Structural Details on mdm2-p53 Interaction
2005; Elsevier BV; Volume: 280; Issue: 46 Linguagem: Inglês
10.1074/jbc.m508578200
ISSN1083-351X
AutoresSeung‐Wook Chi, Si‐Hyung Lee, Do‐Hyoung Kim, Min-Jung Ahn, Kim Js, Jin-Young Woo, Takuya Torizawa, Masatsune Kainosho, Kyou‐Hoon Han,
Tópico(s)Epigenetics and DNA Methylation
ResumoMdm2 is a cellular antagonist of p53 that keeps a balanced cellular level of p53. The two proteins are linked by a negative regulatory feedback loop and physically bind to each other via a putative helix formed by residues 18-26 of p53 transactivation domain (TAD) and its binding pocket located within the N-terminal 100-residue domain of mdm2 (Kussie, P. H., Gorina, S., Marechal, V., Elenbaas, B., Moreau, J., Levine, A. J., and Pavletich, N. P. (1996) Science 274, 948-953). In a previous report we demonstrated that p53 TAD in the mdm2-freee state is mostly unstructured but contains two nascent turns in addition to a "preformed" helix that is the same as the putative helix mediating p53-mdm2 binding. Here, using heteronuclear multidimensional NMR methods, we show that the two nascent turn motifs in p53 TAD, turn I (residues 40-45) and turn II (residues 49-54), are also capable of binding to mdm2. In particular, the turn II motif has a higher mdm2 binding affinity (∼20 μm) than the turn I and targets the same site in mdm2 as the helix. Upon mdm2 binding this motif becomes a well defined full helix turn whose hydrophobic face formed by the side chains of Ile-50, Trp-53, and Phe-54 inserts deeply into the helix binding pocket. Our results suggest that p53-mdm2 binding is subtler than previously thought and involves global contacts such as multiple "non-contiguous" minimally structured motifs instead of being localized to one small helix mini-domain in p53 TAD. Mdm2 is a cellular antagonist of p53 that keeps a balanced cellular level of p53. The two proteins are linked by a negative regulatory feedback loop and physically bind to each other via a putative helix formed by residues 18-26 of p53 transactivation domain (TAD) and its binding pocket located within the N-terminal 100-residue domain of mdm2 (Kussie, P. H., Gorina, S., Marechal, V., Elenbaas, B., Moreau, J., Levine, A. J., and Pavletich, N. P. (1996) Science 274, 948-953). In a previous report we demonstrated that p53 TAD in the mdm2-freee state is mostly unstructured but contains two nascent turns in addition to a "preformed" helix that is the same as the putative helix mediating p53-mdm2 binding. Here, using heteronuclear multidimensional NMR methods, we show that the two nascent turn motifs in p53 TAD, turn I (residues 40-45) and turn II (residues 49-54), are also capable of binding to mdm2. In particular, the turn II motif has a higher mdm2 binding affinity (∼20 μm) than the turn I and targets the same site in mdm2 as the helix. Upon mdm2 binding this motif becomes a well defined full helix turn whose hydrophobic face formed by the side chains of Ile-50, Trp-53, and Phe-54 inserts deeply into the helix binding pocket. Our results suggest that p53-mdm2 binding is subtler than previously thought and involves global contacts such as multiple "non-contiguous" minimally structured motifs instead of being localized to one small helix mini-domain in p53 TAD. p53 is known to be implicated in more than 50% of all human cancers and probably represents one of the proteins that is most critically associated with cancer (1.Vogelstein B. Lane D. Levine A.J. Nature. 2000; 408: 307-310Crossref PubMed Scopus (5905) Google Scholar, 2.Sherr C.J. Cell. 2004; 116: 235-246Abstract Full Text Full Text PDF PubMed Scopus (795) Google Scholar). Understanding how this "hub" in the cancer protein network interacts with other members of the network is not only important for gaining insights into fundamental principles underlying tumorigenesis but also for efficient development of anticancer agents (3.Chene P. Nat. Rev. Cancer. 2003; 3: 102-109Crossref PubMed Scopus (591) Google Scholar, 4.Garcia-Echeverria C. Chene P. Blommers M.J.J. Furet P. J. Med. Chem. 2000; 43: 3205-3208Crossref PubMed Scopus (220) Google Scholar, 5.Issaeva N. Bozko P. Enge M. Protopopova M. Verhoef L.G. Masucci M. Pramanik A. Selivanova G. Nat. Med. 2004; 10: 1321-1328Crossref PubMed Scopus (665) Google Scholar, 6.Vassilev L.T. Vu B.T. Graves B. Carvajal D. Podlaski F. Filipovic Z. Kong N. Kammlott U. Lukacs C. Klein C. Fotouhi N. Liu E.A. Science. 2004; 303: 844-848Crossref PubMed Scopus (3902) Google Scholar). Ironically, establishing a structure-function relationship for p53 has been possible only for ∼30% of its amino acid residues; namely, for those forming globular domains, a DNA binding domain (7.Cho Y. Gorina S. Jeffrey P.D. Pavletich N.P. Science. 1994; 265: 346-355Crossref PubMed Scopus (2195) Google Scholar) and an oligomerization domain (8.Clore G.M. Omichinski J.G. Sakaguchi K. Zambrano N. Sakamoto H. Appella E. Gronenborn A.M. Science. 1994; 265: 386-391Crossref PubMed Scopus (287) Google Scholar, 9.Jeffrey P.D. Gorina S. Pavletich N.P. Science. 1995; 267: 1498-1502Crossref PubMed Scopus (444) Google Scholar). This fact can be attributed to a rather interesting finding that a large fraction (∼70%) of amino acid residues in p53 does not participate in forming a well defined tertiary structure, a common feature shared by many intrinsically unstructured proteins (IUPs) 2The abbreviations used are: IUP, intrinsically unstructured protein; TAD, transactivation domain; mdm2, mouse double minute-2; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; HSQC, heteronuclear single quantum coherence; TOCSY, total shift correlation spectroscopy; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight. (10.Wright P.E. Dyson H.J. J. Mol. Biol. 1999; 293: 321-331Crossref PubMed Scopus (2389) Google Scholar, 11.Dunker A.K. Obradovic Z. Romero P. Garner E.C. Brown C.J. Genome Inform. Ser. Workshop Genome Inform. 2000; 11: 161-171PubMed Google Scholar, 12.Lee H. Mok K.H. Muhandiram R. Park K.H. Suk J.E. Kim D.H. Chang J. Sung Y.C. Choi K.Y. Han K.H. J. Biol. Chem. 2000; 275: 29426-29432Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 13.Bell S. Klein C. Muller L. Hansen S. Buchner J. J. Mol. Biol. 2002; 322: 917-927Crossref PubMed Scopus (219) Google Scholar, 14.Kumar R. Thompson E.B. Mol. Endocrinol. 2003; 17: 1-10Crossref PubMed Scopus (160) Google Scholar, 15.Mucsi Z. Hudecz F. Hollosi M. Tompa P. Friedrich P. Protein Sci. 2003; 12: 2327-2336Crossref PubMed Scopus (34) Google Scholar, 16.Fuxreiter M. Simon I. Friedrich P. Tompa P. J. Mol. Biol. 2004; 338: 1015-1026Crossref PubMed Scopus (462) Google Scholar). IUPs are an interesting class of proteins that maintain their function despite the lack of a well defined globular structure. Structurally, IUPs are in a similar state as folding intermediates but are distinct from the latter in that they are not in an artificially denatured state. Although some IUPs totally lack any structural elements, others have minimal secondary structural elements (12.Lee H. Mok K.H. Muhandiram R. Park K.H. Suk J.E. Kim D.H. Chang J. Sung Y.C. Choi K.Y. Han K.H. J. Biol. Chem. 2000; 275: 29426-29432Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 16.Fuxreiter M. Simon I. Friedrich P. Tompa P. J. Mol. Biol. 2004; 338: 1015-1026Crossref PubMed Scopus (462) Google Scholar, 17.Hua Q.X. Jia W.H. Bullock B.P. Habener J.F. Weiss M.A. Biochemistry. 1998; 37: 5858-5866Crossref PubMed Scopus (88) Google Scholar, 18.Zitzewitz J.A. Ibarra-Molero B. Fishel D.R. Terry K.L. Matthews C.R. J. Mol. Biol. 2000; 296: 1105-1116Crossref PubMed Scopus (114) Google Scholar, 19.Paker D. Rivera M. Zor T. Henrion-Caude A. Radhakrishnan I. Kumar A. Shapiro L.H. Wright P.E. Montminy M. Brindle P.K. Mol. Cell. Biol. 1999; 19: 5601-5607Crossref PubMed Scopus (117) Google Scholar). One subgroup of IUPs consists of unstructured or flexible domains consisting of more than ∼50 amino acid residues within large mother proteins (12.Lee H. Mok K.H. Muhandiram R. Park K.H. Suk J.E. Kim D.H. Chang J. Sung Y.C. Choi K.Y. Han K.H. J. Biol. Chem. 2000; 275: 29426-29432Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 20.Uversky V.N. Protein Sci. 2002; 11: 739-756Crossref PubMed Scopus (1544) Google Scholar, 21.Dunker A.K. Brown C.J. Lawson J.D. Iakoucheva L.M. Obradovic Z. Biochemistry. 2002; 41: 6573-6582Crossref PubMed Scopus (1515) Google Scholar). Because of their flexible nature, structural features of IUPs can be characterized in a quantitative manner only by NMR spectroscopy since local structural or dynamic features are well reflected in NMR parameters such as NOEs, coupling constants, temperature coefficient of the backbone amide protons, and hydrogen exchange rate of labile protons as well as relaxation behavior (12.Lee H. Mok K.H. Muhandiram R. Park K.H. Suk J.E. Kim D.H. Chang J. Sung Y.C. Choi K.Y. Han K.H. J. Biol. Chem. 2000; 275: 29426-29432Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 22.Dyson H.J. Wright P.E. Adv. Protein Chem. 2002; 62: 311-340Crossref PubMed Scopus (214) Google Scholar, 23.Vise P.D. Baral B. Latos A.J. Daughdrill G.W. Nucleic Acids Res. 2005; 33: 2061-2077Crossref PubMed Scopus (83) Google Scholar). Recent NMR investigations on p53 TAD pointed out that the full-length transcriptional activation domain in p53 (∼70 residues at the N terminus of p53) is intrinsically unstructured under physiological conditions (12.Lee H. Mok K.H. Muhandiram R. Park K.H. Suk J.E. Kim D.H. Chang J. Sung Y.C. Choi K.Y. Han K.H. J. Biol. Chem. 2000; 275: 29426-29432Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 13.Bell S. Klein C. Muller L. Hansen S. Buchner J. J. Mol. Biol. 2002; 322: 917-927Crossref PubMed Scopus (219) Google Scholar, 24.Dawson R. Muller L. Dehner A. Klein C. Kessler H. Buchner J. J. Mol. Biol. 2003; 3: 1131-1141Crossref Scopus (201) Google Scholar). When carefully analyzed, the p53 TAD was found to have three minimally structured motifs, a helix and two nascent turns, although it did not have a globular structure (12.Lee H. Mok K.H. Muhandiram R. Park K.H. Suk J.E. Kim D.H. Chang J. Sung Y.C. Choi K.Y. Han K.H. J. Biol. Chem. 2000; 275: 29426-29432Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). The helix formed by residues 18-26 can be most clearly identified and preexists even in the absence of any target protein. This helix coincides with the amphipathic helix that was reported to be induced upon mdm2 binding (25.Kussie P.H. Gorina S. Marechal V. Elenbaas B. Moreau J. Levine A.J. Pavletich N.P. Science. 1996; 274: 948-953Crossref PubMed Scopus (1845) Google Scholar). During the last several years extensive structural studies on the helix motif of p53 TAD have been carried out including a detailed structural investigation of the structure of the p53 helix-mdm2 complex (25.Kussie P.H. Gorina S. Marechal V. Elenbaas B. Moreau J. Levine A.J. Pavletich N.P. Science. 1996; 274: 948-953Crossref PubMed Scopus (1845) Google Scholar, 26.Schon O. Friedler A. Bycroft M. Freund S.M. Fersht A.R. J. Mol. Biol. 2002; 323: 491-501Crossref PubMed Scopus (274) Google Scholar, 27.Schon O. Friedler A. Freund S. Fersht A.R. J. Mol. Biol. 2004; 336: 197-202Crossref PubMed Scopus (50) Google Scholar, 28.Stoll R. Renner C. Hansen S. Palme S. Klein C. Belling A. Zeslawski W. Kamionka M. Rehm T. Muhlhahn P. Schumacher R. Hesse F. Kaluza B. Voelter W. Engh R.A. Holak T.A. Biochemistry. 2001; 40: 336-344Crossref PubMed Scopus (262) Google Scholar). Such studies have paved the way for designing various mdm2 inhibitors as potential anticancer therapeutics (3.Chene P. Nat. Rev. Cancer. 2003; 3: 102-109Crossref PubMed Scopus (591) Google Scholar, 4.Garcia-Echeverria C. Chene P. Blommers M.J.J. Furet P. J. Med. Chem. 2000; 43: 3205-3208Crossref PubMed Scopus (220) Google Scholar). On the other hand, keen emphasis on the significance of the helix motif has created a view that the helix must be the only specificity determinant in p53 that governs mdm2-binding or transcriptional activity. Noting a valid assumption that proteins would not form structures without a purpose, be they tertiary or secondary, we have attempted to characterize the potential role of the turn motifs in p53 TAD for p53 function. The results indicate that the helix motif is not the only determinant governing p53-mdm2 binding. Protein Preparation—The full-length 15N-labeled p53 TAD (1-73) was expressed and purified as has been reported previously (12.Lee H. Mok K.H. Muhandiram R. Park K.H. Suk J.E. Kim D.H. Chang J. Sung Y.C. Choi K.Y. Han K.H. J. Biol. Chem. 2000; 275: 29426-29432Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). A recombinant mdm2 construct corresponding to residues 3-109 was expressed in pLM1 vector (29.Uesugi M. Verdine G.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14801-14806Crossref PubMed Scopus (123) Google Scholar). Transformed Escherchia coli BL21(DE3) cells were grown at 37 °C to an A600 of 0.6, and the culture was induced with 0.4 mm isopropyl thio-β-d-thiogalactopyranoside. Then the cells were further cultivated at 20 °C for 16 h. The harvested cell suspension was sonicated in 50 mm Tris-HCl (pH 7.5), 0.4 m NaCl, 1 mm phenylmethylsulfonyl fluoride, 10 mm β-mercaptoethanol and centrifuged for 30 min at 30,000 × g. Proteins in the supernatant were precipitated with ammonium sulfate. For purification the same method was used to purify non-labeled, 15N-labeled, or 13C,15N-labeled mdm2-(3-109) using an SP-Sepharose column, a Q-Sepharose column, and a Hiprep 26/60 Sephacryl S-200 FPLC column (Amersham Biosciences). The molecular weights of the purified proteins were confirmed by MALDI-TOF mass spectrometry. Peptide Preparation—The p53 helix peptide (15.Mucsi Z. Hudecz F. Hollosi M. Tompa P. Friedrich P. Protein Sci. 2003; 12: 2327-2336Crossref PubMed Scopus (34) Google Scholar, 16.Fuxreiter M. Simon I. Friedrich P. Tompa P. J. Mol. Biol. 2004; 338: 1015-1026Crossref PubMed Scopus (462) Google Scholar, 17.Hua Q.X. Jia W.H. Bullock B.P. Habener J.F. Weiss M.A. Biochemistry. 1998; 37: 5858-5866Crossref PubMed Scopus (88) Google Scholar, 18.Zitzewitz J.A. Ibarra-Molero B. Fishel D.R. Terry K.L. Matthews C.R. J. Mol. Biol. 2000; 296: 1105-1116Crossref PubMed Scopus (114) Google Scholar, 19.Paker D. Rivera M. Zor T. Henrion-Caude A. Radhakrishnan I. Kumar A. Shapiro L.H. Wright P.E. Montminy M. Brindle P.K. Mol. Cell. Biol. 1999; 19: 5601-5607Crossref PubMed Scopus (117) Google Scholar, 20.Uversky V.N. Protein Sci. 2002; 11: 739-756Crossref PubMed Scopus (1544) Google Scholar, 21.Dunker A.K. Brown C.J. Lawson J.D. Iakoucheva L.M. Obradovic Z. Biochemistry. 2002; 41: 6573-6582Crossref PubMed Scopus (1515) Google Scholar, 22.Dyson H.J. Wright P.E. Adv. Protein Chem. 2002; 62: 311-340Crossref PubMed Scopus (214) Google Scholar, 23.Vise P.D. Baral B. Latos A.J. Daughdrill G.W. Nucleic Acids Res. 2005; 33: 2061-2077Crossref PubMed Scopus (83) Google Scholar, 24.Dawson R. Muller L. Dehner A. Klein C. Kessler H. Buchner J. J. Mol. Biol. 2003; 3: 1131-1141Crossref Scopus (201) Google Scholar, 25.Kussie P.H. Gorina S. Marechal V. Elenbaas B. Moreau J. Levine A.J. Pavletich N.P. Science. 1996; 274: 948-953Crossref PubMed Scopus (1845) Google Scholar, 26.Schon O. Friedler A. Bycroft M. Freund S.M. Fersht A.R. J. Mol. Biol. 2002; 323: 491-501Crossref PubMed Scopus (274) Google Scholar, 27.Schon O. Friedler A. Freund S. Fersht A.R. J. Mol. Biol. 2004; 336: 197-202Crossref PubMed Scopus (50) Google Scholar, 28.Stoll R. Renner C. Hansen S. Palme S. Klein C. Belling A. Zeslawski W. Kamionka M. Rehm T. Muhlhahn P. Schumacher R. Hesse F. Kaluza B. Voelter W. Engh R.A. Holak T.A. Biochemistry. 2001; 40: 336-344Crossref PubMed Scopus (262) Google Scholar, 29.Uesugi M. Verdine G.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14801-14806Crossref PubMed Scopus (123) Google Scholar), p53 linker peptide (28-37), and several p53 turn peptides were synthesized by a solid phase method with Multiple Peptide Synthesizer APEX 348Ω (Advanced Chemtech). Synthesized p53 turn peptides include the long turn peptide (39-57) (AMDDLMLSPDDIEQWFTED), the turn I peptide (39-48) (AMDDLMLSPD), the turn II peptide (49-54) (DIEQWF), and a Trp analog of the turn II (DWEQWW). The C termini of all the synthesized peptides were amidated. The peptides were purified by reverse phase high performance liquid chromatography using Vydac C18 columns, and the peptide masses were confirmed by MALDI-TOF mass spectrometry. NMR Spectroscopy—NMR spectra were acquired using a Varian Unity INOVA 600 spectrometer, a Bruker DRX 600 MHz spectrometer equipped with a cryoprobe, and a Bruker DRX 800 MHz spectrometer. Aliquots of a concentrated mdm2 stock solution were added in a stepwise fashion to the 15N-labeled p53 TAD during titration. NMR samples containing 0.4 mm 15N-labeled p53 TAD alone or with mdm2 were prepared in 90% H2O, 10% 2H2O, 50 mm tritiated sodium acetate (pH 6.3) and 50 mm NaCl and remained stable for more than 6 months. At each titration point an 1H, 15N heteronuclear single quantum coherence spectroscopy (HSQC) spectrum was collected at 5 °C. The molar ratios of p53 TAD to mdm2 were 1:0.3, 1:0.6, 1:1, and 1:2. Next, 15N-labeled mdm2-(3-109) was titrated with a series of non-labeled p53 peptides including the helix peptide (15-29), the linker peptide (28-37), the long turn peptide (39-57), the turn I peptide (39-48), the turn II peptide (49-54), and the Trp analog of the turn II. During titration, 1H,15N HSQC spectra of 0.2 mm 15N-labeled mdm2-(3-109) in 25 mm Tris-HCl (pH 7.5), 150 mm NaCl, 2 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, 0.1 mm EDTA, 0.1 mm benzamidine, and 0.02% NaN3 were collected at 25 °C. For the titration of mdm2 with p53, the final molar ratio of mdm2 to p53 peptides was 1:3. Backbone sequential assignment of mdm2-(3-109) bound to the p53 helix peptide (15-29) was obtained from three-dimensional HNCA, HNCOCA, 15N-edited total shift correlation spectroscopy (TOCSY), and 15N-edited NOE spectroscopy (NOESY) (τmix = 150 ms). All data were processed and analyzed on a Sun SPARCstation using Varian Vnmr and nmrPipe/nmrDraw software. Transferred NOE Experiment—For transferred NOE experiments the p53 turn (39-57) peptide was dissolved in 90% H2O, 10% 2H2O containing 25 mm Tris-HCl (pH 7.5) and 150 mm NaCl. The NOESY spectrum of 2 mm p53 long turn (39-57) was obtained in the absence or presence of 0.1 mm mdm2-(3-109) at 10 °C with a mixing time of 100 ms. The resonance assignment of the long p53 turn peptide (39-57) was obtained by standard two-dimensional NMR experiments such as TOCSY, double-quantum-filtered correlation spectroscopy, and NOESY. Mixing times of 100 ms for NOESY and 70 ms for TOCSY experiments were used. The three-bond coupling constants, 3JHNHα, for backbone torsion angles were measured using phase-sensitive double-quantum-filtered correlation spectroscopy experiments. The two-dimensional NMR data consist of 2048 complex points in the t2 dimension with 256 complex t1 increments. Spectral widths were 8 kHz in both dimensions. Surface Plasma Resonance—Surface plasma resonance experiments for the turn II peptide (49-54) and the helix peptide (15-29) were carried out using concentrations ranging from 1 to 50 μm in HBS buffer (10 mm HEPES (pH 7.4), 150 mm NaCl, 1 mm EDTA, and 0.005% Tween 20) with a flow rate of 20 μl/min at 25 °C in a BIAcore 2000 instrument (BIAcore AB, Uppsala, Sweden). Mdm2 (3-109) protein in 25 mm sodium acetate (pH 4.5) and 150 mm NaCl was immobilized on a CM5 sensor chip using an amine coupling kit (BIAcore AB). Kinetic measurements were made, and kinetic constants were derived with the BIAe-valuation Version 3.0 software (BIAcore AB) using control flow-subtracted sensograms. Molecular Modeling—A structural model for the complex of mdm2 with p53 turn II motif was generated using the molecular modeling package Insight II on a Silicon Graphics O2 work station. On the basis of transferred NOE data, the turn II peptide of p53 was modeled into an ideal α-helix using the builder and biopolymer modules of Insight II. Initially, the turn II peptide was manually docked on the p53 helix binding groove of the crystal structure of mdm2 (25-109) (Protein Data Bank code 1YCR). The modeled complex structure was based on the contact points from the NMR chemical shift perturbations. The structure was energy-minimized until convergence to 0.01 kcal/mol/Å to remove steric clashes. The DISCOVER module of Insight II and cvff forcefield were used to carry out energy minimization and molecular dynamics. The molecular dynamics simulation was performed for 200 ps at 300 K with the protein backbones fixed. The integration time step was set up to 1 fs. At the end of the dynamics run, energy minimization was performed again until convergence to 0.01 kcal/mol/Å. Figures were drawn by using the programs GRASP and RIBBONS. To test the hypothesis that the turn motifs or any other segments in p53 TAD may bind to mdm2, we first acquired and compared two 1H, 15N HSQC spectra of 15N-labeled p53 TAD, one in the absence and the other in the presence of mdm2-(3-109). The results summarized in Fig. 1 indicate that mdm2 binding affects a large number of resonances in p53 TAD, not just those from the helix; ∼60% (43 of 73) of p53 TAD resonances either completely disappear, broaden, or experience chemical shift perturbation upon mdm2 binding. No further changes in the NMR spectra were observed beyond the titration point of (mdm2/p53 TAD) = 1. The fact that the resonances from the helix-forming residues disappear due to mdm2 binding concurs well with the previous observation that the helix motif binds to mdm2 with an affinity of ∼1 μm (25.Kussie P.H. Gorina S. Marechal V. Elenbaas B. Moreau J. Levine A.J. Pavletich N.P. Science. 1996; 274: 948-953Crossref PubMed Scopus (1845) Google Scholar). On the other hand our data suggest that the residues from the linker region (residues 28-37) between the helix and the turns and those involved in formation of nascent turns may be involved in mdm2 binding as the resonances from these residues also experience spectral change. Binding of p53 TAD (∼8 kDa) to mdm2 (∼12 kDa) would slow down the overall tumbling motion of p53 TAD and consequently shorten its transverse relaxation time (T2), resulting in broader resonances. Further resonance broadening is expected for the helix-forming residues since these residues may exist in an mdm2-bound or an mdm2-unbound state as the mdm2 binding affinity of the helix motif is only of intermediate strength (25.Kussie P.H. Gorina S. Marechal V. Elenbaas B. Moreau J. Levine A.J. Pavletich N.P. Science. 1996; 274: 948-953Crossref PubMed Scopus (1845) Google Scholar, 26.Schon O. Friedler A. Bycroft M. Freund S.M. Fersht A.R. J. Mol. Biol. 2002; 323: 491-501Crossref PubMed Scopus (274) Google Scholar). Resonance broadening due to these two factors seems to be sufficient to make the signals from the helix-forming residues in p53 TAD disappear completely upon titration with mdm2. Similar resonance broadening has been noticed in binding of p53 TAD with hRPA1-168 (23.Vise P.D. Baral B. Latos A.J. Daughdrill G.W. Nucleic Acids Res. 2005; 33: 2061-2077Crossref PubMed Scopus (83) Google Scholar). Immediate neighbors of the helix such as the residues Ser-15-Glu-17 also experience non-negligible chemical shift perturbations. In contrast to the helix-forming resonances, the turn-forming resonances experience only chemical shift change because the turn motifs have weaker mdm2 binding affinities of 20-100 μm (Fig. 5). However, intensities of the turn-forming resonances are also weakened to a noticeable degree due to exchange broadening, as can be seen in Fig. 1B. The addition of a helix peptide to the mixture of mdm2 and p53 TAD makes the disappeared helix-forming resonances of p53 TAD nearly fully reappear, whereas adding a long turn peptide encompassing both turns (residues 39-57) leads to only partial reappearance (data not shown). Interestingly, the resonances from the "linker" residues, i.e. between the helix and the turns, also disappear even though they do not directly participate in mdm2 binding (Figs. 2B and 3B). This linker region was previously shown to have some structural order rather than being disordered. Because their T2 values are as short as those of the helix-forming residues in the mdm2-free state of p53 TAD (12.Lee H. Mok K.H. Muhandiram R. Park K.H. Suk J.E. Kim D.H. Chang J. Sung Y.C. Choi K.Y. Han K.H. J. Biol. Chem. 2000; 275: 29426-29432Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar), additional reduction in T2 due to formation of p53 TAD-mdm2 complex seems to cause their resonances to disappear.FIGURE 2The 1H, 15N HSQC spectra of mdm2-(3-109) titrated with p53 TAD ligands. A, the helix peptide (15-29). B, the linker peptide (28-37). C, the long turn peptide (39-57). D, the turn I peptide (39-48). E, the turn II peptide (49-54). F, the Trp analog of turn II. In each panel, the spectrum shown in black is for the ligand-free state. Color-coded overlaid cross-peaks are for the mdm2-bound state; red, the helix peptide (15-29); blue, the linker peptide (28-37); green, the long turn peptide (39-57), the turn I peptide (39-48), and the turn II peptide (49-54); violet, the Trp analog of turn II. The molar ratio of mdm2 to p53 was 1:3. Note that for the linker peptide and the turn I peptide the perturbation is minimal so that resonances in the absence and the presence of the peptide nearly overlap.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Chemical shift perturbation in mdm2 due to binding of p53 TAD ligands. A, the helix peptide (15-29). B, the linker peptide (28-37). C, the long turn peptide (39-57). D, the turn I peptide (39-48). E, the turn II peptide (49-54). F, the Trp analog of turn II. The Δδ 1cH,15N value is calculated as described earlier (27.Schon O. Friedler A. Freund S. Fersht A.R. J. Mol. Biol. 2004; 336: 197-202Crossref PubMed Scopus (50) Google Scholar) when the molar ratio of mdm2 to p53 is 1:3. Note the different scales of Δδc 1H,15N depending on the panels.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We then examined whether each of the mini-domains in p53 TAD, the helix, the linker, the turn I, and turn II that experience spectral change due to p53 TAD-mdm2 binding is able to bind mdm2. was accomplished by recording series to This a of 1H, 15N HSQC spectra of 15N-labeled mdm2 in the presence of various p53 TAD fragments corresponding to these mini-domains. Shown in Fig. 2 are six HSQC spectra for the helix, the linker, the long turn peptide (residues 39-57), the turn I, the turn II, and a Trp analog of the turn II, respectively. Fig. 3 is a summary of chemical shift perturbation for individual residues in mdm2 due to binding of various p53 TAD peptides. Chemical shift perturbation due to the turn I is small (Δδc < 0.1), indicating that its binding is minimal. Binding of the linker peptide is negligible with Δδc < 0.03, confirming that disappearance of the linker resonances (Fig. 1) is not due to mdm2 binding. These results are depicted in the color-coded structures of mdm2 shown in Fig. 4. In the backside of structures (data not shown) several residues outside of the helix binding pocket are affected due to ligand binding, indicating that there is a long range conformational change within mdm2 upon ligand binding as previously noted (27.Schon O. Friedler A. Freund S. Fersht A.R. J. Mol. Biol. 2004; 336: 197-202Crossref PubMed Scopus (50) Google Scholar). Fast hydrogen exchange rates (disappearing within less than an hour after addition of D2O) of most mdm2 backbone amide protons (data not shown) suggest that the N-terminal domain (3-109) of mdm2 undergoes breathing rather than being rigid despite its globular shape. Several analogs of the turn II with reasonable hydrophobicity have severely reduced mdm2 binding affinities, suggesting that the hexameric sequence integrity DIEWQF of turn II is important for mdm2 binding. One exception is the Trp analog, which shows a tighter binding to mdm2 than the native peptide (Fig. 3). When tested with a larger mdm2 N-terminal domain (residues 10-154), the mdm2 residues affected by binding of the turn II peptide remain the same, indicating that there is no other binding site for the turn motifs beyond residue 109 in mdm2. Together, these results exclude the possibility that binding of the turn motifs to mdm2 is due to nonspecific interactions. Surface plasma resonance experiments have yielded a Kd for the turn II motif of ∼20 μm (Fig. 5), whereas the turn I motif binds only minimally to mdm2. The binding of turn I is so weak that its Kd is estimated to be >100 μm, which is more or less a measurement limit by the surface plasma resonance technique. Because a reasonable correlation exists between the Kd and the Δδc values, one may use the Δδc value of an mdm2 ligand as an indicator for mdm2 binding affinity. The long turn peptide is likely to exhibit a slightly higher affinity than the turn II peptide based on the chemical shift perturbation (compare Fig. 3, C and E). The result that the significantly affected mdm2 residues due to binding of the turn II and the long turn peptide mostly overlap with those affected by helix binding suggests that this turn motif binds to the same helix binding pocket in mdm2. However, the turn-bound mdm2 fraction would be small since the mdm2 binding affinity of the turn region is weaker than that of the helix. One should view the results shown in Fig. 1C as reflecting the presence of a mixture between the helix-bound form of mdm2 and an mdm2 complexed with the turn region (40-55) of p53 TAD. Interestingly, binding of the helix and turn II differentially influenc
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