Local Structural Elements in the Mostly Unstructured Transcriptional Activation Domain of Human p53
2000; Elsevier BV; Volume: 275; Issue: 38 Linguagem: Inglês
10.1074/jbc.m003107200
ISSN1083-351X
AutoresHyun Lee, K. Hun Mok, Ranjith Muhandiram, Kyu-Hwan Park, Jae-Eun Suk, Do‐Hyung Kim, Jun Chang, Young Chul Sung, Kwan Yong Choi, Kyou‐Hoon Han,
Tópico(s)RNA Research and Splicing
ResumoDNA transcription is initiated by a small regulatory region of transactivators known as the transactivation domain. In contrast to the rapid progress made on the functional aspect of this promiscuous domain, its structural feature is still poorly characterized. Here, our multidimensional NMR study reveals that an unbound full-length p53 transactivation domain, although similar to the recently discovered group of loosely folded proteins in that it does not have tertiary structure, is nevertheless populated by an amphipathic helix and two nascent turns. The helix is formed by residues Thr18–Leu26(Thr-Phe-Ser-Asp-Leu-Trp-Lys-Leu-Leu), whereas the two turns are formed by residues Met40–Met44 and Asp48–Trp53, respectively. It is remarkable that these local secondary structures are selectively formed by functionally critical and positionally conserved hydrophobic residues present in several acidic transactivation domains. This observation suggests that such local structures are general features of acidic transactivation domains and may represent "specificity determinants" (Ptashne, M., and Gann, A. A. F. (1997),Nature 386, 569–577) that are important for transcriptional activity. DNA transcription is initiated by a small regulatory region of transactivators known as the transactivation domain. In contrast to the rapid progress made on the functional aspect of this promiscuous domain, its structural feature is still poorly characterized. Here, our multidimensional NMR study reveals that an unbound full-length p53 transactivation domain, although similar to the recently discovered group of loosely folded proteins in that it does not have tertiary structure, is nevertheless populated by an amphipathic helix and two nascent turns. The helix is formed by residues Thr18–Leu26(Thr-Phe-Ser-Asp-Leu-Trp-Lys-Leu-Leu), whereas the two turns are formed by residues Met40–Met44 and Asp48–Trp53, respectively. It is remarkable that these local secondary structures are selectively formed by functionally critical and positionally conserved hydrophobic residues present in several acidic transactivation domains. This observation suggests that such local structures are general features of acidic transactivation domains and may represent "specificity determinants" (Ptashne, M., and Gann, A. A. F. (1997),Nature 386, 569–577) that are important for transcriptional activity. transactivation domain acidic activation domain heteronuclear single quantum coherence total COSY nuclear Overhauser enhancement spectroscopy mouse double minute 2 degree p53 is a tumor suppressor protein involved in the negative feedback of cell proliferation (1Kubbutat M.H.G. Jones S.N. Vousden K.H. Nature. 1997; 387: 299-303Crossref PubMed Scopus (2847) Google Scholar) and is composed of a few discrete functional domains, such as a transactivation domain (TAD)1 (2Fields S. Jang S.K. Science. 1990; 249: 1046-1051Crossref PubMed Scopus (658) Google Scholar), a DNA-binding domain (3Cho Y. Gorina S. Jeffrey P.D. Pavletich N.P. Science. 1994; 265: 346-355Crossref PubMed Scopus (2150) Google Scholar), and an oligomerization domain (4Clore G.M. Ernst J. Clubb E. Omichinski J.G. Poindexter Kennedy W.M. Sakaguchi K. Appella E. Gronenborn A.M. Nat. Struct. Biol. 1995; 2: 321-333Crossref PubMed Scopus (191) Google Scholar, 5Lee W. Harvey T.S. Yin Y. Yau P. Litchfield D. Arrowsmith C.H. Nat. Struct. Biol. 1994; 1: 877-890Crossref PubMed Scopus (234) Google Scholar). The full TAD of p53 consists of the N-terminal 73-residues and has a net charge of –17 because it is rich in acidic amino acid residues, such as Asp and Glu (2Fields S. Jang S.K. Science. 1990; 249: 1046-1051Crossref PubMed Scopus (658) Google Scholar). Hence, the p53 TAD is a member of "acidic" (6Mitchell P.J. Tjian R. Science. 1989; 245: 371-378Crossref PubMed Scopus (2206) Google Scholar) activation domains (AADs), which interact with a variety of target proteins that bind to specific sites on DNA (2Fields S. Jang S.K. Science. 1990; 249: 1046-1051Crossref PubMed Scopus (658) Google Scholar, 7Kussie P.H. Gorina S. Marechal V. Elenbass B. Moreau J. Levine A.J. Pavletich N.P. Science. 1996; 274: 948-953Crossref PubMed Scopus (1798) Google Scholar, 8Lin J. Chen J. Elenbaas B. Levine A.J. Genes Dev. 1994; 8: 1235-1246Crossref PubMed Scopus (581) Google Scholar, 9Chang J. Kim D.-H. Lee S.W. Choi K.Y. Sung Y.C. J. Biol. Chem. 1995; 270: 25014-25019Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 10Ptashne M. Nature. 1988; 335: 683-689Crossref PubMed Scopus (1176) Google Scholar, 11Ptashne M. Gann A.A.F. Nature. 1997; 386: 569-577Crossref PubMed Scopus (941) Google Scholar). The DNA-binding domain and the oligomerization domain of p53 have been subjected to extensive structural investigations, and their structures are well determined (3Cho Y. Gorina S. Jeffrey P.D. Pavletich N.P. Science. 1994; 265: 346-355Crossref PubMed Scopus (2150) Google Scholar, 4Clore G.M. Ernst J. Clubb E. Omichinski J.G. Poindexter Kennedy W.M. Sakaguchi K. Appella E. Gronenborn A.M. Nat. Struct. Biol. 1995; 2: 321-333Crossref PubMed Scopus (191) Google Scholar, 5Lee W. Harvey T.S. Yin Y. Yau P. Litchfield D. Arrowsmith C.H. Nat. Struct. Biol. 1994; 1: 877-890Crossref PubMed Scopus (234) Google Scholar). On the contrary, little is known about the structure of the TAD of p53 except the fact that it is inherently devoid of tertiary structure, as are most AADs (12Sigler P.B. Nature. 1988; 333: 210-212Crossref PubMed Scopus (298) Google Scholar, 13Cho H.S. Liu C.W. Damberger F.F. Pelton J.G. Nelson H.C.M. Wemmer D.E. 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Protein Sci. 1996; 5: 262-269Crossref PubMed Scopus (52) Google Scholar), are useful to some extent to describe the overall structural state of the intact p53 TAD in a target-free environment. However, these models do not suggest the possibility that some specific structural determinants may exist in the unbound state of AADs and mediate recognition of AADs by target proteins. Despite the speculation (11Ptashne M. Gann A.A.F. Nature. 1997; 386: 569-577Crossref PubMed Scopus (941) Google Scholar, 12Sigler P.B. Nature. 1988; 333: 210-212Crossref PubMed Scopus (298) Google Scholar, 17Triezenberg S.J. Curr. Opin. Genet. Dev. 1995; 5: 190-196Crossref PubMed Scopus (350) Google Scholar) that transcriptional activity or TAD-target binding would be governed by some specific structural determinants, such as an amphipathic helix (18Giniger E. Ptashne M. Nature. 1987; 330: 670-673Crossref PubMed Scopus (222) Google Scholar), acquiring direct physical evidence for the putative helical structure in the unbound form of TADs has been mostly unsuccessful (13Cho H.S. Liu C.W. Damberger F.F. Pelton J.G. Nelson H.C.M. Wemmer D.E. Protein Sci. 1996; 5: 262-269Crossref PubMed Scopus (52) Google Scholar, 19Regier J.L. Shen F. Triezenberg S.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 883-887Crossref PubMed Scopus (226) Google Scholar, 20Schmitz M.L. dos Santos Silva M.A. Altmann H. Czisch M. Holak T.A. Baeuerle P.A. J. Biol. Chem. 1994; 269: 25613-25620Abstract Full Text PDF PubMed Google Scholar, 21O'Hare P. Williams G. Biochemistry. 1992; 31: 4150-4156Crossref PubMed Scopus (64) Google Scholar, 22Donaldson L. Capone J.P. J. Biol. Chem. 1992; 267: 1411-1414Abstract Full Text PDF PubMed Google Scholar, 23Dahlman-Wright K. Baumann H. McEwan I.J. Almlof T. Wright A.P.H. Gustafsson J. Hard T. Proc. Natl. Acad. Sci. U. S. 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For the former, no evidence of any secondary structure could be found, even in a hydrophobic solvent such as 80% methanol. Even though the minimal activation domain from the human glucocorticoid receptor was shown to contain multiple helices, this observation was possible only in the presence of a strong helix-promoting solvent (23Dahlman-Wright K. Baumann H. McEwan I.J. Almlof T. Wright A.P.H. Gustafsson J. Hard T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1699-1703Crossref PubMed Scopus (139) Google Scholar). The presence of some secondary structures was noted by CD measurements in the activation domains of GCN4 and GAL4 in a hydrophobic solvent, but the dominant form was controversially found to be antiparallel β-sheet rather than the putative α-helix (24Leuther K.K. Salmeron J.M. Johnston S.A. Cell. 1993; 72: 575-585Abstract Full Text PDF PubMed Scopus (105) Google Scholar, 25Van Hoy M. Leuther K.K. Kodadek T. Johnston S.A. Cell. 1993; 72: 587-594Abstract Full Text PDF PubMed Scopus (121) Google Scholar). The results of NMR studies on short TAD fragments are also controversial. In the case of the VP16 TAD, an unbound 17-residue fragment was reported to have no detectable secondary structure, which becomes helical upon target binding (26Uesugi M. Nyanguile O. Lu H. Levine A.J. Verdine G.L. Science. 1997; 277: 1310-1313Crossref PubMed Scopus (273) Google Scholar). In contrast, a 16-residue p53 TAD fragment was shown to form, even in the absence of a target protein, an ordered structure at 2 °C with two contiguous β-turns, which is very similar to a helix (27Botuyan M.V.E. Momand J. Chen Y. Folding Des. 1997; 2: 331-342Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). For short TAD fragments (<20 residues), helices have been observed only in their target-bound state (7Kussie P.H. Gorina S. Marechal V. Elenbass B. Moreau J. Levine A.J. Pavletich N.P. Science. 1996; 274: 948-953Crossref PubMed Scopus (1798) Google Scholar, 16Uesugi M. Verdine G.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14801-14806Crossref PubMed Scopus (119) Google Scholar, 26Uesugi M. Nyanguile O. Lu H. Levine A.J. Verdine G.L. Science. 1997; 277: 1310-1313Crossref PubMed Scopus (273) Google Scholar). However, helices were detected if a longer fragment, such as the 46-residue cAMP-response element-binding protein kinase-inducible transactivation domain (pKID) (28Hua Q. Jia W. Bullock B.P. Habener J.F. Weiss M.A. Biochemistry. 1998; 37: 5858-5866Crossref PubMed Scopus (86) Google Scholar) or the 88-residue long full-length TAD from the activating transcription factor-2 (29Nagadoi A. Nakazawa K. Uda H. Okuno K. Maekawa T. Ishii S. Nihimura Y. J. Mol. Biol. 1999; 287: 593-607Crossref PubMed Scopus (46) Google Scholar), was examined. In particular, the latter has, unlike typical AADs, extremely well defined secondary structures, such as a zinc finger-like motif and short β-strands in aqueous solution. An interesting exception is the full-length TAD of yeast heat shock transcription factor, which, even as an intact domain, is completely devoid of secondary structure (13Cho H.S. Liu C.W. Damberger F.F. Pelton J.G. Nelson H.C.M. Wemmer D.E. Protein Sci. 1996; 5: 262-269Crossref PubMed Scopus (52) Google Scholar). As the structural features of TADs vary widely, ranging from being totally unstructured to having well defined secondary structures, it is not clear whether one would be able to find a general structural description for unbound TADs. Functionally different from but structurally similar to TADs are several proteins that are also known to be inherently unstructured or loosely folded (30Kriwacki R.W. Hengst L. Tennant L. Reed S.I. Wright P.E. Proc. Natl. Acad. Sci. U. S. 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The observation that these proteins may function without necessarily relying on tertiary structure has in fact suggested establishment of a new view on protein structure (30Kriwacki R.W. Hengst L. Tennant L. Reed S.I. Wright P.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11504-11509Crossref PubMed Scopus (490) Google Scholar, 37Plaxco K.W. Groß M. Nature. 1997; 386: 657-658Crossref PubMed Scopus (114) Google Scholar). The unbound full-length p53 TAD, largely unstructured in aqueous solution, contains a small fraction of secondary structure (9Chang J. Kim D.-H. Lee S.W. Choi K.Y. Sung Y.C. J. Biol. Chem. 1995; 270: 25014-25019Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), as do many other TADs (28Hua Q. Jia W. Bullock B.P. Habener J.F. Weiss M.A. Biochemistry. 1998; 37: 5858-5866Crossref PubMed Scopus (86) Google Scholar, 38Hi R. Osada S. Yumoti N. Osumi T. J. Biol. Chem. 1999; 274: 35152-35158Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 39Tell G. Perrone L. Fabbro D. Pellizzari L. Pucillo C. De Felince M. Acquaviva R. Formisano S. Damante G. Biochem. J. 1999; 329: 395-403Crossref Scopus (32) Google Scholar). In order to fully understand the potential ramification of such minimal secondary structures for transcriptional activity, we have carried out a detailed multidimensional NMR study on a uniformly 15N-labeled full-length p53 TAD. Thorough characterization of the overall structural state of the p53 TAD not only should help in establishing a better structure-function relationship for TADs but also may provide additional insight into the new view on protein structure. The 75-residue human p53 TAD was prepared as described previously (9Chang J. Kim D.-H. Lee S.W. Choi K.Y. Sung Y.C. J. Biol. Chem. 1995; 270: 25014-25019Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). The DNA fragment encoding a GS linker plus residues 1–73 of p53 was amplified by polymerase chain reaction using two primers (5′-GGTCGGATCCATGGAGCCGCAGTCA-3′ and 3′-GGTGAAGCTTACACGGGGGGAGCAGCCTC-5′) and subcloned intoBamHI and HindIII sites of pSK(–) (Stratagene) to construct pSK-p53-TAD (9Chang J. Kim D.-H. Lee S.W. Choi K.Y. Sung Y.C. J. Biol. Chem. 1995; 270: 25014-25019Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). The DNA fragment of the p53 TAD in pSK(–) was subcloned into BamHI and HindIII sites of the Escherichia coli expression vector pGEX-KG. The recombinant DNA containing the p53-TAD DNA fragment was introduced into the E. coli DH5α and subjected to expression as glutathione S-transferase fusion p53-TAD peptides. Cells were grown in 2× YTA medium (16 g/liter tryptone, 10 g/liter yeast extract, 5 g/liter NaCl) with 0.05 mg/ml ampicillin (Sigma) at 37 °C, pH 7.0, for 4 h. 0.3 mmisopropyl-1-thio-β-d-galactopyranoside was added when the A600 reached 0.7, and cells were further grown at 37 °C for 4 h. The finalA600 was approximately 1.2. Cells were centrifuged for 30 min at 8000 rpm. Pellets were resuspended in 50 mm sodium phosphate, pH 7.8, and then reacted with hen egg white lysozyme (0.25 mg/ml, Sigma) for 1 h at 4 °C. The suspension was then sonicated and centrifuged at 4 °C, 12,000 rpm for 20 min. The fusion protein was bound to glutathione-Sepharose affinity resin (Amersham Pharmacia Biotech) and cleaved with thrombin (Roche Molecular Biochemicals) to release the p53-TAD peptide. The peptide was further chromatographically purified using SOURCE 15Q (PE 4.6 mm inner diameter × 100 mm long, Amersham Pharmacia Biotech) ion exchange and C18 218TP1010 (10 mm inner diameter × 250 mm long, 10-μm particle size, Vydac) preparative reversed-phase high pressure liquid chromatography to homogeneity. To obtain the uniformly 15N-labeled p53 TAD, the E. coli was initially grown without labeling for 10 h at 37 °C, pH 7.4, in M9 medium supplemented with 0.4% glucose, 2.0 mg/liter biotin, 2.0 mg/liter thiamin, 1 mmMgSO4, 0.1 mm CaCl2, and 0.05 mg/ml ampicillin. This was used as a 1/20 seed for further culture in the same medium supplemented with 0.1% 15NH4Cl and grown at 37 °C for approximately 12 h or until theA600 reached 0.7. Induction of expression with 0.6 mm isopropyl-1-thio-β-d-galactopyranoside was performed for a period of 4 h. Purification of the15N-labeled peptide was performed as described above. The purified peptide was subjected to amino acid composition analysis and mass spectrometry to confirm its identity. Protein samples in a concentration of ∼0.3 mm were prepared in 90% H2O/10%2H2O or 100% 2H2O containing 50 mm acetate buffer with a final pH of 6.3. All NMR experiments were done at 5 and 25 °C using a Varian Unity 500 or Unity INOVA 600 spectrometer equipped with a triple-resonance probe in order to avoid spectral overlap as much as possible, to monitor temperature dependent structural changes and to calculate temperature coefficients of backbone amide protons. Pulse sequences used were as described previously (40Farrow N.A. Muhandiram R. Singer A.U. Pascal S.M. Kay C.M. Gish G. Shoelson S.E. Pawson T. Forman-Kay J.D. Kay L.E. Biochemistry. 1994; 33: 5984-6003Crossref PubMed Scopus (2013) Google Scholar) except that current15N-1H HSQC spectra consisted of 150 complex t1 increments with spectral widths of 920 Hz in the F1 (15N) dimension and 6600 Hz in the F2 dimension (1H). For the three-dimensional15N-edited TOCSY-HSQC and NOESY-HSQC spectra (41Clore G.M. Gronenborn A. Prog. NMR Spectrosc. 1991; 23: 43-92Abstract Full Text PDF Scopus (247) Google Scholar), 128 complex t1 and 32 complex t2 increments were acquired with spectral widths of 6600 Hz in the F1(1H), 920 Hz in the F2 (15N), and 6600 Hz in the F3 (1H) dimension. Hydrogen-deuterium exchange was monitored by one-dimensional1H methods at 5 °C. Pulsed field gradients were used in all 1H-detected heteronuclear experiments in order to minimize spectral artifacts as well as to select desired coherences using an enhanced-sensitivity approach (42Kay L.E. Keifer P. Saarinen T. J. Am. Chem. Soc. 1992; 114: 10663-10665Crossref Scopus (2433) Google Scholar). Also, water-selective pulses were employed to achieve minimal solvent saturation (43Grzesiek S. Bax A. J. Am. Chem. Soc. 1993; 115: 12593-12594Crossref Scopus (1015) Google Scholar, 44Kay L.E. Xu G.Y. Yamazaki T. J. Magn. Reson. Ser. A. 1994; 109: 129-133Crossref Scopus (417) Google Scholar). Mixing times for TOCSY-HSQC spectra were 55–70 ms, whereas a mixing time of 190 or 250 ms was used for NOESY-HSQC experiments. The three-bond 3JHNHα coupling constants were measured by 3D HNHA technique (45Kuboniwa H. Grzesiek S. Delaglio F. Bax A. J. Biomol. NMR. 1994; 4: 871-878Crossref PubMed Scopus (336) Google Scholar). Temperature coefficients for the backbone amide protons (ΔNH) were calculated using the1H resonance assignments obtained at two temperatures (5 and 25 °C). For measurements of 15N relaxation times a series of 15N-1H HSQC spectra were recorded with seven different relaxation delays. The15N-1H heteronuclear steady-state NOEs were measured from a pair of spectra recorded with and without a proton presaturation; in the former, a net recycle delay of 5 s was used, whereas for the latter, a recycle delay of 2 s was followed by a 3-s proton presaturation period. Data were processed and analyzed on a Sun SPARCstation using Varian Vnmr, nmrPipe/nmrDraw (46Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11570) Google Scholar), and PIPP software (47Garrett D.S. Powers R. Gronenborn A.M. Clore G.M. J. Magn. Reson. 1991; 95: 214-220Crossref Scopus (802) Google Scholar). Resonance assignment for the full-length 73-residue human p53 TAD was achieved using three-dimensional 15N-edited TOCSY-HSQC, NOESY-HSQC techniques (41Clore G.M. Gronenborn A. Prog. NMR Spectrosc. 1991; 23: 43-92Abstract Full Text PDF Scopus (247) Google Scholar, 48Zhang O. Kay L.E. Olivier J.P. Forman-Kay J.D. J. Biomol. NMR. 1994; 4: 845-858Crossref PubMed Scopus (612) Google Scholar), and 1H homonuclear two-dimensional COSY, TOCSY, and NOESY experiments according to the sequential resonance assignment procedure (49Wuthrich K. NMR of Proteins and Nucleic Acids. John Wiley and Sons, New York1986Crossref Google Scholar). The first two N-terminal residues of the recombinant protein, Gly and Ser, were excluded from the amino acid numbering of the peptide as they originated from the N-terminal glutathione-S-transferase fusion linker. Unambiguous assignment of the backbone 15N, amide and aliphatic protons was possible (TableI) for all residues except for 14 prolines and Glu62. The level of achieved resonance assignment, however, was sufficient for subsequent structural characterization of the p53 TAD. Fig. 4 shows an15N-1H HSQC spectrum of the full-length 73-residue human p53 TAD with resonance assignment.Table IResonance assignment for the 73-residue full-length human p53 TAD obtained in 90% H2O/10% 2H2O, pH 6.3 at 5 °CResidue15NNHHαHβHγOthersMet1122.018.714.502.04/2.102.47Glu2121.568.454.282.05/1.912.25Glu3123.398.424.572.02/1.882.28Pro44.57Gln5121.258.244.272.03/1.882.23Ser6118.058.524.423.82Asp7123.848.594.892.78/2.53Pro84.45Ser9116.078.614.403.86Val10121.567.994.172.100.90Glu11126.588.494.562.01/1.862.29Pro12Pro134.42Leu14122.788.494.321.661.60Ser15116.998.484.423.86Gln16122.788.634.332.12/1.942.33Glu17122.328.564.251.892.17Thr18115.318.244.214.091.11Phe19122.478.364.583.13/3.022,6H 7.133,5H 7.114H 7.24Ser20116.998.214.333.86/3.72Asp21122.328.394.542.58Leu22121.258.034.081.531.41δ CH3 0.80Trp23119.737.944.493.302H 7.214H 7.475H 7.076H 7.147H 7.39NH 10.20Lys24120.497.613.991.851.66δ CH2 1.57ɛ CH22.91Leu25120.497.874.251.621.62δ CH3 0.90Leu26123.997.984.581.621.41δ CH3 0.90Pro274.35Glu28119.738.894.181.972.18Asn29118.978.344.742.78/2.65Asn30119.888.334.722.81/2.70Val31120.498.164.072.070.93Leu32125.988.444.391.621.62δ CH3 0.92Ser33118.518.394.723.85/3.77Pro344.44Leu35124.158.474.571.581.58δ CH3 0.90Pro364.43Ser37116.078.494.373.86Gln38121.568.654.212.04/1.932.30Ala39125.988.474.291.36Met40119.888.484.462.08/2.012.60Asp41120.038.574.542.71/2.57Asp42120.498.344.542.67Leu43122.018.204.231.671.58δ CH3 0.87Met44120.498.364.422.062.53Leu45123.548.234.371.621.54Ser46118.668.644.773.87Pro474.41Asp48119.278.334.542.69/2.53Asp49120.348.214.592.66Ile50120.497.994.031.831.37/1.12γ CH30.84δ CH3 0.82Glu51124.308.474.121.882.15Gln52120.958.284.201.842.14Trp53121.868.104.633.01/2.892H 7.104H 7.215H 7.546H 7.097H 7.40NH 10.11Phe54122.018.104.633.172,6H 7.083,5H 7.15Thr55116.998.094.244.071.15Glu56123.698.434.242.01/1.892.24Asp57123.848.604.842.73/2.53Pro58Gly59128.878.494.07Pro604.48Asp61121.258.364.542.63Glu624.27Ala63127.048.424.541.36Pro644.43Arg65122.328.614.311.78/1.721.62CH23.17Met66123.848.664.812.04/1.962.65/2.57Pro674.42Glu68121.258.704.352.13/1.962.40Ala69125.678.454.271.37Ala70125.378.454.571.35Pro71Pro724.45Val73124.307.864.002.050.91 Open table in a new tab Summarized in Fig. 1 are T1and T2 relaxation times of the backbone 15N and15N-1H steady-state heteronuclear NOEs for the unbound 73-residue p53 TAD. Two regions in the full-length p53 TAD have noticeably different local backbone dynamics from the rest of the molecule; a pronounced region near residues 18–30 and another encompassing residues 47–55. The former, interestingly, contains the functionally important conserved hydrophobic residues in the p53 TAD such as Phe19, Leu22, Trp23 and Leu26 (7Kussie P.H. Gorina S. Marechal V. Elenbass B. Moreau J. Levine A.J. Pavletich N.P. Science. 1996; 274: 948-953Crossref PubMed Scopus (1798) Google Scholar, 8Lin J. Chen J. Elenbaas B. Levine A.J. Genes Dev. 1994; 8: 1235-1246Crossref PubMed Scopus (581) Google Scholar, 9Chang J. Kim D.-H. Lee S.W. Choi K.Y. Sung Y.C. J. Biol. Chem. 1995; 270: 25014-25019Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Then, these local structural orders were further analyzed using NOE and chemical shift indices (50Wishart D.S. Sykes B.D. Methods Enzymol. 1994; 239: 363-392Crossref PubMed Scopus (937) Google Scholar) as shown in Fig. 2. The continuity of sequential dNN NOEs indicates that three regions, near residues 18–30, 39–45, and 48–55 of the p53 TAD, have helical propensity. The AGADIR (51Munoz V. Serrano L. Nat. Struct. Biol. 1994; 1: 399-409Crossref PubMed Scopus (612) Google Scholar), a robust algorithm predicting helical content of a protein in the absence of tertiary structure, predicts that the unbound p53 TAD possess three helices, which correspond to the three regions that we found. Among the three predicted regions, the first, encompassing residues Gln16–Leu25, was predicted to be the most helical.Figure 2Summary of interproton NOEs and chemical shift index for a 73-residue full-length p53 TAD.Thickness of bars is proportional to NOE intensities (strong, medium, and weak) that are measured from an NOESY-HSQC spectrum obtained at 5 °C. The hatched portionin the sequential dNN NOEs indicates an ambiguity regarding the presence of the particular NOE due to resonance overlap. Chemical shift index (CSI) values of +1, 0, and –1 are represented by open, half-filled, and filled circles,respectively. A similar pattern of sequential dNN NOEs are obtained at 25 °C (shown at the bottom). Locations of an amphipathic helix (helix I) and two turns (turns I and II) are markedabove the sequence.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Fig. 3 shows details on the chemical shift index analysis for the Hα chemical shift (50Wishart D.S. Sykes B.D. Methods Enzymol. 1994; 239: 363-392Crossref PubMed Scopus (937) Google Scholar), deviation of the three-bond coupling constants3JHNHα from random coil values (52Smith L.J. Bolin K.A. Schwalbe H. MacArthur M.W. Thornton J.M. Dobson C.M. J. Mol. Biol. 1996; 255: 494-506Crossref PubMed Scopus (369) Google Scholar), and the temperature coefficients of the backbone amide protons (ΔNH). In consistent with the AGADIR prediction, an amphipathic helix seems to be formed by residues Thr18–Leu26, whereas the other two regions showing helical propensity form only nascent turns. The HNHA technique has a tendency of underestimating the 3JHNHαvalues by ∼0.5 Hz when compared with other methods (45Kuboniwa H. Grzesiek S. Delaglio F. Bax A. J. Biomol. NMR. 1994; 4: 871-878Crossref PubMed Scopus (336) Google Scholar). As shown in Fig. 3, even if such a tendency is taken into consideration, one finds that the observed 3JHNHα values associated with the helix are not only sufficiently small (4∼6 Hz) (49Wuthrich K. NMR of Proteins and Nucleic Acids. John Wiley and Sons, New York1986Crossref Google Scholar) but also deviate significantly from the random coil values (52Smith L.J. Bolin K.A. Schwalbe H. MacArthur M.W. Thornton J.M. Dobson C.M. J. Mol. Biol. 1996; 255: 494-506Crossref PubMed Scopus (369) Google Scholar). Noticeably small ΔNH values (<3 ppb/K) were observed for the backbone amide protons of residues Lys24–Asn30. Because small ΔNHvalues are typically associated with intramolecular hydrogen bonding (53Dyson H.J. Rance M. Houghten R.A. Lerner R.A. Wright P.E. J. Mol. Biol. 1988; 201: 161-200Crossref PubMed Scopus (643) Google Scholar), these results, in combination with the other NMR parameters, suggest that the backbone amide protons of Leu22–Leu26 are mostly likely involved in hydrogen bonding within the proposed helix. The fact that Leu22 and Trp23 have somewhat larger ΔNH values (4 to ∼5 ppb/K) than the other helix-forming residues seems to indicate "breathing" of the helix at its N terminus. A hydrogen-deuterium exchange experiment showed that backbone amide protons disappeared within 30 min of the addition of2H2O. Such a fast exchange rate is consistent w
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