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• W2051210433 abstract "p53-dependent apoptosis is modulated by the ASPP family of proteins (apoptosis-stimulating proteins of p53; also called ankyrin repeat-, Src homology 3 domain-, and Pro-rich region-containing proteins). Its three known members, ASPP1, ASPP2, and iASPP, were previously found to interact with p53, influencing the apoptotic response of cells without affecting p53-induced cell cycle arrest. More specifically, the bona fide tumor suppressors, ASPP1 and ASPP2, bind to the core domain of p53 and stimulate transcription of apoptotic genes, whereas oncogenic iASPP also binds to the p53 core domain but inhibits p53-dependent apoptosis. Although the general interaction regions are known, details of the interfaces for each p53-ASPP complex have not been evaluated. We undertook a comprehensive biophysical characterization of ASPP-p53 complex formation and mapped the binding interfaces by NMR. We found that the interaction interface on p53 for the proapoptotic protein ASPP2 is distinct from that for the antiapoptotic iASPP. ASPP2 primarily binds to the core domain of p53, whereas iASPP predominantly interacts with a linker region adjacent to the core domain. Our detailed structural analyses of the ASPP-p53 interactions provide insight into the structural basis of the differential behavior of pro- and antiapoptotic ASPP family members. p53-dependent apoptosis is modulated by the ASPP family of proteins (apoptosis-stimulating proteins of p53; also called ankyrin repeat-, Src homology 3 domain-, and Pro-rich region-containing proteins). Its three known members, ASPP1, ASPP2, and iASPP, were previously found to interact with p53, influencing the apoptotic response of cells without affecting p53-induced cell cycle arrest. More specifically, the bona fide tumor suppressors, ASPP1 and ASPP2, bind to the core domain of p53 and stimulate transcription of apoptotic genes, whereas oncogenic iASPP also binds to the p53 core domain but inhibits p53-dependent apoptosis. Although the general interaction regions are known, details of the interfaces for each p53-ASPP complex have not been evaluated. We undertook a comprehensive biophysical characterization of ASPP-p53 complex formation and mapped the binding interfaces by NMR. We found that the interaction interface on p53 for the proapoptotic protein ASPP2 is distinct from that for the antiapoptotic iASPP. ASPP2 primarily binds to the core domain of p53, whereas iASPP predominantly interacts with a linker region adjacent to the core domain. Our detailed structural analyses of the ASPP-p53 interactions provide insight into the structural basis of the differential behavior of pro- and antiapoptotic ASPP family members. Since the discovery of p53 in 1979 (1Lane D.P. Crawford L.V. Nature. 1979; 278: 261-263Crossref PubMed Scopus (1766) Google Scholar, 2Linzer D.I. Levine A.J. Cell. 1979; 17: 43-52Abstract Full Text PDF PubMed Scopus (1248) Google Scholar, 3Linzer D.I. Maltzman W. Levine A.J. Virology. 1979; 98: 308-318Crossref PubMed Scopus (87) Google Scholar), the essential function of this tumor suppressor in preventing uncontrolled cell proliferation after DNA insult has become clear; p53 induces expression of genes to initiate cell cycle arrest or apoptosis (4Vousden K.H. Cell. 2000; 103: 691-694Abstract Full Text Full Text PDF PubMed Scopus (712) Google Scholar, 5Vousden K.H. Lu X. Nat. Rev. Cancer. 2002; 2: 594-604Crossref PubMed Scopus (2727) Google Scholar, 6Lu X. Curr. Opin. Genet. Dev. 2005; 15: 27-33Crossref PubMed Scopus (65) Google Scholar, 7Olivier M. Hussain S.P. Caron de Fromentel C. Hainaut P. Harris C.C. IARC Sci. Publ. 2004; 157: 247-270PubMed Google Scholar). However, the detailed numerous mechanisms by which p53 carries out its function are not fully understood. For example, control of cellular fate by p53 through cell cycle arrest rather than apoptosis (or vice versa) is still a topic of debate, and several types of p53 involvement and modulation have been described (8Balint E.E. Vousden K.H. Br. J. Cancer. 2001; 85: 1813-1823Crossref PubMed Scopus (254) Google Scholar, 9Sionov R.V. Haupt Y. Oncogene. 1999; 18: 6145-6157Crossref PubMed Scopus (501) Google Scholar, 10Haupt S. Berger M. Goldberg Z. Haupt Y. J. Cell Sci. 2003; 116: 4077-4085Crossref PubMed Scopus (956) Google Scholar, 11Liebermann D.A. Hoffman B. Vesely D. Cell Cycle. 2007; 6: 166-170Crossref PubMed Scopus (66) Google Scholar, 12Pietsch E.C. Sykes S.M. McMahon S.B. Murphy M.E. Oncogene. 2008; 27: 6507-6521Crossref PubMed Scopus (239) Google Scholar). An early clue that these two cellular responses can be separated emerged from studies characterizing p53 mutants that were defective in their ability to induce apoptosis but not cell cycle arrest (13Ludwig R.L. Bates S. Vousden K.H. Mol. Cell Biol. 1996; 16: 4952-4960Crossref PubMed Scopus (252) Google Scholar, 14Rowan S. Ludwig R.L. Haupt Y. Bates S. Lu X. Oren M. Vousden K.H. EMBO J. 1996; 15: 827-838Crossref PubMed Scopus (294) Google Scholar, 15Ryan K.M. Vousden K.H. Mol. Cell Biol. 1998; 18: 3692-3698Crossref PubMed Scopus (166) Google Scholar). Since these initial reports, post-translational modifications of p53 and interactions with specific cellular proteins have been found to be more closely associated with one pathway over the other. For example, phosphorylation of Ser46 (16Oda K. Arakawa H. Tanaka T. Matsuda K. Tanikawa C. Mori T. Nishimori H. Tamai K. Tokino T. Nakamura Y. Taya Y. Cell. 2000; 102: 849-862Abstract Full Text Full Text PDF PubMed Scopus (1027) Google Scholar, 17D'Orazi G. Cecchinelli B. Bruno T. Manni I. Higashimoto Y. Saito S. Gostissa M. Coen S. Marchetti A. Del Sal G. Piaggio G. Fanciulli M. Appella E. Soddu S. Nat. Cell Biol. 2002; 4: 11-19Crossref PubMed Scopus (575) Google Scholar, 18Di Stefano V. Rinaldo C. Sacchi A. Soddu S. D'Orazi G. Exp. Cell Res. 2004; 293: 311-320Crossref PubMed Scopus (93) Google Scholar) or acetylation of Lys120 in the DNA binding domain by MYST family acetyltransferases (19Sykes S.M. Mellert H.S. Holbert M.A. Li K. Marmorstein R. Lane W.S. McMahon S.B. Mol. Cell. 2006; 24: 841-851Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar, 20Tang Y. Luo J. Zhang W. Gu W. Mol. Cell. 2006; 24: 827-839Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar) purportedly renders p53 more effective in activating proapoptotic programs. Proteins that bind p53 have also been implicated in promoting p53-dependent apoptosis (21Flores E.R. Tsai K.Y. Crowley D. Sengupta S. Yang A. McKeon F. Jacks T. Nature. 2002; 416: 560-564Crossref PubMed Scopus (725) Google Scholar, 22Shikama N. Lee C.W. France S. Delavaine L. Lyon J. Krstic-Demonacos M. La Thangue N.B. Mol. Cell. 1999; 4: 365-376Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). The ASPP proteins constitute a recently described new family of proteins that bind and modulate p53-dependent apoptosis (23Trigiante G. Lu X. Nat. Rev. Cancer. 2006; 6: 217-226Crossref PubMed Scopus (122) Google Scholar). Their name is based on the domain organization of the proteins (ankyrin repeat, SH3, 3The abbreviations used are: SH3, Src homology 3; ASPP2-CT, the C terminus of ASPP2; p53-CD, the DNA-binding core domain of p53; iASPP-CT, the C terminus of iASPP; p53-PCD, the Pro-rich and DNA-binding core domains of p53; p53-PCD2F, p53-PCD with W91F and W146F; p53-PCD2F-L, p53-PCD2F with linker; p53-PCD2F-L-OD, p53-PCD2F-L with oligomerization domain; p53-PCD2F-L-OD-BD, p53-PCD2F-L-OD with basic domain; p53-L, p53 linker between CD and OD; p53-L-OD-BD, p53 linker, oligomerization and basic domain; TEV, tobacco etch virus; ITC, isothermal titration calorimetry; HSQC, heteronuclear single quantum correlation; TROSY, transverse relaxation optimized spectroscopy; OD, oligomerization domain; BD, basic domain. and proline-rich domain-containing protein) as well as their function (apoptosis-stimulating protein of p53). The founding member of the family, ASPP2, was initially identified as 53BP2 (p53-binding protein 2) in a yeast two-hybrid screen, using the p53 DNA binding core domain as bait (24Iwabuchi K. Bartel P.L. Li B. Marraccino R. Fields S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6098-6102Crossref PubMed Scopus (359) Google Scholar). ASPP1 was identified later in a homology search (25Nagase T. Ishikawa K. Suyama M. Kikuno R. Hirosawa M. Miyajima N. Tanaka A. Kotani H. Nomura N. Ohara O. DNA Res. 1998; 5: 355-364Crossref PubMed Scopus (207) Google Scholar). Functional studies revealed that p53-induced apoptosis was substantially enhanced in the presence of ASPP1 or ASPP2 (26Lopez C.D. Ao Y. Rohde L.H. Perez T.D. O'Connor D.J. Lu X. Ford J.M. Naumovski L. Mol. Cell Biol. 2000; 20: 8018-8025Crossref PubMed Scopus (36) Google Scholar, 27Ao Y. Rohde L.H. Naumovski L. Oncogene. 2001; 20: 2720-2725Crossref PubMed Scopus (30) Google Scholar, 28Samuels-Lev Y. O'Connor D.J. Bergamaschi D. Trigiante G. Hsieh J.K. Zhong S. Campargue I. Naumovski L. Crook T. Lu X. Mol. Cell. 2001; 8: 781-794Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar), and complexes with ASPP1 or ASPP2 increased the affinity of p53 for promoters of proapoptotic genes (28Samuels-Lev Y. O'Connor D.J. Bergamaschi D. Trigiante G. Hsieh J.K. Zhong S. Campargue I. Naumovski L. Crook T. Lu X. Mol. Cell. 2001; 8: 781-794Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar, 29Bergamaschi D. Samuels Y. Sullivan A. Zvelebil M. Breyssens H. Bisso A. Del Sal G. Syed N. Smith P. Gasco M. Crook T. Lu X. Nat. Genet. 2006; 38: 1133-1141Crossref PubMed Scopus (211) Google Scholar). Such preferential activation of apoptosis by ASPP1/2 was also observed for p63 and p73 (30Bergamaschi D. Samuels Y. Jin B. Duraisingham S. Crook T. Lu X. Mol. Cell Biol. 2004; 24: 1341-1350Crossref PubMed Scopus (208) Google Scholar). The third member of ASPP family, iASPP, was originally identified as an inhibitor of NFκB (31Yang J.P. Hori M. Sanda T. Okamoto T. J. Biol. Chem. 1999; 274: 15662-15670Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Compared with ASPP1/2, iASPP inhibits p53-dependent apoptosis. Given the high sequence similarity between all three proteins, it may be possible that iASPP simply functions as a competitive inhibitor of ASPP1/2. However, iASPP specifically inhibits p53-dependent induction of proapoptotic genes (29Bergamaschi D. Samuels Y. Sullivan A. Zvelebil M. Breyssens H. Bisso A. Del Sal G. Syed N. Smith P. Gasco M. Crook T. Lu X. Nat. Genet. 2006; 38: 1133-1141Crossref PubMed Scopus (211) Google Scholar, 32Bergamaschi D. Samuels Y. O'Neil N.J. Trigiante G. Crook T. Hsieh J.K. O'Connor D.J. Zhong S. Campargue I. Tomlinson M.L. Kuwabara P.E. Lu X. Nat. Genet. 2003; 33: 162-167Crossref PubMed Scopus (312) Google Scholar), suggesting a mechanism of inhibition that involves iASPP altering the promoter binding activity of p53. The regions of ASPPs that interact with p53 have been mapped to the C termini, consisting of four ankyrin repeats and an SH3 domain (Fig. 1, A and B). The crystal structure of the complex between the C terminus of ASPP2 (ASPP2-CT) and the DNA binding core domain of p53 (p53-CD; Fig. 1C) reveals that the third and fourth ankyrin repeats and SH3 domain of ASPP2 contact the same p53-CD surface that interfaces with DNA (33Gorina S. Pavletich N.P. Science. 1996; 274: 1001-1005Crossref PubMed Scopus (396) Google Scholar). The crystal structure of the C-terminal region of iASPP (iASPP-CT) shows a very similar structure, and binding of iASPP-CT and ASPP2-CT to p53-CD exhibits comparable affinities (34Robinson R.A. Lu X. Jones E.Y. Siebold C. Structure. 2008; 16: 259-268Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Given the opposing functional outcomes of ASPP2 and iASPP interactions, we investigated whether the detailed molecular interactions of these two proteins with p53 are distinct. Here, we report on biochemical and structural studies of ASPP2-CT and iASPP-CT with various constructs of p53 (Fig. 1C). ASPP-CT binding to p53 that contains the Pro-rich and DNA-binding core domains (p53-PCD) was followed by isothermal titration calorimetry (ITC), fluorescence, and NMR methods. Our results show that both ASPP2-CT and iASPP-CT utilize very similar interaction surfaces to contact p53-PCD. However, in stark contrast to previous reports, we found that ASPP2-CT binds to p53-PCD much more tightly than iASPP-CT (∼60-fold). Extension of these binding studies to several p53 constructs (Fig. 1C) revealed that iASPP-CT and ASPP2-CT exhibit different affinities, suggesting that the molecular interaction determinants are unique to the individual complexes. Although the core domain of p53 is the primary binding target for ASPP2-CT, we found that the p53 linker region is the highest affinity target for iASPP-CT. Therefore, different regions of p53 appear to contribute to the proapopototic and antiapoptotic complexes, respectively, allowing modulation of the functional outcome. Detailed knowledge of the differential interfaces in the ASPP2-p53 and iASPP-p53 complexes will aid in the design of small therapeutic molecules that specifically target either of the two interactions. Protein Expression and Purification—cDNAs encoding human p53 and ASPP2 were purchased from Invitrogen. Constructs coding for residues 56-289 (p53-PCD), 56-322 (p53-PCD-L), 56-362 (p53-PCD-L-OD), 289-322 (p53-L), 289-393 (p53-L-OD-BD), and 56-393 (p53-PCD-L-OD-BD) of p53 (Fig. 1C) were amplified and subcloned into pET21a (EMD Chemicals, Inc., San Diego, CA) using NdeI and XhoI sites. The final protein products included an N-terminal Met and Leu-Glu-His6 at their C termini. Gene constructs coding for residues 289-322 of p53 (p53-L), 925-1128 of ASPP2 (ASPP2-CT), and 623-828 of iASPP (iASPP-CT) were amplified and subcloned into pET32a (EMD Chemicals) using EcoRI and XhoI sites. A TEV protease recognition sequence (ENLYFQS) was created between the BamHI and EcoRI sites in pET32, at the C terminus of thioredoxin. Final ASPP proteins, after TEV protease cleavage, contained Ser-Glu-Phe and Leu-Glu-His6 at their N termini and C termini, respectively. p53-L only possessed the three extra residues (Ser-Glu-Phe) at the N terminus. All p53 mutant constructs, p53-PCD-W91F/W146F, p53-PCD-L-OD-W91F/W146F, p53-PCD-L-OD-BD-W91F/W146F, p53-PCD-L-OD-BD-W91F/W146F/L344R, and p53-PCD-L-OD-BD-W91F/W146F/L344P (Fig. 1C), were created using QuikChange site-directed mutagenesis kits (Stratagene, La Jolla, CA). Nucleotide sequences were verified for the entire coding regions of all constructs. Proteins were expressed in Escherichia coli Rosetta 2 (DE3), cultured in Luria-Bertani medium, using 0.4 mm isopropyl 1-thio-β-d-galactopyranoside for induction and growth at 18 °C for 16 h. Soluble forms of His-tagged proteins were first purified using 5 ml of Ni2+-nitrilotriacetic acid columns, and aggregated material was removed by gel filtration column chromatography using Hi-Load Superdex200 16/60 (GE Healthcare) equilibrated with a buffer containing 25 mm sodium phosphate, pH 7.5, 50 mm NaCl, 1 mm dithiothreitol, and 0.02% sodium azide. The thioredoxin portions of the fusion proteins were cleaved from p53-L, ASPP2-CT, and iASPP-CT by incubating with TEV protease (ratio of 20:1) overnight at 4 °C. The expression plasmid PRK709 for TEV protease was obtained from Addgene Inc. (Cambridge, MA), and TEV protease was purified as described by Nallamsetty et al. (35Nallamsetty S. Kapust R.B. Tozser J. Cherry S. Tropea J.E. Copel T.D. Waugh D.S. Protein Expression Purif. 2004; 38: 108-115Crossref PubMed Scopus (109) Google Scholar). All final purifications of p53 proteins were performed over a Hi-Trap SP column (GE Healthcare) at pH 6.5 using a 0-1 m NaCl gradient. ASPP2-CT and iASPP-CT were purified over a Hi-Trap QP column (GE Healthcare) at pH 7.5 and a 0-1 m NaCl gradient. Buffer exchange was carried out using Amicon concentrators (Millipore, Billerica, MA), and proteins were stored at 4 °C in solution or as lyophilized powders at -80 °C. The p53-L peptides were purified over a C4 reverse phase column using an acetonitrile (5-80%) gradient with 0.1% formic acid. For isotopic labeling, proteins were expressed in modified minimal medium using 15NH4Cl and [U-13C6]glucose or [U-13C6, 2H7]-glucose as sole nitrogen and/or carbon sources, respectively. For deuterium labeling, growth media were prepared with 99.9% 2H2O. U-13C,15N,2H-Labeled proteins with selective Val/Leu-methyl and Phe/Tyr-12C,1H-protonation were prepared as described for the isotopic labeling above, except that U-13C5,15N,2H2(2,3)-labeled valine and/or unlabeled tyrosine and phenylalanine were added prior to induction (36Rosen M.K. Gardner K.H. Willis R.C. Parris W.E. Pawson T. Kay L.E. J. Mol. Biol. 1996; 263: 627-636Crossref PubMed Scopus (263) Google Scholar, 37Smith B.O. Ito Y. Raine A. Ben-Tovim L. Nietlispach D. Broadhurst W. Terada T. Kelly M. Oschkinat H. Shibata T. Yokoyama S. Laue E.D. J. Biomol. NMR. 1996; 8: 360-368Crossref PubMed Scopus (53) Google Scholar). The molecular masses of all purified proteins were confirmed by mass spectrometry, and isotope labeling efficiency was estimated to be greater than 98% for all labeled proteins. Protein concentrations were determined using theoretical extinction coefficients based on amino acid sequences using the ExPASy Proteomics Server (available on the World Wide Web). The concentration of p53-L was determined by amino acid analysis. ITC—Typically, p53 protein or ASPP2-CT, at concentrations of 20-40 μm, stirred at 307 rpm in the sample cell, were titrated with aliquots of 240-400 μm solutions of ASPP2-CT or p53 at 12 °C in the calorimeter (MicroCal Inc., Northampton, MA). Titrations were carried out in 25 mm sodium phosphate, pH 7.5, 1 mm dithiothreitol, and 0.02% sodium azide with varying amounts of NaCl (50-150 mm). In some titrations, 25 mm sodium phosphate, pH 7.5, was replaced by 25 mm Tris-HCl, pH 7.5. All samples were extensively buffer-exchanged using Amicon concentrators (Millipore) prior to the titrations. 10-μl aliquots were used for injections (the first injection was 3 μl) at 250-s intervals (initial delay, 60 s). Titrations into the buffers were used for base-line corrections. Data analyses were performed using Origin 7 software (OriginLab Corp., Northampton, MA). NMR Spectroscopy—All NMR experiments were conducted at 17 °C using protein samples in 25 mm sodium phosphate buffer, pH 7.5, 50 or 150 mm NaCl, 1 mm dithiothreitol, and 0.02% sodium azide, using Bruker Avance 900-, 800-, 700-, and 600-MHz spectrometers, equipped with 5-mm, triple resonance and z axis gradient cryoprobes. For backbone chemical shift assignments of ASPP2-CT and iASPP-CT, two-dimensional 1H-15N HSQC, and three-dimensional HNCACB and HN(CO)CACB experiments were performed on U-13C,15N,2H-labeled proteins or U-13C,15N,2H-labeled ones with selective Val/Ile/Leu-13C1H3 - and Phe/Tyr-12C,1H-protonation. Binding site mapping and ligand titration studies were conducted using 1H-15N TROSY-HSQC experiments on U-15N,2H- or U-13C,15N,2H-labeled samples after adding aliquots of unlabeled partner protein. A total of 5-8 1H-15N TROSY-HSQC spectra were acquired for each titration. Titration curves were plotted for five TROSY-HSQC resonances that exhibited reasonable shifts and no peak overlap or broadening. Dissociation constants were calculated by nonlinear best fitting the 1HN titration curves using KaleidaGraph (Synergy Software, Reading, PA), averaging over the five curves. All spectra were processed with NMRPipe (38Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11566) Google Scholar) and analyzed using NMRDraw and NMRView (39Johnson B.A. Methods Mol. Biol. 2004; 278: 313-352PubMed Google Scholar). Fluorescence Spectroscopy—Fluorescence titrations were carried out using a QuantaMaster Spectrofluorometer (Photon Technology International, Inc., Birmingham, NJ). Typically, solutions contained 1-2 μm ASPP2-CT or iASPP-CT in 25 mm sodium phosphate, pH 7.5, 1 mm dithiothreitol, 0.02% sodium azide, 50-150 mm NaCl, and varying amounts of nonfluorescent p53 proteins (0-8 μm). Trp fluorescence was excited at 295 nm, and emission spectra were recorded by scanning from 300 to 380 nm with a step size of 1 nm and integration of 1 s. Trp fluorescence experiments were carried out at 12 °C, the temperature of the emission maximum. Any background fluorescence of p53 was subtracted from the fluorescence of ASPPCT-p53 mixtures at the emission maximum. The net increase of Trp fluorescence was plotted against the concentration of added p53 proteins, and the binding isotherms were nonlinear best fitted using Prism (GraphPad Software, La Jolla, CA) to extract dissociation constants. Binding of ASPP2-CT and iASPP-CT to p53-PCD; Affinities and Binding Site Mapping by NMR—The interactions between the C-terminal domains of ASPP2 and iASPP (ASPP2-CT (residues 925-1128) and iASPP-CT (residues 623-828)) and p53 that comprised the Pro-rich and core domains of p53 (p53-PCD; residues 56-289) were investigated by NMR chemical shift mapping. Backbone resonance assignments of ASPP2-CT and iASPP-CT based on three-dimensional HNCACB and HN(CO)CACB triple resonance experiments were essentially complete for ASPP2-CT (188 of 195 nonproline residues) and almost complete for iASPP-CT (168 of 193 nonproline residues). The binding sites for p53 on ASPP2-CT (Fig. 2, A and B) and iASPP-CT (Fig. 2, D and E) were determined using 1H-15N TROSY-HSQC spectroscopy following the amide resonances upon the addition of increasing amounts of p53-PCD. For ASPP2-CT, both fast (Thr1028, Trp993, and Met1021, each exhibiting small chemical shift changes upon binding) and intermediate-slow (Val1109, Ser1024, and Tyr1108, each exhibiting larger chemical shift changes upon binding) exchange was observed (insets in Fig. 2A). In contrast, most of the iASPP-CT resonances exhibited fast exchange upon p53-PCD binding (inset in Fig. 2D), indicating that the binding of iASPP-CT to p53-PCD is weaker than that of ASPP2-CT. Identification of the bound positions was straightforward for all resonances in the fast exchange regime but was difficult for those exhibiting intermediate-slow exchange; ASPP2-CT resonances exhibiting intermediate-slow exchange were frequently too broad to be observed at the mid-point of titration (insets in Fig. 2A; yellow). In these cases, the chemical shift differences between the free (black) and the first titration point (green) and between the last two titration points (cyan and red) were carefully investigated. Even if changes were very small, this approach allowed us to identify the direction of the shifts and to unambiguously distinguish bound from free frequencies for most residues in both ASPP-CT (Fig. 2B) and iASPP-CT (Fig. 2E). Several resonances in the ASPP2-CT spectrum exhibited significant spectral perturbations upon p53-PCD binding (Fig. 2B); for 23 residues, chemical shift changes larger than the mean plus one S.D. (0.068 ppm) are noted. Structural mapping of the affected residues by NMR reveal them to be clustered in the SH3 domain and in the loops between ankyrin repeats 2 and 3 and repeats 3 and 4. These NMR results are in good agreement with the x-ray model of the ASPP2-CT·p53-CD complex (33Gorina S. Pavletich N.P. Science. 1996; 274: 1001-1005Crossref PubMed Scopus (396) Google Scholar). Extraction of a dissociation constant from the titration curves of selected ASPP2-CT resonances (Fig. 2C) yielded very similar values, with an average Kd of 1.3 ± 0.2 μm (also see Table 1).TABLE 1Summary of dissociation constants for ASPP2-CT and iASPP-CT binding to various constructs of p53 determined by NMR, ITC, or fluorescencep53 variantsASPP variantsKd (NMR)Kd (ITC)Kd (fluorescence)μmμmμmp53-PCDASPP2-CT1.3 ± 0.2aKd values were obtained in the presence of 50 mm NaCl.1.5 ± 0.1aKd values were obtained in the presence of 50 mm NaCl.p53-PCDiASPP-CT76.7 ± 10.0aKd values were obtained in the presence of 50 mm NaCl.—bITC and fluorescence experiments were not performed for these pairs due to weak interaction.p53-PCD2FASPP2-CT2.2 ± 0.6cKd values were obtained in the presence of 150 mm NaCl.2.3 ± 0.3aKd values were obtained in the presence of 50 mm NaCl.2.8 ± 0.6aKd values were obtained in the presence of 50 mm NaCl.p53-PCD2FiASPP-CT107.5 ± 12.1cKd values were obtained in the presence of 150 mm NaCl.—bITC and fluorescence experiments were not performed for these pairs due to weak interaction.—bITC and fluorescence experiments were not performed for these pairs due to weak interaction.p53-PCD2F-LASPP2-CT2.2 ± 0.9cKd values were obtained in the presence of 150 mm NaCl.3.0 ± 0.5cKd values were obtained in the presence of 150 mm NaCl.p53-PCD2F-LiASPP-CT11.1 ± 1.7cKd values were obtained in the presence of 150 mm NaCl.7.7 ± 5.9cKd values were obtained in the presence of 150 mm NaCl.p53-PCD2F-L-OD (t)ASPP2-CT2.7 ± 0.6cKd values were obtained in the presence of 150 mm NaCl.p53-PCD2F-L-OD (t)iASPP-CT7.6 ± 1.9cKd values were obtained in the presence of 150 mm NaCl.p53-PCD2F-L-OD-BD (t)ASPP2-CT1.7 ± 0.2, 33 ± 8cKd values were obtained in the presence of 150 mm NaCl.,dThe titration curve was best fitted to two sequential binding events.1.9 ± 0.3cKd values were obtained in the presence of 150 mm NaCl.p53-PCD2F-L-OD-BD (t)iASPP-CT—eITC data using iASPP-CT were not analyzed, because the sample precipitated during the experiment.4.6 ± 1.2cKd values were obtained in the presence of 150 mm NaCl.p53-PCD2F-L-OD(L344R)-BD (d)ASPP2-CT1.9 ± 0.2, 29 ± 7cKd values were obtained in the presence of 150 mm NaCl.,dThe titration curve was best fitted to two sequential binding events.1.7 ± 0.3cKd values were obtained in the presence of 150 mm NaCl.p53-PCD2F-L-OD(L344R)-BD (d)iASPP-CT—eITC data using iASPP-CT were not analyzed, because the sample precipitated during the experiment.3.3 ± 1.5cKd values were obtained in the presence of 150 mm NaCl.p53-PCD2F-L-OD(L344P)-BD (m)ASPP2-CT1.5 ± 0.7cKd values were obtained in the presence of 150 mm NaCl.1.4 ± 0.3, 18 ± 5cKd values were obtained in the presence of 150 mm NaCl.,dThe titration curve was best fitted to two sequential binding events.1.6 ± 0.2cKd values were obtained in the presence of 150 mm NaCl.p53-PCD2F-L-OD(L344P)-BD (m)iASPP-CT4.3 ± 1.1cKd values were obtained in the presence of 150 mm NaCl.—eITC data using iASPP-CT were not analyzed, because the sample precipitated during the experiment.3.0 ± 0.6cKd values were obtained in the presence of 150 mm NaCl.p53-LASPP2-CT39.7 ± 4.8cKd values were obtained in the presence of 150 mm NaCl.p53-LiASPP-CT15.9 ± 2.0cKd values were obtained in the presence of 150 mm NaCl.p53-L-OD-BD (t)ASPP2-CT38.9 ± 5.0cKd values were obtained in the presence of 150 mm NaCl.p53-L-OD-BD (t)iASPP-CT17.8 ± 0.9cKd values were obtained in the presence of 150 mm NaCl.a Kd values were obtained in the presence of 50 mm NaCl.b ITC and fluorescence experiments were not performed for these pairs due to weak interaction.c Kd values were obtained in the presence of 150 mm NaCl.d The titration curve was best fitted to two sequential binding events.e ITC data using iASPP-CT were not analyzed, because the sample precipitated during the experiment. Open table in a new tab The effects of p53-PCD binding, as seen in the spectral data of iASPP-CT, were similar to those observed for ASPP2-CT in some ways but distinct in others. As in ASPP2-CT, resonances of residues residing in the SH3 domain and the junction between ankyrin repeats 3 and 4 of iASPP-CT exhibited significant perturbations upon interaction with p53-PCD (Fig. 2E), and 18 residues experienced chemical shift changes larger than the overall mean value plus 1 S.D. (0.031 ppm; Fig. 2E). However, all perturbations were much smaller than those observed for ASPP2-CT, and the large effects that were observed for residues residing between ankyrin repeats 3 and 4 of ASPP2-CT were significantly smaller in iASPP-CT. In addition, no effects were observed for residues connecting ankyrin repeats 2 and 3 in iASPP-CT. The NMR binding site mapping data clearly show that the interface in the iASPP-CT·p53-PCD complex is different from that in the ASPP2·p53-PCD complex; iASPP-CT mainly uses the SH3 domain to interact with p53-PCD, whereas ASPP2-CT uses both SH3 and ankyrin repeats. The results of our NMR binding experiments provide the first structural data on the p53/iASPP interface. The dissociation constant extracted from the NMR titration curves of selected iASPP-CT residues (Fig. 2F) for the iASPP-CT·p53-PCD complex was Kd = 76.7 ± 10.0 μm. This value reveals that the affinity of iASPP-CT to p53-PCD is ∼60-fold weaker than that of ASPP2-CT to p53-PCD. Our binding constants are in sharp contrast to previous reports that stated similar Kd values of 20-30 nm for both ASPP2-CT and iASPP-CT binding to the core domain of p53 (p53-CD), as determined by surface plasmon resonance or enzyme-linked immunosorbent assay (33Gorina S. Pavletich N.P. Science. 1996; 274: 1001-1005Crossref PubMed Scopus (396) Google Scholar, 34Robinson R.A. Lu X. Jones E.Y. Siebold C. Structure. 2008; 16: 259-268Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). This discrepancy cannot be easily explained based on the use of different lengths of proteins. The previously employed ASPP2-CT and iASPP-CT protein constructs are very similar to those used here, with ASPP2-CT and iASPP-CT comprising residues 905-1128 and 625-828 (34Robinson R.A. Lu X. Jones E.Y. Siebold C. Structure. 2008; 16: 259-268Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) compared with 925-1128 and 623-828, respectively. It seems unlikely that the minor N-terminal variations have any impact on the interaction, since our NMR data clearly show that no interaction with p53 at the N termini of A" @default.
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• W2051210433 title "Insight into the Structural Basis of Pro- and Antiapoptotic p53 Modulation by ASPP Proteins" @default.
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