NMR Spectroscopy Reveals the Solution Dimerization Interface of p53 Core Domains Bound to Their Consensus DNA
2001; Elsevier BV; Volume: 276; Issue: 52 Linguagem: Inglês
10.1074/jbc.m107516200
ISSN1083-351X
AutoresChristian Klein, Eckart Planker, Tammo Diercks, Horst Kessler, Klaus‐Peter Künkele, Kurt Lang, Silke Hansen, Manfred Schwaiger,
Tópico(s)RNA Research and Splicing
ResumoThe p53 protein is a transcription factor that acts as the major tumor suppressor in mammals. The core DNA-binding domain is mutated in about 50% of all human tumors. The crystal structure of the core domain in complex with DNA illustrated how a single core domain specifically interacts with its DNA consensus site and how it is inactivated by mutation. However, no structural information for the tetrameric full-length p53-DNA complex is available. Here, we present novel experimental insight into the dimerization of two p53 core domains upon cooperative binding to consensus DNA in solution obtained by NMR. The NMR data show that the p53 core domain itself does not appear to undergo major conformational changes upon addition of DNA and elucidate the dimerization interface between two DNA-bound core domains, which includes the short H1 helix. A NMR-based model for the dimeric p53 core-DNA complex incorporates these data and allows the conclusion that the dimerization interface also forms the actual interface in the tetrameric p53-DNA complex. The significance of this interface is further corroborated by the finding that hot spot mutations map to the H1 helix, and by the binding of the putative p53 inhibitor 53BP2 to this region via one of its ankyrin repeats. Based on symmetry considerations it is proposed that tetrameric p53 might link non-contigous DNA consensus sites in a sandwich-like manner generating DNA loops as observed for transcriptionally active p53 complexes. The p53 protein is a transcription factor that acts as the major tumor suppressor in mammals. The core DNA-binding domain is mutated in about 50% of all human tumors. The crystal structure of the core domain in complex with DNA illustrated how a single core domain specifically interacts with its DNA consensus site and how it is inactivated by mutation. However, no structural information for the tetrameric full-length p53-DNA complex is available. Here, we present novel experimental insight into the dimerization of two p53 core domains upon cooperative binding to consensus DNA in solution obtained by NMR. The NMR data show that the p53 core domain itself does not appear to undergo major conformational changes upon addition of DNA and elucidate the dimerization interface between two DNA-bound core domains, which includes the short H1 helix. A NMR-based model for the dimeric p53 core-DNA complex incorporates these data and allows the conclusion that the dimerization interface also forms the actual interface in the tetrameric p53-DNA complex. The significance of this interface is further corroborated by the finding that hot spot mutations map to the H1 helix, and by the binding of the putative p53 inhibitor 53BP2 to this region via one of its ankyrin repeats. Based on symmetry considerations it is proposed that tetrameric p53 might link non-contigous DNA consensus sites in a sandwich-like manner generating DNA loops as observed for transcriptionally active p53 complexes. transactivation domain DNA-binding domain tetramerization domain dithiothreitol fluorescence correlation spectroscopy transverse relaxation-opimized spectroscopy nuclear Overhauser enhancement spectroscopy p53-binding protein 2 6-carboxytetramethylrhodamine The tumor suppressor gene p53 is the most frequent site of genetic alterations found in human tumors (1Hollstein M. Hergenhahn M. Yang Q. Bartsch H. Wang Z.Q. Hainaut P. Mutat. Res. 1999; 431: 199-209Crossref PubMed Scopus (171) Google Scholar) and acts as the major tumor suppressor in mammals. In addition to non-transcriptional functions, p53 acts primarily as a transcriptional activator, that regulates the expression of several genes involved in cell cycle arrest, cellular senescence, anti-angiogenesis, and apoptosis (reviewed in Refs. 2Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6727) Google Scholar, 3Vousden K.H. Cell. 2000; 103: 691-694Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar, 4Vogelstein B. Lane D. Levine A.J. Nature. 2000; 408: 307-310Crossref PubMed Scopus (5798) Google Scholar). Recently, two homologues of p53, p63 and p73, were discovered, coding for a variety of different isoforms. These three p53 family members play distinct roles in differentiation, development, and tumor suppression (reviewed in Ref. 5Yang A. McKeon F. Nat. Rev. Mol. Cell. Biol. 2000; 1: 199-207Crossref PubMed Scopus (425) Google Scholar). p53 possesses a modular architecture with an N-terminal transactivation domain (TAD),1 a strongly conserved core DNA-binding domain (DBD), a tetramerization domain (TD), and a regulatory C terminus (6Arrowsmith C.H. Morin P. Oncogene. 1996; 12: 1379-1385PubMed Google Scholar, 7Arrowsmith C.H. Cell Death Differ. 1999; 6: 1169-1173Crossref PubMed Scopus (94) Google Scholar). Tetrameric p53 binds specifically to a DNA consensus sequence consisting of two consecutive palindromic 10-bp half-sites, where each half-site is formed by two head-to-head quarter-sites (8El-Deiry W.S. Kern S.E. Pietenpol J.A. Kinzler K.W. Vogelstein B. Nat. Genet. 1992; 1: 45-49Crossref PubMed Scopus (1744) Google Scholar, 9Funk W.D. Pak D.T. Karas R.H. Wright W.E. Shay J.W. Mol. Cell. Biol. 1992; 12: 2866-2871Crossref PubMed Scopus (677) Google Scholar, 10Cho Y. Gorina S. Jeffrey P.D. Pavletich N.P. Science. 1994; 265: 346-355Crossref PubMed Scopus (2145) Google Scholar, 11Wang Y. Schwedes J.F. Parks D. Mann K. Tegtmeyer P. Mol. Cell. Biol. 1995; 15: 2157-2165Crossref PubMed Scopus (146) Google Scholar, 12Waterman J.L. Shenk J.L. Halazonetis T.D. EMBO J. 1995; 14: 512-519Crossref PubMed Scopus (129) Google Scholar). The isolated TD forms a symmetric dimer of dimers (13Lee 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, 14Clore G.M. Ernst J. Clubb R. Omichinski J.G. Kennedy W.M. Sakaguchi K. Appella E. Gronenborn A.M. Nat. Struct. Biol. 1995; 2: 321-333Crossref PubMed Scopus (191) Google Scholar, 15Jeffrey P.D. Gorina S. Pavletich N.P. Science. 1995; 267: 1498-1502Crossref PubMed Scopus (438) Google Scholar), and contrasting models have been proposed that describe how the DBDs of each dimer are attached to DNA, namely with either consecutive or alternating arrangements (16McLure K.G. Lee P.W. EMBO J. 1998; 17: 3342-3350Crossref PubMed Scopus (203) Google Scholar). The p53 DBD comprises several hot spot regions for mutation that inactivate p53 in more than half of all human tumors (1Hollstein M. Hergenhahn M. Yang Q. Bartsch H. Wang Z.Q. Hainaut P. Mutat. Res. 1999; 431: 199-209Crossref PubMed Scopus (171) Google Scholar). Therefore, wild-type and mutant p53 DBDs have been the focus of various studies (17Bargonetti J. Manfredi J.J. Chen X. Marshak D.R. Prives C. Genes Dev. 1993; 7: 2565-2574Crossref PubMed Scopus (248) Google Scholar, 18Pavletich N.P. Chambers K.A. Pabo C.O. Genes Dev. 1993; 7: 2556-2564Crossref PubMed Scopus (443) Google Scholar, 19Balagurumoorthy P. Sakamoto H. Lewis M.S. Zambrano N. Clore G.M. Gronenborn A.M. Appella E. Harrington R.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8591-8595Crossref PubMed Scopus (119) Google Scholar, 20Bullock A.N. Henckel J. DeDecker B.S. Johnson C.M. Nikolova P.V. Proctor M.R. Lane D.P. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14338-14342Crossref PubMed Scopus (349) Google Scholar, 21Bullock A.N. Henckel J. Fersht A.R. Oncogene. 2000; 19: 1245-1256Crossref PubMed Scopus (325) Google Scholar). The crystal structure of the p53 DBD in complex with DNA (10Cho Y. Gorina S. Jeffrey P.D. Pavletich N.P. Science. 1994; 265: 346-355Crossref PubMed Scopus (2145) Google Scholar) showed that almost all known mutations affect residues that are in direct contact with DNA or maintain the tertiary structure. However, only one out of three p53 DBDs is bound to DNA sequence specifically in this crystal structure. Recently, the crystal structure of free mouse p53 DBD has been solved (22Zhao K. Chai X. Johnston K. Clements A. Marmorstein R. J. Biol. Chem. 2001; 276: 12120-12127Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), and NMR studies provided further insight into the folding of wild-type and mutant p53 DBDs (23Wong K.B. DeDecker B.S. Freund S.M. Proctor M.R. Bycroft M. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8438-8442Crossref PubMed Scopus (178) Google Scholar). Owing to its prominent role in tumorigenesis, the restoration of wild-type p53 activity for tumor therapy has gained widespread attraction (24Hupp T.R. Lane D.P. Ball K.L. Biochem. J. 2000; 352: 1-17Crossref PubMed Scopus (131) Google Scholar). Several studies have used structural information in attempts to rescue mutated or to stabilize the wild-type p53 DBD conformation (25Wieczorek A.M. Waterman J.L. Waterman M.J. Halazonetis T.D. Nat. Med. 1996; 2: 1143-1146Crossref PubMed Scopus (83) Google Scholar, 26Brachmann R.K. Yu K. Eby Y. Pavletich N.P. Boeke J.D. EMBO J. 1998; 17: 1847-1859Crossref PubMed Scopus (139) Google Scholar, 27Nikolova P.V. Wong K.B. DeDecker B. Henckel J. Fersht A.R. EMBO J. 2000; 19: 370-378Crossref PubMed Scopus (141) Google Scholar, 28Nikolova P.V. Henckel J. Lane D.P. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14675-14680Crossref PubMed Scopus (192) Google Scholar, 29Matsumura I. Ellington A.D. Protein Sci. 1999; 8: 731-740Crossref PubMed Scopus (28) Google Scholar, 30Xirodimas D.P. Lane D.P. J. Biol. Chem. 1999; 274: 28042-28049Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Based on the allosteric model of p53 regulation (31Hupp T.R. Lane D.P. Curr. Biol. 1994; 4: 865-875Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 32Hupp T.R. Sparks A. Lane D.P. Cell. 1995; 83: 237-245Abstract Full Text PDF PubMed Scopus (448) Google Scholar, 33Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2168) Google Scholar), peptides derived from the C terminus of p53 have been devised (34Selivanova G. Iotsova V. Okan I. Fritsche M. Strom M. Groner B. Grafstrom R.C. Wiman K.G. Nat. Med. 1997; 3: 632-638Crossref PubMed Scopus (316) Google Scholar, 35Selivanova G. Kawasaki T. Ryabchenko L. Wiman K.G. Semin. Cancer Biol. 1998; 8: 369-378Crossref PubMed Scopus (54) Google Scholar, 36Kim A.L. Raffo A.J. Brandt-Rauf P.W. Pincus M.R. Monaco R. Abarzua P. Fine R.L. J. Biol. Chem. 1999; 274: 34924-34931Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) to restore the wild-type activity of mutant p53. Recently, low molecular weight compounds have been reported to stabilize the wild-type conformation of human p53 and show an anti-tumor activityin vivo (37Foster B.A. Coffey H.A. Morin M.J. Rastinejad F. Science. 1999; 286: 2507-2510Crossref PubMed Scopus (688) Google Scholar). Several studies have disclosed that four p53 DBDs bind cooperatively to the DNA consensus sequence (11Wang Y. Schwedes J.F. Parks D. Mann K. Tegtmeyer P. Mol. Cell. Biol. 1995; 15: 2157-2165Crossref PubMed Scopus (146) Google Scholar, 17Bargonetti J. Manfredi J.J. Chen X. Marshak D.R. Prives C. Genes Dev. 1993; 7: 2565-2574Crossref PubMed Scopus (248) Google Scholar, 18Pavletich N.P. Chambers K.A. Pabo C.O. Genes Dev. 1993; 7: 2556-2564Crossref PubMed Scopus (443) Google Scholar, 19Balagurumoorthy P. Sakamoto H. Lewis M.S. Zambrano N. Clore G.M. Gronenborn A.M. Appella E. Harrington R.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8591-8595Crossref PubMed Scopus (119) Google Scholar, 21Bullock A.N. Henckel J. Fersht A.R. Oncogene. 2000; 19: 1245-1256Crossref PubMed Scopus (325) Google Scholar,38Wang Y. Reed M. Wang P. Stenger J.E. Mayr G. Anderson M.E. Schwedes J.F. Tegtmeyer P. Genes Dev. 1993; 7: 2575-2586Crossref PubMed Scopus (221) Google Scholar). The crystal structure of the p53 DBD-DNA complex is compatible with a model where four p53 DBDs bind to the consensus DNA without steric clashes (Fig. 7B in Ref. 10Cho Y. Gorina S. Jeffrey P.D. Pavletich N.P. Science. 1994; 265: 346-355Crossref PubMed Scopus (2145) Google Scholar) and bend the DNA (19Balagurumoorthy P. Sakamoto H. Lewis M.S. Zambrano N. Clore G.M. Gronenborn A.M. Appella E. Harrington R.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8591-8595Crossref PubMed Scopus (119) Google Scholar, 39Nagaich A.K. Zhurkin V.B. Sakamoto H. Gorin A.A. Clore G.M. Gronenborn A.M. Appella E. Harrington R.E. J. Biol. Chem. 1997; 272: 14830-14841Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 40Appella E. Nagaich A.K. Zhurkin V.B. Harrington R.E. J. Protein Chem. 1998; 17: 527-528Google Scholar, 41Nagaich A.K. Zhurkin V.B. Durell S.R. Jernigan R.L. Appella E. Harrington R.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1875-1880Crossref PubMed Scopus (99) Google Scholar, 42Lebrun A. Lavery R. Weinstein H. Protein Eng. 2001; 14: 233-243Crossref PubMed Scopus (18) Google Scholar). Yet, no structural information is available for the intact tetrameric p53 bound to DNA owing mainly due to the difficulties in obtaining suitable protein samples. Here, we present the results of NMR experiments that allow for the first time the experimental identification of the actual dimerization interface between two p53 DBDs cooperatively bound to their consensus DNA in aqueous solution. This dimerization interface resides in the short H1 helix previously suggested to be involved in protein-protein interaction (10Cho Y. Gorina S. Jeffrey P.D. Pavletich N.P. Science. 1994; 265: 346-355Crossref PubMed Scopus (2145) Google Scholar). Based on NMR data, we have created a consistent model for the dimeric p53 DBD-DNA complex. The findings that inactivating hot spot p53 mutations map in the dimerization interface (43Walker D.R. Bond J.P. Tarone R.E. Harris C.C. Makalowski W. Boguski M.S. Greenblatt M.S. Oncogene. 1999; 18: 211-218Crossref PubMed Scopus (166) Google Scholar) and that the putative p53 inhibitor 53BP2 binds to this region via one of its ankyrin repeats (44Gorina S. Pavletich N.P. Science. 1996; 274: 1001-1005Crossref PubMed Scopus (395) Google Scholar) further support this model. We therefore conclude that the experimentally identified region forms the actual p53 interface in the tetrameric p53-DNA complex. Based on the symmetry of the dimeric p53 DBD-DNA complex a sandwich-like model (12Waterman J.L. Shenk J.L. Halazonetis T.D. EMBO J. 1995; 14: 512-519Crossref PubMed Scopus (129) Google Scholar) is discussed for the intact tetrameric p53-DNA complex. This model is characterized by tetrameric p53 binding as a dimer of dimers to two separate juxtaposed DNA consensus sites, implying an inherent ability of p53 to link DNA strands, e.g. in transcriptionally active complexes (45Stenger J.E. Tegtmeyer P. Mayr G.A. Reed M. Wang Y. Wang P. Hough P.V. Mastrangelo I.A. EMBO J. 1994; 13: 6011-6020Crossref PubMed Scopus (109) Google Scholar). It brings into accord the symmetry of the p53 TD with the structural requirements of p53 DBD binding to the palindromic DNA consensus sequence without assuming a conformational switch upon DNA binding. All chemicals used were of analytical grade and obtained from major commercial suppliers. TAMRA-labeled DNA oligonucleotides were purchased from MWG-BIOTECH and TIB Molbiol.15NH4Cl and [13C6]glucose was obtained from Martek Biosciences. C-terminal (residues 361–382: GSRAHSSHLKSKKGQSTSRHKK-NH2) and N-terminal (residues 79–94: APAAPTPAAPAPAPSWPLS-NH2) p53 peptides were synthesized using standard Fmoc peptide chemistry and purified by reversed phase high performance liquid chromatography. Residues 94–312 of human p53 coding for the wild-type p53 DBD (10Cho Y. Gorina S. Jeffrey P.D. Pavletich N.P. Science. 1994; 265: 346-355Crossref PubMed Scopus (2145) Google Scholar, 21Bullock A.N. Henckel J. Fersht A.R. Oncogene. 2000; 19: 1245-1256Crossref PubMed Scopus (325) Google Scholar, 23Wong K.B. DeDecker B.S. Freund S.M. Proctor M.R. Bycroft M. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8438-8442Crossref PubMed Scopus (178) Google Scholar) were amplified from plasmid pT7.7Hup53 (46Midgley C.A. Fisher C.J. Bartek J. Vojtesek B. Lane D. Barnes D.M. J. Cell Sci. 1992; 101: 183-189Crossref PubMed Google Scholar) by polymerase chain reaction and cloned into a modified pQE40 vector (Qiagen). p53 DBDs were expressed as inclusion bodies inEscherichia coli BL21 co-transfected with pUBS520 (47Brinkmann U. Mattes R.E. Buckel P. Gene (Amst.). 1989; 85: 109-114Crossref PubMed Scopus (336) Google Scholar). For the preparation of unlabeled p53 DBD, bacteria were grown in Luria broth medium. For preparation of uniformly U-15N- and U-13C,15N-labeled p53 DBDs, bacteria were grown at 37 °C in M9 minimal medium containing antibiotics, minerals, and vitamins supplemented with 2 g/liter 15NH4Cl and 4 g/liter [13C6]glucose as only nitrogen and carbon source up to an optical density of 0.8, followed by overnight induction at 37 °C with 1 mmisopropyl-d-thiogalactoside. After induction, cells were harvested by centrifugation, resuspended, and ruptured by high-pressure dispersion. Inclusion bodies were isolated, washed, and solubilized in 100 mm Tris, pH 7.5, 6 m guanidine HCl, and 10 mm DTT as described previously (48Lilie H. Schwarz E. Rudolph R. Curr. Opin. Biotechnol. 1998; 9: 497-501Crossref PubMed Scopus (394) Google Scholar). In the following, p53 DBD was refolded according to standard procedures (48Lilie H. Schwarz E. Rudolph R. Curr. Opin. Biotechnol. 1998; 9: 497-501Crossref PubMed Scopus (394) Google Scholar) and purified as published (21Bullock A.N. Henckel J. Fersht A.R. Oncogene. 2000; 19: 1245-1256Crossref PubMed Scopus (325) Google Scholar, 23Wong K.B. DeDecker B.S. Freund S.M. Proctor M.R. Bycroft M. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8438-8442Crossref PubMed Scopus (178) Google Scholar). Due to the high purity (>80%) of the inclusion body preparation, no additional Heparin Hi-Trap column was necessary during the purification. Refolded and concentrated p53 DBD was dialyzed into 50 mm potassium phosphate, pH 6.8, 50 mmKCl, and 5 mm DTT, loaded onto a SP-Sepharose Fast Flow cation exchange column (Amersham Bioscience, Inc.) and eluted with a linear KCl gradient. The final purification was achieved by size exclusion chromatography on a High Load 26/60 Superdex 75 column (Amersham Bioscience, Inc.) in 50 mm potassium phosphate, pH 6.8, 150 mm KCl, and 5 mm DTT. The purity of p53 DBD was >98%. 5% (v/v) 2H2O was added to the p53 DBD and samples were concentrated using 5 K Ultrafree 4 Centrifugal Filter Devices (Millipore) to 200–500 μm, dialyzed into 50 mm potassium phosphate, pH 6.8, and 5 mm DTT containing 40–100 mm KCl, flash-frozen in liquid nitrogen, and stored at −80 °C. Final yields were some 60 mg of uniformly U-15N-labeled and some 45 mg of uniformly U-13C,15N-labeled p53 DBD per 1-liter culture. Electrospray mass-spectrometry confirmed the identity and complete isotopic labeling of p53 DBD, as well as cleavage of the N-terminal methionine after translation. The protein concentration was measured spectrophotometrically according to Bradford (49Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215575) Google Scholar) or using an extinction coefficient of ε280 nm = 15,930 m−1cm−1 (50Edelhoch H. Biochemistry. 1967; 6: 1948-1954Crossref PubMed Scopus (3003) Google Scholar). SDS-polyacrylamide gel electrophoresis was performed with 12.5% gels. The monomeric state of all p53 DBD preparations was confirmed by analytical size exclusion chromatography with a TSK gel G 3000SW (TosoHaas) analytical gel filtration at a flow-rate of 0.5 ml/min in 50 mm potassium phosphate, pH 7.0, 150 mm KCl, and 5 mm DTT. Specific DNA binding activity of the p53 DBDs was confirmed by electrophoretic mobility shift assays (51Hupp T.R. Meek D.W. Midgley C.A. Lane D.P. Cell. 1992; 71: 875-886Abstract Full Text PDF PubMed Scopus (863) Google Scholar). The15N,1H-HSQC spectrum of the refolded U-15N-labeled p53 DBD was identical to the one published recently (23Wong K.B. DeDecker B.S. Freund S.M. Proctor M.R. Bycroft M. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8438-8442Crossref PubMed Scopus (178) Google Scholar). Quantitative analysis of the DNA binding properties of p53 DBD was performed with a ConfoCor fluorescence correlation spectrometer (Carl Zeiss Jena and Evotec OAI). 5′-TAMRA-labeled CON2x5 and CON4x5 DNA oligonucleotides containing one or two 10-mer p53 consensus half-sites, 16-meric CON2x5 (5′-CCTAGACATGCCTAAT-3′) and 26-meric CON4x5 (5′-CCTAGACATGCCTAGACATGCCTAAT-3′) (18Pavletich N.P. Chambers K.A. Pabo C.O. Genes Dev. 1993; 7: 2556-2564Crossref PubMed Scopus (443) Google Scholar), were annealed with complementary oligonucleotides. The concentrations of the annealed double-stranded DNA oligonucleotides were determined using FCS and adjusted to an equimolar ratio in relation to quarter-sites (3 nm for CON2x5, 1.5 nm for CON4x5). Measurements were performed at 20 °C in 50 mm potassium phosphate, pH 7.0, 50 mm KCl, 5 mm DTT, and 0.1% Triton X-100 in the presence of 1 nm supercoiled, nonspecific pBluescript (pBS) DNA (Stratagene) to suppress nonspecific DNA binding. Experimental autocorrelation curves were fitted using the FCS-plus 1.0 software package (Evotec OAI). For the determination of apparent binding constants by the program Prism 3.0, the values were fitted to the equation: f = [DBD)]/([DBD] +Kd), for [DNA] ≪ [DBD] and [DNA] ≪Kd, where f is the fraction of complexed TAMRA-labeled oligonucleotide and Kd is the apparent equilibrium binding constant. NMR investigations on p53 DBD were carried out on Bruker DMX750 and DMX600 spectrometers equipped with a triple channel (1H, 13C, 15N) and quadruple channel (1H, 13C, 15N,31P) inverse probe head, respectively. Water suppression was achieved by magic angle gradients (52Mattiello D.L. Warren W.S. Mueller L. Farmer B.T. J. Am. Chem. Soc. 1996; 118: 3253-3261Crossref Scopus (56) Google Scholar), either employing heteronuclear or homonuclear WATERGATE (53Piotto M. Saudek V. Sklenar V. J. Biomol. NMR. 1992; 2: 661-665Crossref PubMed Scopus (3522) Google Scholar) gradient echoes. Standard sample conditions for p53 DBD are given above; the standard temperature was 293 K. NMR diffusion experiments were carried out using first-order compensation for linear convection effects (54Jerschow A. Muller N. J. Magn. Reson. 1998; 134: 17-29Crossref PubMed Scopus (47) Google Scholar), bipolar square-shaped diffusion-encoding pulsed magnetic field gradients (2-ms duration, strength ranging from 3 to 57 G/cm), and a WATERGATE suppression scheme serving at the same time as a longitudinal eddy current compensation delay. The diffusion mixing time was set to 125 ms. Saturation transfer difference experiments (55Mayer M. Meyer B. Angew. Chem. Int. Ed. Engl. 1999; 38: 1784-1788Crossref PubMed Scopus (1352) Google Scholar) were performed by selectively irradiating either on imino protons of DNA (around 12.2–13.8 ppm) or on methyl protons of p53 DBD (at 0.28 ppm), using a series of 120° Gauss pulses for 3 s. The WATERGATE suppression scheme was concomitantly used as a T2 filter (20-ms duration) to suppress residual signals from the complexed state. Residues were assigned according to Wong et al. (23Wong K.B. DeDecker B.S. Freund S.M. Proctor M.R. Bycroft M. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8438-8442Crossref PubMed Scopus (178) Google Scholar). DNA oligonucleotides CON4x5 and CON2x5 (sequence see above) were annealed, diluted into the NMR buffer, and titrated into the NMR sample. Generally, a 1.2–1.5 mexcess of consensus quarter-sites relative to the p53 DBD was necessary to achieve stochiometric binding. The pH of the solution was verified during measurements by monitoring the 31P NMR chemical shift of the phosphate buffer. Hydrodynamic calculations were performed with the DIFFC module of the DASHA software package (version 3.48b) (56Orekhov V.Y. Nolde D.E. Golovanov A.P. Korzhnev D.M. Arseniev A.S. Appl. Magnetic Res. 1995; 9: 581-588Crossref Scopus (80) Google Scholar), using the published crystal structure of the p53 DBD (PDB-ID: 1TSR, chain B) and the modeled structure of the dimeric p53 DBD-DNA complex described in this article (see below). The bead model (representing molecules as sets of bead-like spherical friction centers) was used with beads centered in all CA, CG, and CZ (side chains of Arg, Tyr, Phe, and Trp) atoms of the protein as well as in all C2 (nucleobases), C3′ (ribose), and phosphor atoms of the DNA. Beads were scaled equally and a hydration shell of 0.5 atoms thickness was added. Molecular modeling was performed with the program X-PLOR (57Brünger A.T. A System for X-ray Crystallography and NMR. Yale University Press, New Haven, CT1992Google Scholar) using the parallhdg force field. The crystal structure of the p53 DBD-DNA complex (PDB-ID: 1TSR) was the basis of the calculations (10Cho Y. Gorina S. Jeffrey P.D. Pavletich N.P. Science. 1994; 265: 346-355Crossref PubMed Scopus (2145) Google Scholar). From chain B, which is the only subunit bound specifically to the DNA in the crystal structure, a starting structure of the dimer was generated by applying a C2-symmetry operation to the monomer. First, the structure was optimized with fixed internal atom coordinates until no clashes between the subunits could be detected. Second, the internal atom coordinates were tethered according to chemical shift changes observed by the NMR experiment, i.e. atoms, which show only little shift changes were fixed, and no restraint was put on atoms, which show the strongest shift changes. The system was minimized using 300 steps of conjugate gradient, relaxed using 5000 steps with a time step of 3 fs, and a temperature of 300 K and finally another 300-step minimization was performed. Figures were created with InsightII (MSI Inc.). The allosteric model of p53 regulation proposes that the C-terminal regulatory domain of p53 interacts with the p53 DBD and keeps it in a latent state incapable of specific interaction with DNA. Upon cellular stress the C terminus is subjected to phosphorylation and acetylation, releasing the inhibitory interaction such that p53 can bind to its specific consensus sites and activate the corresponding target genes (31Hupp T.R. Lane D.P. Curr. Biol. 1994; 4: 865-875Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 32Hupp T.R. Sparks A. Lane D.P. Cell. 1995; 83: 237-245Abstract Full Text PDF PubMed Scopus (448) Google Scholar, 33Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2168) Google Scholar). In addition, several publications have suggested that an N-terminal region of p53 participates in its regulation (36Kim A.L. Raffo A.J. Brandt-Rauf P.W. Pincus M.R. Monaco R. Abarzua P. Fine R.L. J. Biol. Chem. 1999; 274: 34924-34931Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar,58Hansen S. Lane D.P. Midgley C.A. J. Mol. Biol. 1998; 275: 575-588Crossref PubMed Scopus (36) Google Scholar, 59Muller-Tiemann B.F. Halazonetis T.D. Elting J.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6079-6084Crossref PubMed Scopus (77) Google Scholar). The published NMR assignment for p53 DBD (23Wong K.B. DeDecker B.S. Freund S.M. Proctor M.R. Bycroft M. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8438-8442Crossref PubMed Scopus (178) Google Scholar) allowed us to probe for the postulated interaction between the C terminus and the p53 DBD (60Selivanova G. Ryabchenko L. Jansson E. Iotsova V. Wiman K.G. Mol. Cell. Biol. 1999; 19: 3395-3402Crossref PubMed Scopus (129) Google Scholar) by NMR spectroscopy and to map possible shift differences upon the protein structure. The experiments were carried out by titrating U-15N-labeled p53 DBD (residues 94–312) with the C-terminal peptide (residues 361–382, phosphorylated and unphosphorylated) under varying conditions (i.e. varied temperatures, pH, and ionic strengths). These NMR experiments included protein-detected (15N,1H-HSQC) and peptide-detected methods (1H spectra, diffusion-ordered spectroscopy, inversion recovery, and saturation transfer difference experiments with selective saturation of p53 DBD methyl groups). None of the performed experiments, however, produced any indication of molecular interactions between the C-terminal peptide and p53 DBD. In addition, no direct interactions were found between a peptide covering the N-terminal polyproline-rich part of p53 (residues 76–94) (59Muller-Tiemann B.F. Halazonetis T.D. Elting J.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6079-6084Crossref PubMed Scopus (77) Google Scholar) and the U-15N-labeled p53 DBD. Likewise, no contacts were found between the C-terminal peptide (residues 361–382) and the polyproline-rich region of a U-15N-labeled N-terminal extended p53 DBD construct (residues 40–312) (36Kim A.L. Raffo A.J. Brandt-Rauf P.W. Pincus M.R. Monaco R. Abarzua P. Fine R.L. J. Biol. Chem. 1999; 274: 34924-34931Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), which proved to be highly flexible in the NMR spectra. We then verified the hypothesis that consensus DNA might be involved in the interaction between the C terminus and the p53 DBD. While no direct interaction of the C-terminal peptide with p53 DBD in complex with DNA consensus oligonucleotide could be detected, we observed clear evidence of weak unspecific interactions between the peptide and the oligonucleotide as titratable NMR shift changes of the isolated DNA imino protons (data not shown). Earlier studies have demonstrated that p53 DBD cooperatively binds to DNA consensus sequences covering one or two consensus half-sites, whereas binding to a single quarter-site cannot be detected by electrophoretic mobility shift assays (11Wang Y. Schwedes J.F. Parks D. Mann K. Tegtmeyer P. Mol. Cell. Biol. 1995; 15: 2157-2165Crossref PubMed Scopus (146) Google Scholar, 16McLure K.G. Lee P.W. EMBO
Referência(s)