Molecular Modeling of Nucleic Acids - American Chemical Society

Chapter 12

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on March 30, 2016 | http://pubs.acs.org Publication Date: November 26, 1997 | doi: 10.1021/bk-1998-0682.ch012

NMR Studies of the Binding of an SPXX-Containing Peptide from High-Molecular-Weight Basic Nuclear Proteins to an A-T Rich DNA Hairpin Ning Zhou and Hans J. Vogel Department of Biological Sciences, Universityof Calgary, Calgary, Alberta T2N 1N4, Canada

The amino acid sequence SPXX in nuclear proteins has been identified as a motif that recognizes the minor groove of A-T rich DNA. Phosphorylation of the serine residue by proline-directed kinases diminishes its DNA binding capacity. To learn more about this abundant protein-DNA interaction motif, we have studied the synthetic 14-mer peptide MRSRSPSRSKSPMR (derived from sperm chromatin of winter flounder), and its binding to a d(T6C4A6) DNA hairpin by one dimensional NMR titration experiments and two dimensional NOESYand TOCSY NMR experiments. Our results show that the first equivalent of the peptide binds to the hairpin loop, while the second equivalent interacts with the minor groove of the hairpin stem with an extended structure. These observations suggest that binding of SPXX motifs to DNA loops or single-sranded regions may be significant.

In studies of protein-DNA interactions, it has long been recognized that contact of the protein with the D N A bases is important in providing sequence discrimination. Bases located in the major groove show a greater degree of sequence dependence in terms of their hydrogen-bonding and van der Waals interaction patterns than those in the minor groove. Therefore it is not surprising that most sequence specific DNA-binding proteins characterized thus far make contact with their target D N A in the major groove . Minor groove binding proteins identified earlier, such as the nucleolins and the H U family proteins , appear to lack a high degree of sequence specificity in their binding to D N A and show only some preference for A.T rich or G.C rich sites. The protein motifs used for binding to the minor groove are still not well characterized. Putative α-helical motifs and proline-rich motifs have been proposed (for a review, see 4). 1

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© 1998 American Chemical Society

Leontis and SantaLucia; Molecular Modeling of Nucleic Acids ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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Several recent developments have, however, expanded and changed our notions about minor groove binding. For example, the TATA-box sequences in promoter regions are recognized by the TATA-box binding protein (TBP); its binding is the first step in transcription initiation. The structure of T B P ' and its complexes with the 8basepair TATA-box sequences ' were solved by X-ray crystallography. TBP utilizes an antiparallel β-sheet structural element to contact the minor groove of the T A T A sequence, distorting the B - D N A structure considerably. Specific hydrogen bonding, as well as hydrophilic and hydrophobic interactions between the T B P protein and bases in the minor groove were identified. TBP represents the first example of an antiparallel β-sheet motif, which binds to the minor groove to confer sequence specificity. More recently the structures of the DNA-binding H M G (high-mobilitygroup) domain of mouse LEF-1 and human SRY, complexed with their respective cognate D N A , were determined by N M R spectroscopy ' . The H M G domain of these two proteins utilizes a concave surface formed mainly by three α-helices to bind in the D N A minor groove; a large number of specific interactions between hydrophobic amino acid sidechains on the surface and the D N A bases were identified. The binding also causes large scale kinking of D N A . Along a separate line of research, Dervan, Wemmer and their co-workers have succeeded in designing minor groove binding peptides that could not only distinguish G.C or C.G from A.T basepairs but also were able to discriminate a G.C basepair from a C.G basepair ' . These peptides contain imidazole and pyrrole rings. Hydrogen bonding between the imidazole nitrogens of the peptides and guanine amino groups in the D N A minor groove were shown to be critical in determining this specificity. This work has set the stage for the design of other minor groove binding molecules to target specific base sequences. Another interesting group of minor groove binding ligands is comprised of carbohydrate-based c o m p o u n d s . These compounds display a different kind of specificity for oligopyrimidine sequences. The progress in these unrelated projects pointed to the diverse ways in which DNA-protein binding specificity could be achieved. It also highlighted the importance of sequence dependent D N A structural features, such as groove width and backbone flexibility, in conferring binding specificity, especially for proteins recognizing the minor groove of B-DNA. 5

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The amino acid sequence S P X X has been proposed by Suzuki and others ' as a minor groove binding motif that recognizes A.T rich D N A segments. In this sequence X often represents a basic amino acid Κ or R. The S P X X sequence was identified primarily through footprinting experiments and competition experiments with the minor groove binding drug Hoechst 33258. The high frequency with which this sequence is found in chromosomal proteins and other regulatory D N A binding proteins seems to support a function in D N A binding. It was proposed on the basis of N M R experiments that two joint S P X X motifs formed a crescent-shaped structure that binds into the narrow minor groove of A.T rich D N A in a way similar to the drug netropsin . Phosphorylation of the serine residue in S P X X was shown to diminish its D N A binding , therefore serine phosphorylation by proline-directed protein kinases in this motif can serve as a modulation of D N A binding. A study on an octamer (SPRK)2, however, found that it did not bind specifically to A.T rich D N A . In order to further investigate the interaction of the proposed S P X X motif with DNA, we have studied the interaction of a proline-rich, serine-rich 14-mer peptide (MRSRSPSRSKSPMR), with an A-T rich D N A hairpin. This peptide corresponds to a repeated sequence found in the high molecular weight basic nuclear proteins that are present in the sperm chromatin of winter flounder ; it contains two S P X X sequences. The oligo-DNA used in this study is d(T6C4A6). A D N A hairpin was chosen, because of its greater thermodynamic stability for the relatively short basepaired oligonucleotide. The interaction between the peptide and the D N A was studied by ^H, 17

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Binding of an SPXX-Containing Peptide

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P and C N M R spectroscopy. Our work led to the surprising conclusion that the peptide binds with a higher affinity to the hairpin loop than to the minor groove in the A.T stem.


Experimental. The peptide was synthesized by the Peptide Synthesis Facility at Queens University. The D N A was provided by the Nucleic Acid Synthesis Facility at the University of Calgary. The peptide MRSRSPSRSKSPMR was dissolved in aqueous (90%/10% H 2 O / D 2 O ) solution to a final concentration of 5 mM, the pH of the solution was adjusted to 6.5. The oligo-nucleotide d(T6C4A6) was dissolved in aqueous (90%/10% H 2 O / D 2 O ) solution with lOOmM phosphate buffer (pH 6.5) and 150mM KC1 to a concentration of 2.5mM. Aliquots of the peptide stock solution were added to the d(T6C4A6) solution for the titration experiments. N M R experiments were carried out at 15°C on a Bruker AMX-500 spectrometer equipped with a 5 mm inverse detection probe and an X-32 computer. A l l ID spectra were recorded with a 5500 Hz spectral width and 8k data points. The water resonance was suppressed either by a pre-saturation irradiation or by using a tailored jump-return excitation pulse . Phase-sensitive detection in the t l dimension of 2D experiments was achieved using the time-proportional phase incremental scheme . For natural abundance ^ H , l ^ C correlation experiments, the 2D H M Q C pulse sequence as described by Bax et a l . was used. A proton spectral width of 5000 Hz and a carbon-13 spectral width of 25000 Hz were used, with 2K X 512 data points in the t2 and t l dimensions respectively. ^H, 31p correlation experiments were obtained using a hetero-TOCSY sequence , with an isotropic mixing time of 67 ms. The spectral width was set to 4000 Hz for protons and 607 Hz for phosphorus, with typically 2K X 256 data points in the two dimensions respectively. The proton 2D DQF-COSY and TOCSY experiments were recorded with standard pulse sequences; the data size was 2K X 512, with spectral width of 5500 Hz in both dimensions. The N O E S Y sequence with a jump-return excitation pulse was used for optimal imino proton detection . Mixing times from 100 to 300 ms were used. The spectral widths were 11000 Hz in both dimensions. 2D data processing consisted of zero-filling once and apodization by a 90°-shifted sine-square-bell function in both dimensions before Fourier transformation. Proton and carbon-13 chemical shifts are referenced to TSP as an internal standard. Phosphorus31 chemical shifts are referenced to 85% phosphoric acid as an external standard. 21

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Results and Discussion. Our first task was to characterize the NMR spectra of the oligo-nucleotide d(T6C4A6); this was accomplished by recording NOESY and TOCSY experiments. Below 30°C in aqueous solution (pH 6.5), it forms a hairpin structure with a stem consisting of T6.A6 and a loop formed by the four C residues. The NOEs observed between neighboring A H2 protons (Fig. 1A), between the Τ imino protons and the A H2 protons (Fig. IB), and between neighboring Τ imino protons (Fig. 1C) indicate that Watson-Crick A.T basepairs were formed holding the T6.A6 stem together. However, in addition, crossstrand NOEs between the Τ Η Γ protons and the A H2 protons were detected (see Fig.l, Scheme). These cross-strand NOEs are characteristic of oligo-(dA).oligo-(dT) sequences; they are particularly strong when the A.T basepairs have a high propeller twist ' , and have been linked to D N A bending and bendability ' . The N O E patterns observed for the d(T6C4A6) molecule resemble those reported for a longer homologous hairpin [d(T8C4A8)] which we have characterized in a previous study . Further, the first C residue in the loop had an abnormal proton chemical shift profile 25

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Figure 1. Contour plots of a N O E S Y experiment for the d(T6C4A6) hairpin in 90%/10% H 2 O / D 2 O solution. The d(T6C4A6) hairpin sequence and the numbering of the residues are shown in the scheme. The bars connecting the Τ and the A residues in the scheme represent observed cross-strand NOEs between the Τ Hl'and the A H 2 protons. (A) The region showing the sequential NOE crosspeaks between the H 2 protons of neighboring A residues (labeled with the two residue numbers involved). (B) The region for the cross peaks between the A H 2 and the Τ imino protons. The crosspeaks are labeled with the Τ residue numbers involved. (C) The region for the Τ imino protons. The crosspeaks between neighboring Τ imino protons are labeled with the two corresponding residue numbers.


12. ZHOU & VOGEL


during thermal melting (data not shown), which is again similar to that of the corresponding C residue in the longer hairpin. We have previously modeled the structure of the d(T8C4A8) hairpin using our 2D N M R data and restrained molecular dynamics simulation . The stem of this hairpin is bent with a slightly narrower minor-groove compared to a standard B-DNA conformation, and the first C residue in the loop is not stacked but exposed to the solvent. The present measurements on the shorter d(T6C4A6) hairpin demonstrate that it has a similar structure as the longer d(T8C4A8) hairpin. No regular secondary structure can be detected through 2D N M R experiments for the 14-mer peptide. The N O E S Y experiments showed few crosspeaks (data not shown) which is characteristic of a flexible peptide backbone. Upon addition of the peptide to the d(T6C4A6) sample, the peptide amide signals appear at shifted resonance positions and with increased linewidths, indicating that binding takes place (Fig. 2). Addition of aliquots of the peptide sample up to one equivalent to the D N A sample causes no significant changes of the imino proton signals; surprisingly only further additions of peptide causes a noticeable change in the chemical shifts of these Τ imino protons (Fig. 3). The hairpin stem T-imino protons are involved in the basepairing of the stem; their N M R signals are sensitive to ligand binding in the D N A grooves . To further characterize the changes that occurred in the 1:1 peptide-DNA sample, the chemical shifts of the D N A base aromatic protons, aromatic and deoxyribose carbons and phosphorus were monitored and compared with that of the hairpin. The 31p chemical shift changes (Fig. 3A) for the 1:1 peptide:DNA sample are only observed for the loop region and for the two A residues closest to the loop. The changes in the chemical shifts of the aromatic l ^ C give a similar profile (Fig. 3A), here only the aromatic C6 of the four loop C residues experienced chemical shift changes. In addition, the deoxyribose C T of the two A residues close to the loop experienced small chemical shift changes as well, also the C T resonances of the two A residues at the end of the hairpin changed; but the loop C C T resonances did not (Fig. 3A). The chemical shift changes for the aromatic proton are plotted in Figure 3B. The loop C H6 as well as the A H8 protons in the 1:1 peptide-DNA sample undergo some changes; while the Τ H6 and the A H2 proton chemical shifts remained almost identical. These results indicate that up to a 1:1 ratio, the peptide interacts primarily with the loop region of the hairpin. At peptide to D N A ratios higher than 1, both A H2 and Τ imino protons showed significant chemical shift changes, while changes in the loop region were minimal (Fig. 3B and 2C). Since all of the A H2 protons are situated in the minor groove of the stem, these changes suggest that at higher than 1:1 peptide to D N A ratios, binding of the peptide to the minor groove of the stem takes place. Except for all the amide proton signals of the peptide, which show chemical shift changes in the peptide/DNA samples, most of the non-solvent-exchangeable proton signals do not display significant chemical shift changes in these samples. The few that had been identified include the N-terminal Met α-proton and several Arg δ protons. Since it is likely that the peptide undergoes exchange among different bound and free forms, its proton chemical shift do not give a direct indication of the conformation of the bound peptide. However, in NOESY experiments recorded for the 2:1 peptide-hairpin sample, a significantly increased number of crosspeaks involving the peptide protons were detected (in comparison with a corresponding experiment for the sample of the isolated peptide in aqueous solution). This resulted from an increase of the correlation time of the peptide in the bound form(s). A n analysis of the NOESY patterns indicated that the peptide backbone was extended. Therefore an extended peptide which interacts with the convex minor groove of the bent stem through some of its amide and arginine sidechain groups is consistent with these N M R results. 27


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ppm 8.5 7.5 6.5 Figure 2. (A) The aromatic proton region of the N M R titration experiments of d(T6C4A6) with the peptide MRSRSPSRSKSPMR. The equivalent amount of the peptide present in the sample is indicated by the number beside each spectrum. (B) Upper trace, the amide and aromatic region of the peptide MRSRSPSRSKSPMR proton spectrum. Lower trace, the same region of the spectrum of the 1:1 peptide D N A hairpin sample. (C) The imino proton region of the d(T6C4A6) with varying equivalent amounts of the 14-mer peptide present as indicated by the number beside each spectrum. The assignments of the imino protons are indicated by residue numbers.


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Figure 3. Diagrams showing the chemical shift changes observed in the peptide-DNA hairpin samples relative to the isolated D N A hairpin sample. (A) (upper panel) Phosphorus-31 chemical shift changes of the 5'- Ρ for each residue of the 1:1 peptidehairpin sample; and (lower panel) carbon-13 chemical shift changes of the aromatic C6/C8 (open bar) and the deoxyribose C F (thatched bar) signals in the 1:1 peptidehairpin sample. (B) Aromatic proton chemical shift changes for the 1:1 (upper panel) and the 2:1 (lower panel) peptide-hairpin samples. The changes for the H6/H8 protons (open bar), the A H2 and the C H5 protons (thatched bar) are plotted against the hairpin sequence.



ZHOU & VOGEL


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Taken together, our results indicate first of all that the SPXX-containing peptide interacts preferentially with the single-stranded hairpin C loop region. Secondly we find that at higher than 1:1 peptide-DNA ratios, the peptide binds to the minor groove of the T.A stem in an extended conformation. These results indicate that binding of SPXX motifs to loop regions might be more prevalent than previously considered.


Acknowledgments. We are indebted to Dr. Peter Davies (Queens University) for providing the peptide and to Dr. Richard Pon (University of Calgary) for synthesizing the D N A hairpin. Financial support by the Alberta Heart and Stroke Foundation is gratefully acknowledged. HJV is a Scientist of the Alberta Heritage Foundation for Medical Research. References 1. Steitz, T.A. Q. Rev. Biophys. 1990, 23, 205.. 2. Lapeyre, B., Bourlion, H. and Amalric, F. Proc. Natl. Acad. Sci. USA 1987, 84, 1472. 3. White, S. W., Appelt, K., Wilson, K. S. and Tanaka, I Prot. Struct. Fund. Genet. 1989, 5, 281. 4. Churchill, M. E. A. and Travers, A. A. Tr. Biochem. Sc. 1991, 16, 92. 5. Nikolov, D. B., Hu, S., Lin, J., Gasch, Α., Hoffmann, Α., Horikoshi, M., Chua, N., Roeder, R. G. and Burley, S. K. Nature 1992, 360, 40. 6. Chasman, D. I., Flaherty, Κ. M. Sharp, P. A. and Kornberg, R. D. Proc. Natl. Acad. Sci. USA 1993, 90, 8174. 7. Kim, Y., Geiger, J., Hahn S. and Sigler, P. Nature 1993, 365, 512. 8. Kim, J. L., Nikolov, D. B. and Burley S. K. Nature 1993, 365, 520. 9. Love, J. J., Li, X., Case, D. Α., Giese, K., Grosschedl, R. and Wright, P. E. Nature 1995, 376, 791. 10. Werner, M. H., Huth, J. R., Gronenborn, A. M. and Clore, G. M. Cell 1995, 81, 705. 11. Geierstanger, Β. H., Mrksich, M. Dervan, P. Β. and Wemmer, D. E. Science 1994, 266, 646. 12. Trauger, J. W., Baird, Ε. E. and Dervan, P. B. Nature 1996, 382, 559. 13. Kahne, D. Chem. & Biol. 1995, 2, 7. 14. Suzuki, M. J. Mol. Biol. 1989, 207, 61. 15. Churchill, M. E. A. and Suzuki, M. EMBO J. 1989, 8, 4189. 16. Hill, C. S., Packman, L. C. and Thomas, J. O. EMBO J. 1990, 9, 805. 17. Suzuki, M., Gerstein, M. and Johnson, T. Protein Eng. 1993, 6, 565. 18. Green, G. R., Lee, H. and Poccia, D. L. J. Biol. Chem. 1993, 268, 11247. 19. Geierstanger, B. H., Volkman, B. F., Kremer, W. and Wemmer, D. E. Biochemistry 1994, 33, 5347. 20. Kennedy, B. P. and Davies, P. L. J. Biol. Chem. 1984, 260, 4338. 21. Plateau P. and Gueron, M. J. Am. Chem. Soc. 1982, 104, 7310. 22. Marion, D. and Wuthrich, K. Biochem. Biophys. Res. Commun. 1983, 113, 967. 23. Bax, Α., Griffey, R. H. and Hawkins, B. L. J. Magn. Reson. 1983, 55, 301. 24. Kellogg, G. W. J. Magn. Reson. 1992, 98, 176. 25. Kintanar, Α., Klevit, R. E. and Reid, B. R. Nucleic Acids Res. 1987, 15, 5845. 26. Aymami, J., Coll, M., Frederick, C. Α., Wang, A. H.-J. and Rich, A. Nucleic Acids Res. 1989, 17, 3229. 27. Zhou N. and Vogel, H. J. Biochemistry 1993, 32, 637. 28. Feigon, J. Denny, W. A . , Leupin, W. and Kearns, D. R. J. Med. Chem. 1984, 27, 450. Leontis and SantaLucia; Molecular Modeling of Nucleic Acids ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

Molecular Modeling of Nucleic Acids - American Chemical Society

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