E.R.Kellenbach, M.L.Remerowski, D.Eib, R.Boelens, G.A.van der Marel', H.van den Elst1. J.H.van Boom1 and R.Kaptein*. Department of Chemistry, University ...
Nucleic Acids Research, Vol. 20, No. 4 653 -657
Synthesis of isotope labeled oligonucleotides and their use in an NMR study of a protein-DNA complex E.R.Kellenbach, M.L.Remerowski, D.Eib, R.Boelens, G.A.van der Marel', H.van den Elst1 J.H.van Boom1 and R.Kaptein* Department of Chemistry, University of Utrecht, Padualaan 8, 3584 CH Utrecht and 1Gorlaeus Laboratory, University of Leiden PO Box 9502, 2300 RA Leiden, The Netherlands Received January 3, 1992; Revised and Accepted January 28, 1992
ABSTRACT The synthesis of an oligonucleotide labeled with 13C at the thymine methyls and 15N at the exocyclic amino groups of the cytosines is described. 13CH31 and 15NH40H were used as sources of the labels. The labeled oligonucleotide was characterized by several NMR techniques. The duplex possesses a labeled functional group in the major groove at every base pair which makes it a very suitable probe for the study of sequence-specific protein-DNA interaction. The labeled thymine methyl group facilitates the detection of hydrophobic contacts with aliphatic side-chains of proteins. This is demonstrated in an NMR study of a complex between the glucocorticoid receptor DNAbinding domain and the labeled oligomer, which revealed a hydrophobic contact between a thymine methyl group and the methyl groups of a valine residue. There are indications for small differences between the solution structure the X-ray structure of the complex. INTRODUCTION Hydrogen bonding and hydrophobic contacts between the functional groups of the DNA major groove and protein sidechains are major determinants in the sequence-specific recognition of DNA by proteins (1). The bulky thymine methyl group plays a pivotal role in the hydrophobic interaction with amino acid sidechains (2), as has been documented for various proteins (3,4). Deletion of this methyl group through substitution by deoxyuridine in synthetic operators results in a dramatic decrease of the affinity of several DNA-binding proteins for these operators (5,6). In 'H-NMR spectra of protein-DNA complexes it is difficult to observe cross-peaks between the thymine methyl group and aliphatic protein side-chains due to severe overlap of resonances in a crowded part of the spectrum. In addition, these peaks can be obscured by their proximity to the diagonal. Isotope labeling combined with heteronuclear editing techniques will help to overcome these problems (7). To our knowledge, the synthesis of 13C-methyl-thymidine has not been reported before. We have *
To whom correspondence should be addressed
labeled thymidine with carbon-13 at the thymine methyl using relatively inexpensive 13CH31. The labeled nucleoside was subsequently incorporated into a DNA fragment. An additional benefit of carbon-13 labeling of DNA is that isotope edited NMR spectra can be obtained in D20 while, in contrast, heteronuclear filtered NMR spectra using nitrogen-15 are recorded in H20 because detection is usually achieved through the exchangeable protons. It was shown recently that thymine methyl groups play a crucial role in the sequence specific recognition of DNA by steroid hormone receptors (8). As part of our program aimed at understanding sequence-specific DNA recognition of the glucocorticoid receptor (9-12), we have applied this method to the synthesis of the stronger binding half-site of the consensus glucocorticoid response element (GRE, 13) with two additional GC base pairs at the termini to increase the stability of the duplex: 5'd(GC*T3G4*TS*T67*T8GC)3' *5'd(GCA_8GG7A_6A-_C_4A3GC)3' The sequence of the half-site GRE is in bold letters. Numbering of the oligonucleotide is according to Luisi et al. (14). In addition to the labeling of the thymidine methylgroup, nitrogen-15 labeling of the exocyclic cytosine amino groups in both strands of the GRE was achieved using a method described previously (11). This method involves the use of a 4-triazolo deoxyuridine building block. After completion of the synthesis, the triazolo group is displaced by 15N labeled ammonia to yield 4-15N-cytosine during the deprotection of the oligonucleotide by treatment with 6N 15NH40H. The labeled thymidines are indicated by an asterisk while the labeled cytosines have been underlined. Although it was recently demonstrated that nitrogen-15 resonances of the imino nitrogens in DNA can be detected at natural abundance using proton detected heteronuclear correlation experiments (15-17), efforts to observe the amino nitrogen resonances with this technique have not been successful. This stresses the need for labeling the cytosine amino group. Moreover, these groups are exposed in the major groove and are available for hydrogen-bonding. The imino protons, on the other hand, are located in the centre of the double helix and involved in Watson -Crick base pairing and are, therefore, not
654 Nucleic Acids Research, Vol. 20, No. 4
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MATERIALS AND METHODS Methyl-'3C-thymidine was synthesized from 5-bromodeoxyuridine (1) as outlined in Scheme I (DMT stands for dimethoxytrityl, DHP for dihydropyranyl, THP for tetrahydropyranyl, THF for tetrahydrofuran, CNE for cyanoethyl and R for diisopropyl) by a method used previously for " C and 14C incorporation (18-22). The hydroxyl groups of 1 were protected (21) by converting them to their tetrahydropyranyl (THP) derivatives (2). The 5 position and the imino nitrogen were metallated by treatment with butyllithium in dry tetrahydrofuran to give 3. Subsequently, 3 was methylated at the 5 position using 13CH3I 99% 13C (Isotec Inc.). The reaction was quenched by adding a methanol/water mixture and the THP groups removed with acid (80% AcOH/H20, 17 h) yielding 4. The reaction mixture was concentrated under reduced pressure and the residue was dissolved in water, extracted with dichloromethane, and concentrated. The crude product was purified to remove the main
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Figure 2: 'H-13C HMQC spectra in D20 of the free GRE (A) and the GRE complexed with the GR-DBD (B).
impurity, deoxyuridine, by preparative reverse phase FPLC on Pharmacia Prp RPC-HR16/10 column using 10% methanol/water as the eluant to yield crystalline '3C labeled thymidine (4). IH NMR showed more than 98% isotope incorporation. Overall yield was 35% based on 13CH3I. The labeled thymidine 4 Was subsequently converted to its a
Nucleic Acids Research, Vol. 20, No. 4 655 LO
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Figure 3: Slices perpendicular to f, -axis of a 3D HMQC-NOE spectrum at the methyl carbon-13 chemical shift frequency of thymine 6 (A), thymine 5 (B), thymine 8 (C) and thymine 3 (D).
dimethoxytrityl amidite derivative 5 and used in a conventional solid phase DNA synthesis (23,24) towards the DNA fragment 5'd(GC*TG*T*TC*TGC)3' (Pharmacia Gene Assembler at a 10 ltmol scale; the labeled thymidines are indicated by an asterisk). The oligomer was purified by ion-exchange FPLC (Pharmacia Hiload 16/10 Q-Sepharose High Performance column) and gel filtration (Pharmacia Hiload 16/120 Sephadex 75 prep grade column) to yield a homogeneous fragment (8.5 mg). The complimentary strand 5'd(GCAGAACAGC)3' was synthesized and purified in the same way with a yield of 18.3 mg. Nitrogen-15 labeling of the exocyclic amino groups of the cytosines in both strands was achieved as reported previously (1 1). The labeled cytosines have been underlined. The cytosine amino groups of the additional GC base pairs at the 3' and 5' termini were not labeled because they are not part of the GRE. The strands were mixed 1:1 to obtain a duplex and purified from any residual single stranded material by gel filtration, lyophilized and dissolved in an appropriate buffer for the NMR experiments (see below). The DNA-binding domain of the glucocorticoid receptor (GRDBD) was expressed and purified as described before (25) except that an AccelCM ion-exchange column instead of a phosphocellulose column was used in the purification. The complex between the half-GRE and the GR-DBD was prepared by adding 0.4 ml of a 2 mM solution of the protein to 0.4 ml of a 2 mM solution of the DNA. During the mixing of the protein and the DNA of the sample some precipitation occurred which was removed by centrifugation. This was followed by concentration of the complex on an AmiconT flow cell to
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reduce the volume of the sample to 0.4 ml. Final concentration of the sample was about 1 mM. All NMR spectra were obtained on a Bruker AMX-500 spectrometer using a 5 mm broadband heteronuclear reverse probe. NMR experiments were performed at 300 K, pH 6.5 in 99.5% D20 or 95% H20/ 5% D20 containing 50 mM sodium phosphate buffer, 200 mM KCl and 0.1 mM NaN3. In the case of protein-DNA complexes, the sodium phosphate concentration was 10 mM and 0.1 mM DTE was added to the samples to prevent oxidation of the cysteines. 2D 'H-13C HMQC (26, 27) and 3D HMQC-NOE spectra (28) were recorded using standard pulse sequences. For the 'H-13C HMQC experiments, 32 increments of 32 scans were collected resulting in a total measuring time of about 20 minutes.
656 Nucleic Acids Research, Vol. 20, No. 4 The IH-15N HMQC experiment was recorded in about 1 hour with 128 increments and 32 scans. The IH-13C HMQC-NOE was recorded with 16 increments in the w,, (HMQC) direction and 160 increments in the c2 (NOE) direction 48 transients of 1024 data points were collected for each increment in a total measuring time of 48 hours. Spectra were processed using the 'TRITON' NMR software package developed at Utrecht University. Chemical shifts are reported in ppm and were calculated relative to DSS for IH and 13C and to liquid NH3 for '5N.
RESULTS AND DISCUSSION Thymidine was 13C labeled at the tfiymine methyl group in three steps starting from 5-bromo-deoxyuridine using 13CH31 as the source of the label in a 35 % yield based on '3CH31 (Scheme I). The labeled thymidine was subsequently incorporated into a halfGRE in which the cytosine amino groups were nitrogen-15 labeled by a previously reported method (11). Isotope incorporation of the duplex was corroborated by proton detected heteronuclear correlation spectroscopy (26,27). In Figure 1, the IH-15N HMQC spectrum of the labeled GRE in H20 is shown which correlates the cytosine amino nitrogen frequencies with the amino proton frequencies. The small spectral width and, consequently, high resolution in the f, direction causes the homonuclear proton-proton coupling to be visible as fine-structure in the cross-peaks in this direction (27,29)). The amino protons were assigned from a homonuclear 2D NOE experiment in water (30). Figure 2A shows the 'H-13C HMQC spectrum of the DNA fragment in D20 displaying the correlation between the IH and 13C chemical shifts of the thymine methyl groups in the GRE. The assignments were obtained from a homonuclear 2D NOE experiment in D20 (31). Although several groups have synthesized isotopically labeled (oligo)nucleotides for NMR studies of ligand-DNA interaction (32 -38), there are no reports yet on their actual application in these studies. To demonstrate the potential of the labeled oligonucleotide in NMR studies of protein DNA interaction, we prepared a 1:1 complex of the half-GRE with the glucocorticoid receptor DNA-binding domain (GR-DBD). Figure 2B shows the 13C-IH HMQC spectrum of the labeled GRE complexed with the GR-DBD. The methyl groups of thymines 3 and 5 show the largest changes in chemical shift, while for thymines 6 and 8 they are smaller. This agrees well with biochemical data on the role of the thymines in the complex, which indicate that the methyls of thymines 3 and 5 make important contributions to the free energy of binding of the intact glucocorticoid receptor to the GRE, while the methyls of thymine 6 and 8 are not contacted by the protein (8). It is interesting to note that although there is a relatively large change in carbon-13 chemical shift of the thymine 5 methyl, the proton chemical shift is virtually unchanged. Besides monitoring of carbon-13 chemical shifts, the carbon-13 label allows selective detection of crosspeaks originating from the thymine methyl groups. Figure 3 shows four NOE planes of a 3D 13C-'H HMQC-NOE spectrum (28) in D20 of the complex at the carbon-13 chemical shift of the four different methyl groups in the oligonucleotide. In the plane of thymine 5 (Figure 4), an NOE cross-peak between the methyl group of thymine 5 and the fy methyl groups of V462 could be detected. Due to its proximity to the diagonal this crosspeak is much more easily observed in the 13C edited spectrum than in the nonnal 2D NOE spectrum. From interference methods
and base-substitution experiments Truss et al. (8) concluded that the methyl group of thymine 5 is contacted by the GR. V462 belongs to the three amino acid residues that discriminate between GRE's and ERE's, while thymine 5 belongs to the two base pairs that distinguish the GRE from the ERE (39-41). Thus, the hydrophobic contact between V462 and thymine 5 could play an important role in the discrimination between GRE's and ERE's. A more complete analysis of the complex between the GR-DBD and the half-GRE has been presented elsewhere (42). Recently, the X-ray structure of the complex between the GRDBD and DNA was published (14). The contact between V462 and thymine 5 described here is also observed in the crystal structure of the complex. However, in contrast to the crystal structure, where only one of the two V462 methyl groups contacts the thymine 5 methyl, the equal intensity of the cross-peaks in figure 4 suggests that in solution both methyls contact thymine 5. This difference between the solution and crystal structure may be due to the fact that for the crystal structure determination an oligonucleotide with a four base-pair instead of the natural three base-pair spacing was used, resulting in a dimeric complex in which only one of the monomers is able to bind specifically to the DNA (14). In conclusion, the efficient stable isotope labeling of major groove functional groups of DNA described here, in combination with sensitive detection and editing methods, will simplify the analysis of NMR spectra of protein-DNA complexes and thus provide more insight into the mechanisms underlying the sequence specificity of protein-DNA recognition.
ACKNOWLEDGEMENT This work was supported by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for the Advancement of Pure Research (ZWO). M.L.R. was supported by a NSF-NATO post-doctoral fellowship award # RCD-9050102. We would like to thank Dr. K.Y.Yamamoto for the gift of the E. coli strain carrying the plasmid containing the cDNA for the GR-DBD.
REFERENCES 1. Steitz, T.A. (1990) Q. Rev. Biophys. 3, 205-280. 2. Ivarie, R. (1987) Nucleic Acids Res. 15, 9975-9983. 3. Takeda, Y., Sarai, A. and Rivera, V. M.(1989) Proc. Natl. Acad. Sci. USA 86,439-443. 4. Li, R., Knight, J., Bream, G., Stenlund, A.and Botchan, M. (1989) Genes Dev. 3, 510-526. 5. Caruthers, M. (1980) Acc. Chem. Res. 13, 155-160. 6. Dubendorff, J. W., deHaseth, P. L., Rosendahl, M. S., and Caruthers, M. H.(1987)J. Biol. Chem. 262, 892-898. 7. Otting, G., Wothrich, K. (1990) Q. Rev. Biophys. 23, 39-96. 8. Truss, M., Chalepakis, G., and Beato, M. (1990) Proc. Natl. Acad. Sci. USA 87, 7180-7184 9. Hiird, T., Kellenbach, E. , Boelens, R., Maler, B. A., Dahlman, K., Freedman, L. P., Carlstedt-Duke, J. , Yamamoto, K. R. , Gustafsson J.-A. and Kaptein, R. (1990a) Science 249, 157-160. 10. Hiird, T. , Kellenbach, E. , Boelens, R., Dahlman, K., Carlstedt-Duke, J., Freedman, L. P.Maler, B. A., Hyde, E. I., Gustafsson, J-A, Yamamoto, K. R., and Kaptein, R. (1990b) Biochemistry 29, 9015-9023. 11. Kellenbach, E. R., van den Elst, H, Boelens, R., van der Marel, G. A., van Boom, J. H., and Kaptein, R.(1991a) submitted for publication. 12. Kellenbach, E., H&d, T., Boelens, R., Dahlman, K., Carlstedt-Duke, J., Gustaffson, J.-A ., van der Marel, G. A., van Boom, J. H., Maler, B., Yamamoto, K. R., and Kaptein, R. (199lb) J. Biomol. NMR 1, 105-110. 13. Tsai, S.-Y., Carlstedt-Duke, J., Wiegel, N. L., Dahlman, K., Gustaffson, J.-A., Tsai, M. J., and O'Malley, B. W. (1988) Cell 55, 361-369.
Nucleic Acids Research, Vol. 20, No. 4 657 14. Luisi, B. F., Xu, W. X., Otwinowski, Z., Freedman, L. P., Yamamoto, K. R., and Sigler, P. B.(1991) Nature 352, 497-505. 15. Ashcroft, J., Live, D. H., Patel, D. J., and Cowbum, D., (1991) Biopolymers 31, 45-55. 16. Live, D. H. (1990) in Frontiers of NMR in Molecular Biology, UCLA Symposium Series in Molecular and Cellular Biology, New Series, Vol. 109 (Live, D. H., Armitage, I., and Patel, D.J., Eds) Wiley-Liss, New York, pp 259-270. 17. Live, D. H., Radhakrishnan, I., Misra, V., and Patel, D. J. (1991) J. Am. Chem. Soc 113, 4687-4688. 18. Pichat, L., Masse, B., Deschamps, J., and Duray, Ph. (1971) Bull. Soc. Chim. Franc. 6, 2102-2109. 19. Sundoro-Wu, B. M., Schmall, B., Conti, P. S., Dahl, J. B., Drumm, P., and Jacobsen, J. K. (1984) Int. J. Appl. Radiat. Isot. 35, 705-708. 20. Labar, D., and Cogneau M.(1987) Nucl. Med 26, 196. 21. Martiat, Ph., Ferrant, A., Labar, D., Cogneau, M., Bol, A., Michel, C., Michaux, J. L., and Sokal, G. (1988) J. Nucl. Med. 29, 1633-1637. 22. Poupeye, E., Counsell, R. E., De Leenheer, A., Slegters, G., and Goethals, P.(1989) Appl. Radiat. Isot. 40, 57-61. 23. Matteucci, M. D., and Caruthers, M. H. (1981) J. Am. Chem. Soc. 103, 3185-3191. 24. Engels, J. W., and Uhlman, E. (1989) Angew. Chem. Int. Ed. Engl. 28, 716. 25. Freedman, L. P., Luisi, B. F., Korszun, Z. R., Basavappa, B., Sigler, P. B., and Yamamoto, K. R. (1988) Nature 334, 543-546. 26. Bax, A., Soarks, S. W., and Torchia, D. A. (1989) in Methods in Enz)ynology 176, (Oppenheimer, N. J., James, T.L., Eds), Academic Press, New York., pp 134-151. 27. Bax, A., Ikura, M., Kay, L., Torchia, D. A., Tschudin, R. (1990) J. Magn. Reson.86, 304-318. 28. Fesik, S. W. and Zuiderweg, E. R. P. (1988) J. Magn. Reson. 78, 588-595. 29. Norwood, T. J., Boyd, J., Heritage, J. E., Soffe, N., and Campbell, I. D. (1990) J. Magn. Reson. 87, 488-501. 30. Boelens, R., Scheek, R. M. , Dijkstra, K. and Kaptein, R. (1985) J. Magn. Reson. 62, 378-386. 31. Scheek, R. M., Russo, N., Boelens, R., Kaptein, R., and van Boom, J. H. (1983) J. Am. Chem. Soc. 105, 2914-2916. 32. Gao, X., and Jones, R.A.. (1987) J. Am. Chem. Soc. 109, 1275-1278. 33. Kupferschmidt, G., Schmidt, J., Schmidt, Th., Fera, B., Buck, F., Ruterjans, H. (1987) Nucleic. Acids Res. 15, 6225-6238. 34. Fera, B., Singrun, B., Kupferschmidtt, G., Schmidt, J., Buck, F., and Ruterjans, H. (1987) Nucleosides and Nucleotides 6, 477-481. 35. Massefski, W., Redfield, A., Sarma, U. D., Bannerji, A., Roy S. (1990) J. Am. Chem .Soc. 112, 5350-5351. 36. Gaffney, B.L., Kung, P.-P., and Jones, R. A. (1990) J. Am. Chem. Soc. 112, 6748-6749. 37. Rhee, Y.-S., and Jones, R. A. (1990) J. Am. Chem. Soc. 112, 8174-8175 38. Goswami, B., and Jones, R. A.(1991) J. Am. Chem. Soc. 113, 644-647. 39. Mader, S., Kumar, V., de Verneuil, H and Chambon, P. (1989) Nature, 338, 271-274. 40. Umesono, K., and Evans, R. M. (1989) Cell, 57 1139-1146. 41. Danielsen, M., Hinck, L., and Ringold, G. M. (1989) Cell 57, 1131-1138. 42. Remerowski, M. L., Kellenbach, E. R., Boelens, R., van der Marel, G. A., van Boom, J. H., Maler, B. A., Yamamoto K. R., and Kaptein, R. (1991) Biochemistry 30, 11620-11624. Figure Captions
