Effects of Common Cancer Mutations on Stability and DNA Binding of Full-length p53 Compared with Isolated Core Domains

Common cancer mutations of p53 tend either to lower the stability or distort the core domain of the protein or weaken its DNA binding affinity. We have previously analyzed in vitro the effects of mutations on the core domain of p53. Here, we extend those measurements to full-length p53, using either the wild-type protein or a biologically active superstable construct that is more amenable to accurate biophysical measurements to assess the possibilities of rescuing different types of mutations by anticancer drugs. The tetrameric full-length proteins had similar apparent melting temperatures to those of the individual domains, and the structural mutations lowered the melting temperature by similar amounts. The thermodynamic stability of tetrameric p53 is thus dictated by its core domain. We determined that the common contact mutation R273H weakened binding to the gadd45 recognition sequence by ∼700-1000 times. Many mutants that have lowered melting temperatures should be good drug targets, although the common R273H mutant binds response elements too weakly for simple rescue. Common cancer mutations of p53 tend either to lower the stability or distort the core domain of the protein or weaken its DNA binding affinity. We have previously analyzed in vitro the effects of mutations on the core domain of p53. Here, we extend those measurements to full-length p53, using either the wild-type protein or a biologically active superstable construct that is more amenable to accurate biophysical measurements to assess the possibilities of rescuing different types of mutations by anticancer drugs. The tetrameric full-length proteins had similar apparent melting temperatures to those of the individual domains, and the structural mutations lowered the melting temperature by similar amounts. The thermodynamic stability of tetrameric p53 is thus dictated by its core domain. We determined that the common contact mutation R273H weakened binding to the gadd45 recognition sequence by ∼700-1000 times. Many mutants that have lowered melting temperatures should be good drug targets, although the common R273H mutant binds response elements too weakly for simple rescue. The tumor suppressor protein p53 is a transcription factor that plays a critical role in the network of signals that control the fate of a cell (1Vogelstein B. Lane D. Levine A.J. Nature. 2000; 408: 307-310Crossref PubMed Scopus (5822) Google Scholar). In about half of human tumors, p53 is inactivated as a result of point missense mutations in the sequence-specific DNA binding core domain of the protein (2Beroud C. Soussi T. Nucleic Acids Res. 1998; 26: 200-204Crossref PubMed Scopus (167) Google Scholar, 3Hainaut P. Hollstein M. Adv. Cancer Res. 2000; 77: 81-137Crossref PubMed Scopus (852) Google Scholar). Six “hot spots” are most frequently associated with cancer: Arg175, Gly245, Arg248, Arg249, Arg273, and Arg282 (3Hainaut P. Hollstein M. Adv. Cancer Res. 2000; 77: 81-137Crossref PubMed Scopus (852) Google Scholar) (Fig. 1). From quantitative folding and DNA binding studies of numerous cancer-associated core domain mutants, three phenotypes have been broadly categorized: (i) DNA contact mutations with only minor effects on folding and stability, such as R273H; (ii) mutations that disrupt the local structure, mainly in proximity to the DNA binding surface, which destabilize the protein by ≤2 kcal/mol relative to wild type, such as G245S; and (iii) highly destabilizing mutations, such as R175H, which destabilize the protein by >3 kcal/mol (4Bullock 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 (354) Google Scholar, 5Bullock A.N. Henckel J. Fersht A.R. Oncogene. 2000; 19: 1245-1256Crossref PubMed Scopus (329) Google Scholar, 6Bullock A.N. Fersht A.R. Nat. Rev. Cancer. 2001; 1: 68-76Crossref PubMed Scopus (488) Google Scholar). The structural effects of the mutations R249S and R273H have recently been elucidated by x-ray crystallography (7Joerger A.C. Ang H.C. Veprintsev D.B. Blair C.M. Fersht A.R. J. Biol. Chem. 2005; 280: 16030-16037Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). p53 has several domains: the N-terminal transactivation domain (residues 1-63) (8Fields S. Jang S.K. Science. 1990; 249: 1046-1049Crossref PubMed Scopus (658) Google Scholar), the proline-rich regulatory domain (residues 64-92) (9Müller-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, 10Walker K.K. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15335-15340Crossref PubMed Scopus (393) Google Scholar), the DNA binding core domain (residues 94-312) (11Cho Y. Gorina S. Jeffrey P.D. Pavletich N.P. Science. 1994; 265: 346-355Crossref PubMed Scopus (2150) Google Scholar), the tetramerization domain (residues 324-355) (12Jeffrey P.D. Gorina S. Pavletich N.P. Science. 1995; 267: 1498-1502Crossref PubMed Scopus (442) Google Scholar), and the C-terminal domain (residues 360-393) (13Ahn J. Prives C. Nat. Struct. Biol. 2001; 8: 730-732Crossref PubMed Scopus (102) Google Scholar). The core domain and the C-terminal domain bind DNA. The isolated core domain (p53C) 3The abbreviations used are: p53C, p53 core domain (residues 94-312); T-p53C, thermostable variant of p53 core domain (residues 94-312) containing the four point mutations M133L, V203A, N239Y, and N268D; T-p53CT, thermostable p53 truncation mutant (residues 94-360, comprising the core and tetramerization domains); T-p53FL, thermostable variant of p53 full-length protein; DTT, dithiothreitol; KD, dissociation constant; AUC, analytical ultracentrifugation; DSC, differential scanning calorimetry. binds specifically to a double-stranded DNA consensus site containing two copies of the 10-base pair “half-site” motif 5′-PuPuPuC(A/T)(T/A)GPy-PyPy-3′ (Pu = A/G, Py = T/C) that can be separated by up to 13 bases (14El-Deiry W.S. Kern S.E. Pietenpol J.A. Kinzler K.W. Vogelstein B. Nat. Genet. 1992; 1: 45-49Crossref PubMed Scopus (1748) Google Scholar). p53C monomers bind specific DNA to give a 4:1 complex (15Balagurumoorthy 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, 16Weinberg R.L. Veprintsev D.B. Fersht A.R. J. Mol. Biol. 2004; 341: 1145-1159Crossref PubMed Scopus (191) Google Scholar). Binding to a minimal tetrameric p53 construct comprising the core and tetramerization domains is cooperative with a stoichiometry of two dimers per DNA molecule (16Weinberg R.L. Veprintsev D.B. Fersht A.R. J. Mol. Biol. 2004; 341: 1145-1159Crossref PubMed Scopus (191) Google Scholar). The C-terminal domain, its post-translational modifications and its role as a regulatory domain have been extensively studied and debated (13Ahn J. Prives C. Nat. Struct. Biol. 2001; 8: 730-732Crossref PubMed Scopus (102) Google Scholar, 17Hupp T.R. Meek D.W. Midgley C.A. Lane D.P. Cell. 1992; 71: 875-886Abstract Full Text PDF PubMed Scopus (865) Google Scholar, 18Espinosa J.M. Emerson B.M. Mol. 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Biol. 2001; 8: 730-732Crossref PubMed Scopus (102) Google Scholar, 25Bakalkin G. Selivanova G. Yakovleva T. Kiseleva E. Kashuba E. Magnusson K.P. Szekely L. Klein G. Terenius L. Wiman K.G. Nucleic Acids Res. 1995; 23: 362-369Crossref PubMed Scopus (157) Google Scholar) with a strong electrostatic component (26Friedler A. Veprintsev D.B. Freund S.M. von Glos K.I. Fersht A.R. Structure. 2005; 13: 629-636Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). A new approach in cancer therapy is to use drugs that can rescue the activity of mutant p53 (6Bullock A.N. Fersht A.R. Nat. Rev. Cancer. 2001; 1: 68-76Crossref PubMed Scopus (488) Google Scholar, 27Friedler A. Hansson L.O. Veprintsev D.B. Freund S.M. Rippin T.M. Nikolova P.V. Proctor M.R. Rüdiger S. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 937-942Crossref PubMed Scopus (234) Google Scholar, 28Wiman K.G. Cell Death Differ. 2006; 13: 921-926Crossref PubMed Scopus (126) Google Scholar, 29Bykov V.J. Selivanova G. Wiman K.G. Eur. J. Cancer. 2003; 39: 1828-1834Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). To assess further the feasibility of rescue drug therapy, we have quantitatively analyzed the effect of p53 cancer mutations on the stability of full-length protein and the effect of the contact mutation R273H on p53-DNA interactions. We used an engineered thermostable mutant of p53 core domain (T-p53C), containing the mutations M133L/V203A/N239Y/N268D (30Nikolova P.V. Henckel J. Lane D.P. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14675-14680Crossref PubMed Scopus (195) Google Scholar, 31Joerger A.C. Allen M.D. Fersht A.R. J. Biol. Chem. 2004; 279: 1291-1296Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) to produce p53 mutant proteins suitable for accurate biophysical measurements. Protein Expression and Purification—T-p53C, T-p53CT, and mutants of these constructs were purified as previously described (16Weinberg R.L. Veprintsev D.B. Fersht A.R. J. Mol. Biol. 2004; 341: 1145-1159Crossref PubMed Scopus (191) Google Scholar, 31Joerger A.C. Allen M.D. Fersht A.R. J. Biol. Chem. 2004; 279: 1291-1296Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The plasmid for human p53 (residues 1-393) subcloned into the polylinker region of vector pET-24a(+) (Novagen) using the NdeI and EcoRI restriction sites was kindly provided by C. Blair. Additional point mutations were introduced using the QuikChange site-directed mutagenesis kit (Stratagene). The expression vector was transformed into Escherichia coli BL21 for overexpression. Expression cultures were incubated was to give a of and protein expression was with were by The cell of was in of DTT, and and using an The was a and with a The were with DTT, and a and with a The were purified further on a in DTT, and The purified p53 proteins were as by Protein were and in for further DNA and were and purified to described (16Weinberg R.L. Veprintsev D.B. Fersht A.R. J. Mol. Biol. 2004; 341: 1145-1159Crossref PubMed Scopus (191) Google Scholar, R.L. Veprintsev D.B. Bycroft M. Fersht A.R. J. Mol. Biol. 2005; PubMed Scopus Google Scholar). The were on the of the with the DNA was to The gadd45 sequence was the recognition W.S. T. B.S. Vogelstein B. A.J. Cell. 1992; 71: Full Text PDF PubMed Scopus Google Scholar), and by the and in with the consensus sequence (14El-Deiry W.S. Kern S.E. Pietenpol J.A. Kinzler K.W. Vogelstein B. Nat. Genet. 1992; 1: 45-49Crossref PubMed Scopus (1748) Google Scholar). DNA that a p53 recognition was by the and for were using a of and protein to protein in DTT, and of were incubated for to The of p53, were in the of on a with a and by The were analyzed as previously described (4Bullock 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 (354) Google Scholar). were using a with an active cell of to were a of Protein were into a of This was used for core domain, protein was full-length protein was A of was to the The were analyzed with The apparent is in in analytical were using a and a To DNA binding of gadd45 DNA and monomers were up in and To the stoichiometry and dissociation for protein binding to and were and the were analyzed using The apparent dissociation per was as = and in This is to of p53 to specific gadd45 as by analytical of were determined by in and DTT, a of were with double-stranded DNA that the specific recognition the gadd45 and were analyzed as described for have been (7Joerger A.C. Ang H.C. Veprintsev D.B. Blair C.M. Fersht A.R. J. Biol. Chem. 2005; 280: 16030-16037Abstract Full Text Full Text PDF PubMed Scopus (146) Google in a new measurements were on a with a and by The and used were and and the for and were and The used was with an of for The of tetrameric constructs were The of DNA were The were in DTT, with a of as described by (16Weinberg R.L. Veprintsev D.B. Fersht A.R. J. Mol. Biol. 2004; 341: 1145-1159Crossref PubMed Scopus (191) Google Scholar) to by binding were in the of to binding gadd45 in and minimal in binding were into the using and protein were to use using to the of and von von PubMed Scopus Google Scholar). p53 was into a containing and the was for the and were using an of were on the protein (i) of p53 the for DNA is the measurements are of p53 that are the dissociation for into dimers can be that protein is the and to DNA. The binding to that of the simple which was previously used to the sequence-specific DNA binding of (15Balagurumoorthy 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, 16Weinberg R.L. Veprintsev D.B. Fersht A.R. J. Mol. Biol. 2004; 341: 1145-1159Crossref PubMed Scopus (191) Google Scholar). (ii) of p53 or the for binding to measurements are of p53 that approach or the of The of point was and to give the We the effects of structural mutations and and the mutation on the stability of by and the the wild-type protein (4Bullock 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 (354) Google Scholar, 5Bullock A.N. Henckel J. Fersht A.R. Oncogene. 2000; 19: 1245-1256Crossref PubMed Scopus (329) Google Scholar). the core domain by kcal/mol in wild and kcal/mol in The structural mutations R175H, and that destabilize the wild by kcal/mol stability in T-p53C, in on of p53 core domain mutants were in The of 13 is which was used to the wild and mutant the = for mutations in the wild-type are and are are for mutations in the wild-type are 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 (354) Google and P.V. DeDecker B. Henckel J. Fersht A.R. J. 2000; 19: PubMed Scopus Google are A.C. Ang H.C. Veprintsev D.B. Blair C.M. Fersht A.R. J. Biol. Chem. 2005; 280: 16030-16037Abstract Full Text Full Text PDF PubMed Scopus (146) Google in a new In the full-length the which was used to the of the core domain by is by the strong in the N-terminal We studied the effects of mutations on the stability of full-length p53 by differential scanning The is the melting temperature an apparent p53 with temperature A.N. Henckel J. Fersht A.R. Oncogene. 2000; 19: 1245-1256Crossref PubMed Scopus (329) Google Scholar). the are the relative destabilizing effects of the the apparent of the thermodynamic (Fig. A and The apparent are for the stability of p53 in and A. The apparent of was the as The apparent of was to The apparent of was further to The highly destabilizing mutations, and the to and for full-length p53 were with the for full-length p53 into dimers is p53 D.B. Freund S.M. A. S.E. H. J.M. Blair C.M. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 2006; PubMed Scopus Google Scholar), the protein should as shown effects of on the apparent were relative as the on The apparent of wild-type full-length p53 was to that of wild-type core domain In full-length T-p53FL, the cancer mutations and R273H the relative effect on stability as in core domain (Fig. of the R273H mutation had effect on the stability, and the apparent to The structural mutation the apparent to The mutation which was more destabilizing the further the apparent to We have previously the DNA binding of p53 core domains by analytical studies (7Joerger A.C. Ang H.C. Veprintsev D.B. Blair C.M. Fersht A.R. J. Biol. Chem. 2005; 280: 16030-16037Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). The of using the similar was We analyzed the binding to tetrameric p53 using two constructs to specific DNA binding to the core and binding to the (i) a minimal tetrameric construct of the core and tetramerization domains and (ii) the full-length protein R.L. Freund S.M. Veprintsev D.B. Bycroft M. Fersht A.R. J. Mol. Biol. 2004; 342: 801-811Crossref PubMed Scopus (80) Google Scholar, A. Veprintsev D.B. Freund S.M. von Glos K.I. Fersht A.R. Structure. 2005; 13: 629-636Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The DNA binding were using an of to electrostatic the DNA binding which is and numerous that are in DNA binding (16Weinberg R.L. Veprintsev D.B. Fersht A.R. J. Mol. Biol. 2004; 341: 1145-1159Crossref PubMed Scopus (191) Google Scholar). types of double-stranded DNA were that the pair specific recognition the gadd45 that a p53 recognition (16Weinberg R.L. Veprintsev D.B. Fersht A.R. J. Mol. Biol. 2004; 341: 1145-1159Crossref PubMed Scopus (191) Google Scholar, R.L. Veprintsev D.B. Bycroft M. Fersht A.R. J. Mol. Biol. 2005; PubMed Scopus Google Scholar). We analyzed the binding for and using the as previously described (16Weinberg R.L. Veprintsev D.B. Fersht A.R. J. Mol. Biol. 2004; 341: 1145-1159Crossref PubMed Scopus (191) Google Scholar) (Fig. The for binding to gadd45 DNA was for and for (Fig. with for binding and DNA (16Weinberg R.L. Veprintsev D.B. Fersht A.R. J. Mol. Biol. 2004; 341: 1145-1159Crossref PubMed Scopus (191) Google Scholar), and a cooperative binding for and binding gadd45 DNA. DNA Binding of the and p53 is in a (Fig. The for the minimal tetrameric construct into dimers is p53 D.B. Freund S.M. A. S.E. H. J.M. Blair C.M. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 2006; PubMed Scopus Google Scholar). p53 mutants that bind DNA weakly are as the protein of the of p53 for for different constructs and mutants of p53 were the gadd45 recognition sequence with of and DNA about more weakly with of and In the minimal tetrameric mutant gadd45 about more weakly the two types of DNA and gadd45 with of several more DNA of (Fig. and of tetrameric p53 to specific gadd45 DNA and as by a of Binding are in of is the protein The specific and DNA used are gadd45 and The are described to be binding to the to be binding to the to be binding to the in a new binding for minimal tetrameric and binding to double-stranded that either the recognition the gadd45 or a sequence of bases (16Weinberg R.L. Veprintsev D.B. Fersht A.R. J. Mol. Biol. 2004; 341: 1145-1159Crossref PubMed Scopus (191) Google Scholar, R.L. Veprintsev D.B. Bycroft M. Fersht A.R. J. Mol. Biol. 2005; PubMed Scopus Google Scholar). binding gadd45 binding binding gadd45 binding DNA. were in of DTT, and DNA Binding of the full-length protein gadd45 DNA with of about more and T-p53CT, strong for gadd45 and about more to gadd45 DNA and The R273H mutation the binding of gadd45 to the full-length protein to of and binding is mainly to binding to the R.L. Freund S.M. Veprintsev D.B. Bycroft M. Fersht A.R. J. Mol. Biol. 2004; 342: 801-811Crossref PubMed Scopus (80) Google Scholar, A. Veprintsev D.B. Freund S.M. von Glos K.I. Fersht A.R. Structure. 2005; 13: 629-636Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), the dissociation for DNA was and The is for which is a weakly binding mutant of and for binding to specific and (Fig. binding for full-length mutants and binding to double-stranded that either the recognition the gadd45 or a sequence of bases (16Weinberg R.L. Veprintsev D.B. Fersht A.R. J. Mol. 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The use of construct to produce full-length p53 mutants for accurate biophysical measurements. cancer mutations had the relative effects on the stability of the full-length protein as in isolated core domain (Fig. A and This that the core domain is in the stability of p53, and that stability core domain into similar relative stability in the full-length of R273H on DNA Binding of R273H contact mutation the binding of the gadd45 response by the for the wild-type minimal tetrameric core and tetramerization domain construct to The contact mutation DNA with studies in which transactivation is for the mutant R273H Oncogene. 8: Google Scholar, A.M. Oncogene. 8: Google Scholar, J.A. T. S. W.S. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar) and of the R273H mutant with or more wild-type monomers are active A. Mol. 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Binding to and are in to the binding of DNA to isolated core domains with p53 that is in dimers and is that as the dissociation of tetrameric p53 into dimers is about the binding of response elements with in the region are in a p53 in which is mainly the of to for the binding of gadd45 DNA to full-length that the p53 dimers have to to to bind to the response elements that have be shown that for binding response elements such as gadd45 to wild-type proteins the is the of the of the dissociation of the into and the DNA the = (Fig. T-p53CT, the of the dissociation for the to into the with the DNA binding a of for gadd45 and of weakly binding such as we can the dissociation of DNA the by the of tetrameric p53 in using the dissociation and the the tetrameric p53 The for gadd45 and was to be the can be that the binding to the is weakened by on the mutation of The of binding of DNA to tetrameric constructs be quantitatively with the binding to core domains of in which to different of The effects of mutation can be The for core domains binding gadd45 are for and for (7Joerger A.C. 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