The dialyzed solution was lyophilized, dissolved in *H,O, incubated at 50°C for 30 min for substituting exchangeable protons by deuterons, and lyophilized again. Then the residue was dissolved in a small volume of 2H,0, and the composition of the solution was adjusted to 0.1 M NaCl and 0.1 mM dithiothreitol by adding *H20 solution of 1 M NaCl and 1 mM dithiothreitol. The pH values were varied with 0.1 M NaOZH and 0.1 M *HCl in the range from 5.5 to 8.0. The nmr measurements at pH below 5.5 EXPERIMENTAL PROCEDURES were impossible because of the precipitation of the enzyme, while the measurements of CZproton signals Materials. EF-Tu.GDP and EF-Ts were purified from T. thewnophilus HB8 as previously described of histidine residues at pH above 8.0 were difficult because of hydrogen-deuterium exchange (20) and (12). EF-Tu.GTP was prepared from EF-Tu.GDP of significant signal overlapping. Protein concenby incubation with phosphoenolpyruvate and pyruvate kinase (17). Nueleotide-free EF-Tu was pre- tration was estimated from the absorbance at 280 nm pared as follows. Purified EF-Tu.GDP (-100 mg) using Agum = 0.8 for EF-Tu.GDP and EF-Tu. was extensively dialyzed against 10 mM imidazole- GTF, and A.jS~g’ml = 0.6 for free EF-Tu (14, 21). Photoo&&ion. EF-Tu was photooxidized using HCl (pH 5.0), 1 mM EDTA, and 5 mM 2-mercaptoethanol. It was then applied on an SP-Sephadex C-50 rose bengal as a photosensitizer (22). In a typical experiment, 0.5 ml of the reaction mixture containing column (2 x 40 cm) equilibrated with the above buffer, and the column was further washed with 1 liter of the 20 pM free EF-Tu or EF-Tu.GDP and 10 pM rose same buffer. The fraction containing free EF-Tu was bengal in 20 mM Tris-HCl (pH 7.5), 10 mM magnesium obtained by stepwise elution with 100 mM imidazole- acetate, and 6 rn~ &mercaptoethanol (buffer TMM’) was ice-cooled, aerated by continuous stirring, and HCl (pH 8.0), 1 mM EDTA, 5 mM 2-mercaptoethanol, was illuminated by a 375-W tungsten lamp from a and 200 mM NH&l. Pyruvate kinase and phosphoenolpyruvate were distance of 15 cm. Aliquots of 10 ~1 were withdrawn purchased from Boehringer. GDP and GTP were at appropriate intervals and diluted 10 times in buffer TMM in the dark. Then the activities of EF-Tu, i.e., obtained from Yamasa Shoyu Company, and [VH]GDP was purchased from the Amersham Radiochemi- GDP binding activity and the activity to promote cal Centre. All nucleotides were purified by Dowex 1 Phe-tRNA binding to ribosomes, were analyzed. For the photooxidation of the ternary aminoacylcolumn chromatography (18). L-[UJ4C]Phe-tRNA complex, the reaction mixture was also prepared as previously described (17). Rose tRNA.EF-Tu’GTP bengal was purchased from Wako Pure Chemical in the final volume of 0.1 ml contained 2 pM EF-Tu, Industries. Deuterated chemicals, Na02H, *HCl, 1 pM rose bengal, 6 pM [14C]Phe-tRNA, 0.1 PM EF-Ts, 30 pM GTP and its regenerating system (0.6 mM and 99.75% 2H,0 were obtained from E. Merck. phosphoenolpyruvate and 1.5 pg/ml pyruvate kinase) Assays of EF-Tu. The [3H]GDP binding activities of EF-Tu’GDP and free EF-Tu were measured by in 50 mM Tris-HCl (pH 7.5), 8 mM magnesium acetate, the nitrocellulose membrane filter procedure (15). 5 mM 2-mereaptoethanol, and 150 mM NH&l. PhotoThe activity for promoting the binding of [14C]Phe- oxidation reaction was then started as described above and the binding activity of the ternary comtRNA to ribosomes was assayed as described before (3). Nuclear magnetic resonance measurements. Proton plex to ribosomes were measured for the aliquots nmr spectra of T. thermophilus EF-Tu were recorded withdrawn at appropriate intervals. For determination of the amino acid composition, on Bruker WH270 pulse FT spectrometer at 50°C. photooxidized EF-Tu was precipitated with 5% (v/v) Accumulation of as many as 1000-2000 free induction decays over 8192 data points was necessary for trichloroacetic acid and was hydrolyzed in constant l-l.5 mM EF-Tu solution. Convolution difference boiling HCl in a sealed tube at 110°C for 24 h. The technique (19) was used to obtain well-resolved spec- amino acid composition of the hydrolyzate was detertra, where accumulated free induction decays were mined with a Hitachi Model 835-50amino acid analyzer. multiplied by a function, e-*lif - 0.9t-‘O”‘. The solvent peak was suppressed by homo-gated decoupling. RESULTS AND DISCUSSION The pH values were measured at 50°C using Radiometer PHM26 pH meter equipped with a long, thin Well-resolved proton nmr spectra were combination electrode from Nisshin Rika (Catalog observed for T. themophilus EF-Tu, with
HISTIDINE
RESIDUES IN T. thewnophilus EF-Tu
the molecular weight of 49,000. Figure 1 shows the convolution difference spectra of EF-Tu. GDP in the low field region at several pHs. Especially in the region of CZ proton resonances of histidine residues (from 8.6 to 7.5 ppm), as many as 10 signals are resolved. It must be noted that relative intensities of signals do not correspond to the number of protons because of the convolution difference method and of the selective spin diffusion (23). Nontitratable signals at 7.9 and 6.0 ppm are assigned to C8 and Cl,, protons of free GDP, respectively. The pH titration curves were obtained for nucleotide-free EF-Tu, EF-Tu *GDP, and EF-Tu .GTP by plotting the chemical shifts of low field signals versus pH (Fig. 2). Here, filled symbols represent relatively well-defined titration curves for C, proton resonances of histidine residues. First, the effect of the interaction of GDP with EF-Tu was studied by comparing the nmr titration curves of free EF-Tu and EF-Tu*GDP. As shown in Figs. 2A and B, the titration curves with filled circles and triangles are affected little by the GDP binding. However, the titration curve with filled squares shows a drastic shift to lower field on interaction of EF-Tu with GDP. Since either protonation or deprotonation of imidazole rings cannot be completed in this pH range from 5.5 to 8.0, it is difficult to determine the accurate pK, values of histidine residues. Nevertheless, the apparent pK, of the titration curve with filled squares in Fig. 2A appears to be lower than 6.0 and that in Fig. 2B is higher than 7.0. This large change in pK, value is quite reasonable if the histidine residue is directly involved in the binding with phosphate group of nucleotides; the electrostatic interaction between the cationic imidazolium group and the anionic phosphate group increases the p K, of the imidazolium group. Similar changes in pK, values have been definitely observed for His 12 and His 119 of ribonuclease A (20, 24); the pK, values of these histidine residues are increased by l-2 pH unit on the binding with 3’-CMP inhibitor. It is, however, difficult to ascertain that the signals with filled squares in Figs. 2A and B are due to the same histidine residue of free EF-Tu and EF-Tu *GDP,
235
Cherrkal Shii (ppm) FIG. 1. Lowfield region of the convolution difference proton nmr spectra of T. therrnophilus EF-Tu.GDP at various pHs. Chemical shifts are given in parts per million (ppm) from sodium 2,2-dimethyl-2-silapentane5-sulfonate.
because the dissociation rate of E. eoli EF-Tu *GDP is as low as 3.4 x lop4 s-l (25) and accordingly the exchange of EF-Tu between the nucleotide-free state and the GDP-binding state is supposed to be extremely slow. Nevertheless, the relative intensities of C, proton signals of the histidine residue (tilled squares in Figs. 2A and B) were observed as a function of the added amount of GDP. As shown in Fig. 3, the signal intensity of the histidine proton of free EF-Tu (open circle) is lowered while the signal intensity of EF-TunGDP (filled circle) is raised by the addition of GDP, but there are no other prominent signals which are appreciably affected by the addition of GDP. This observation suggests that at least one histidine residue of EF-Tu is directly involved in the binding with GDP. Furthermore the effect of the ligand change from GDP to GTP was studied by
236
NAKANO ET AL.
comparing the pH titration curves in Figs. 2B and C. The titration curves with filled circles are little affected by the ligand substitution. However, the titration curve with filled squares is most significantly affected
8.6
and its apparent pK, is decreased by 0.3-0.4 pH unit upon the ligand substitution from GDP to GTP, again suggesting that this resonance is due to the histidine residue in the nucleotide binding site of EF-Tu. B 8.6
I
28.4. a
78.4. E
E 8.2. 5 $.E 8.0. Q, 6 7.8. 7.6
7.6 t
6
I
eI
7
6
PH
PH
;; 8.4. B
7.6. I
7
6 PH
FIG. 2. Proton nmr titration curves of EF-Tu in *H,O solution at 50°C. Samples in a volume of 0.2 ml commonly contained 0.1 M NaCl and 0.1 mM dithiothreitol. Filled symbols represent relatively well-defined titration curves. (A) Titration curves of nucleotide-free EF-Tu. The solution contained 1.5 mM EF-Tu. (B) Titration curves of EF-Tu.GDP. The solution contained 1.5 mM EF-Tu, 2.5 mM GDP, and 10 mM MgCl,. (C) Titration curves of EF-Tu.GTP. The sample solution was prepared from that of EF-Tu.GDP by converting GDP to GTP enzymatically. Phosphoenolpyruvate and pyruvate kinase were added to the solution of EF-Tu.GDP to final concentrations of 10 mM and 0.5 mg/ml, respectively, and then the solution was incubated at 50°C for 30 min.
HISTIDINE
RESIDUES IN 2’. thermophilus EF-Tu
In addition, the titration curve with filled triangle is also affected; its p K, is increased by about 0.2 pH unit upon the ligand change from GDP to GTP. This resonance may be due to another histidine residue in the nucleotide binding site or to a histidine residue in another site which is affected indirectly by the ligand substitution. As described above, several histidine residues are found near the cysteine residue essential for aminoacyl-tRNA binding of E. coli EF-Tu (16), so that the latter histidine residue of T. thermophilus EF-Tu is possibly located in the neighborhood of aminoacyl-tRNA binding site, where the microenvironment is affected by the ligand substitution from GDP to GTP. Photooxidation experiments on T. thermophilus EF-Tu were useful for elucidating the role of histidine residues in the activity of EF-Tu. The amino acid analyses on the photooxidized EF-Tu *GDP showed that only histidine residues were modified by photooxidation. As shown in Fig. 4, the GDP binding activity of EF-Tu was appreciably reduced upon photooxidation of nucleotide-free EF-Tu for 60 min (filled circle). However, if GDP was present during photooxidation of EF-Tu, the GDP binding activity of EF-Tu was much less affected (open circle). These observations
237
Time( min)
FIG. 4. Time courses of photooxidative inactivation of EF-Tu. EF-Tu was photooxidized for the times indicated in the form of either free EF-Tu (0, n ), EF-Tu. GDP (0, q ), or the ternary Phe-tRNA.EF-Tu.GTP (A), and the activities to bind GDP (0,O) or to promote Pbe-tRNA binding to ribosomes (Cl, W, A) were measured. For details, see under Experimental Procedures.
indicate that the GDP binding site is protected by the nucleotide from photooxidation, supporting the suggestion from nmr analyses that histidine residue(s) is possibly essential for the binding with guanine nucleotides. Furthermore, the possible role of histidine residues for the aminoacyl-tRNA binding was also studied. If free EF-Tu (filled square in Fig. 4) or EF-Tu . GDP (open square) was photooxidized in the absence of aminoacyl-tRNA, the activity of EF-Tu to promote the binding of aminoacyl-tRNA to ribosomes was lost much faster than the GDP binding activity. However, the former activity was little affected if photooxidation experiments were carried out in the presence of GTP and aminoacyltRNA (open triangle in Fig. 4), indicating that the binding site for aminoacyl-tRNA and/or ribosomes was protected from photoI I oxidation by the binding with aminoacyl0 0.5 1.5 1.0 tRNA. These observations suggest that GOP (mM) histidine residue(s) is also essential for FIG. 3. Relative intensities of the signals tentatively the binding with aminoacyl-tRNA and/or assigned to C, proton of the histidine residue in the ribosomes. GDP binding site of EF-Tu as a function of added In summary, the involvement of histidine GDP. The solution contained 1.2 mM EF-Tu in 0.1 M residue(s) in the binding of T. thermophilus NaCl and 0.1 mM dithiothreitol at pH 6.7, 50°C. RelaEF-Tu with guanine nucleotides was revealed tive intensities of the two signals at 7.6 (0) and 8.4 ppm (0) are represented as peak height ratios to the by nmr studies on histidine C, protons and invariant signal at 7.3 ppm. Arrows indicate the point was confirmed by photooxidation experiments. Furthermore, the presence of histiwhere EF-Tu:GDP = 1:l.
238
NAKANO
dine residue(s) in the binding site for aminoacyl-tRNA and/or ribosomes was suggested by photooxidation experiments. These histidine residues may play essential roles in the biochemical functions of T. thermophiZus EF-Tu. ACKNOWLEDGMENT We express onr gratitudes to Dr. T. Oshima, Mitsubishi Kasei Institute of Life Sciences, for his valuable advice and kind supply of Thermus thermophilus HB8, end to Dr. S. Kawashima, Faculty of Medicine, University of Tokyo, for the amino acid analysis of photooxidized EF-Tn. REFERENCES 1. MILLER, D. L., AND WEISSBACH, H. (1977) in Molecular Mechanism of Protein Biosynthesis (Weissbach, H., and Pestka, S., eds.), pp. 323-373, Academic Press, New York. 2. KAZIRO, Y. (1978) Biochim. Biophys. Acta 505, 95- 127. 3. ARAI, K., KAWAKITA, M., KAZIRO, Y., MAEDA, T., AND OHNISHI, S. (1974) J. Biol. Chem. 249, 3311-3313. 4. ARAI, K., MAEDA, T., KAWAKITA, M., OHNISHI, S., AND KAZIRO, Y. (1976)J. Biochem. 80,1047-1055. 5. WILSON, G. E., COHN, M., AND MILLER, D. (1978) J. Biol. Chem. 253, 5764-5768. 6. CRANE, L. J., AND MILLER, D. L. (1974) Biochemistry 13, 933-939. 7. &AI, K., AELAI, T., GWAKITA, M., AND KAZIRO, Y. (1975) J. Biochem. 77, 1095-1106. 8. ARAI, K., ARAI, T., KAWAKITA, M., AND KAZlRO, Y. (1977) J. Biochem. 81, 1335-1346. 9. OHTA, S., NAKANISHI, M., TSUBOI, M., ARAI, K., AND KAZIRO, Y. (1977) Eur. J. Biochem. 78, 599-608.
ET AL.
10. K=~BSCH, W., GAS?W. H., S~.JLTZ,G. E., AND 11.
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LEBERMAN,I, R. (1977) J. Mol. Biol. 117, 9991012. MORIKAWA, K., L, LA COUR, T. F. M., NYBORG, J., RASMUSSEN, IV, K. M., MILLER, D. L., AND CLARK, B. F. C. (1978) J. Mol. Biol. 125, 325-338. ARAI, K., OTA, Y., ARAI, N., NAKAMURA, S., HENNEKE, C., OSHIMA, T., AND KAZIRO, Y. (1978) Eur. J. Biochem. 92, 509-520. ARAI, K., ARAI, N., NAKAMURA, S., OSHIMA, T., AND KAZIRO, Y. (1978) Eur. J. Biochem. 92, 521-532. NAKAMURA, S., OHTA, S., ARAI, K., ARAI, N., CSHIMA, T., KAZIRO, Y. (1978) Eur. J. Biochem. 92, 533-543. ARAI, K., KAWAKITA, M., AND KAZIRO, Y. (1972) J. Biol. Chem. 247, 7029-7037. NAKAMURA, S., ARAI, K., TAKAHASHI, K., AND KAZIRO, Y. (1975) Biochem. Biophys. Res. Commun. 66, 1069-1077. ARAI, K., KAWAKITA, M., AND KAZIRO, Y. (1974) J. Biochem. 76, 283-292. KAZIRO, Y., INOUE-YOKOSAWA, N., AND KAWAKITA, M. (1972) J. Biochem. 72, 853-863. CAMPBELL, I. D., DoB~~N, C. M., WILLIAMS, R. J. P., AND XAVIER, A. V. (1973) J. Mugn. Resonance 11, 172-181. MARKLEY, J. L. (1975) Biochemistry 14, 35463554. ARAI, K., KAWAKITA, M., KAZIRO, Y., KONW, T., AND UI, N. (1973) J. Biochem. 73, 1095-1105. TAKAHASHI, K. (1970) J. Biochem. 67, 833-839. AKASAKA, K., KONRAD, M., AND GOODY, R. S. (1978) FEBS Lett. 96, 287-290. GORENSTEIN, D. G., WYRWICZ, A. M., AND BODE, J. (1976) J. Amer. Chem. Sot. 98,23082314. ARAI, K., KAWAKITA, M., AND KAZIRO, Y. (1974) J. Biochem. 76, 293-306.