p53 contains large unstructured regions in its native state.

The human tumor suppressor protein p53 is understood only to some extent on a structural level. We performed a comprehensive biochemical and biophysical structure-function analysis of p53 full-length protein and p53 fragments. The analysis showed that p53 and the fragments investigated form stable functional units. Full-length p53 and the tetrameric fragment N93p53 (residues 93-393) are, however, destabilized significantly compared to the monomeric core domain (residues 94-312) and the monomeric fragment p53C312 (residues 1-312). At the physiological temperature of 37 degrees C and in the absence of modifications or stabilizing partners, wild-type p53 is more than 50% unfolded correlating with a 75% loss in DNA-binding activity. Furthermore the analysis of CD spectra revealed that full-length p53 contains large unstructured regions in its N and C-terminal parts. Our results indicate that full-length p53 is a modular protein consisting of defined structured and unstructured regions. We propose that p53 belongs to the growing family of loosely folded or partially unstructured native proteins. The lack of a rigid structure combined with the low overall stability may allow the physiological interaction of p53 with a multitude of partner proteins and the regulation of its turnover.

[1]  Roger A. Sayle,et al.  DSC: public domain protein secondary structure predication , 1997, Comput. Appl. Biosci..

[2]  N. A. Jacques,et al.  Cell disruption of Escherichia coli by glass bead stirring for the recovery of recombinant proteins. , 1997, Analytical biochemistry.

[3]  K. Kinzler,et al.  Definition of a consensus binding site for p53 , 1992, Nature Genetics.

[4]  P. May,et al.  Twenty years of p53 research: structural and functional aspects of the p53 protein , 1999, Oncogene.

[5]  R. Huber,et al.  High Thermostability and Lack of Cooperative DNA Binding Distinguish the p63 Core Domain from the Homologous Tumor Suppressor p53* , 2001, The Journal of Biological Chemistry.

[6]  C. Pabo,et al.  The DNA-binding domain of p53 contains the four conserved regions and the major mutation hot spots. , 1993, Genes & development.

[7]  D. Lane,et al.  Activating mutations in p53 produce a common conformational effect. A monoclonal antibody specific for the mutant form. , 1990, The EMBO journal.

[8]  P. Argos,et al.  Incorporation of non-local interactions in protein secondary structure prediction from the amino acid sequence. , 1996, Protein engineering.

[9]  G Deléage,et al.  An algorithm for protein secondary structure prediction based on class prediction. , 1987, Protein engineering.

[10]  D. Lane,et al.  Allosteric activation of latent p53 tetramers , 1994, Current Biology.

[11]  Burkhard Rost,et al.  PHD - an automatic mail server for protein secondary structure prediction , 1994, Comput. Appl. Biosci..

[12]  P. Sutphin,et al.  The Physical Association of Multiple Molecular Chaperone Proteins with Mutant p53 Is Altered by Geldanamycin, an hsp90-Binding Agent , 1998, Molecular and Cellular Biology.

[13]  D. Lane,et al.  The N terminus of the murine p53 tumour suppressor is an independent regulatory domain affecting activation and thermostability. , 1998, Journal of molecular biology.

[14]  X. Chen,et al.  A proteolytic fragment from the central region of p53 has marked sequence-specific DNA-binding activity when generated from wild-type but not from oncogenic mutant p53 protein. , 1993, Genes & development.

[15]  C. Arrowsmith,et al.  Thermodynamic analysis of the structural stability of the tetrameric oligomerization domain of p53 tumor suppressor. , 1995, Biochemistry.

[16]  K. Matthews,et al.  Protein-DNA binding correlates with structural thermostability for the full-length human p53 protein. , 2001, Biochemistry.

[17]  J. Gibrat,et al.  Further developments of protein secondary structure prediction using information theory. New parameters and consideration of residue pairs. , 1987, Journal of molecular biology.

[18]  P. V. von Hippel,et al.  Calculation of protein extinction coefficients from amino acid sequence data. , 1989, Analytical biochemistry.

[19]  C. Arrowsmith,et al.  Solution structure of the tetrameric minimum transforming domain of p53 , 1995, Nature Structural Biology.

[20]  Barry Robson,et al.  An algorithm for secondary structure determination in proteins based on sequence similarity , 1986, FEBS letters.

[21]  P. Tegtmeyer,et al.  Interaction of p53 with its consensus DNA-binding site , 1995, Molecular and cellular biology.

[22]  M Bycroft,et al.  Hot-spot mutants of p53 core domain evince characteristic local structural changes. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[23]  P. Hainaut,et al.  Temperature sensitivity for conformation is an intrinsic property of wild-type p53. , 1995, British Journal of Cancer.

[24]  A. Levine,et al.  Structure of the MDM2 Oncoprotein Bound to the p53 Tumor Suppressor Transactivation Domain , 1996, Science.

[25]  C. Klein,et al.  NMR Spectroscopy Reveals the Solution Dimerization Interface of p53 Core Domains Bound to Their Consensus DNA* , 2001, The Journal of Biological Chemistry.

[26]  A. Fersht,et al.  Quantitative analysis of residual folding and DNA binding in mutant p53 core domain: definition of mutant states for rescue in cancer therapy , 2000, Oncogene.

[27]  E Schwarz,et al.  Advances in refolding of proteins produced in E. coli. , 1998, Current opinion in biotechnology.

[28]  M. Uesugi,et al.  The α-helical FXXΦΦ motif in p53: TAF interaction and discrimination by MDM2 , 1999 .

[29]  L. Kay,et al.  Latent and active p53 are identical in conformation , 2001, Nature Structural Biology.

[30]  Ted R. Hupp,et al.  Strategies for manipulating the p53 pathway in the treatment of human cancer , 2000 .

[31]  Christophe Geourjon,et al.  SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments , 1995, Comput. Appl. Biosci..

[32]  N. Sreerama,et al.  Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. , 2000, Analytical biochemistry.

[33]  K. Sakaguchi,et al.  Calcium-dependent Interaction of S100B with the C-terminal Domain of the Tumor Suppressor p53* , 1999, The Journal of Biological Chemistry.

[34]  J. Gibrat,et al.  Secondary structure prediction and protein design. , 1990, Biochemical Society symposium.

[35]  N. Pavletich,et al.  Crystal structure of the tetramerization domain of the p53 tumor suppressor at 1.7 angstroms , 1995, Science.

[36]  H. Dyson,et al.  Intrinsically unstructured proteins: re-assessing the protein structure-function paradigm. , 1999, Journal of molecular biology.

[37]  Alan R. Fersht,et al.  Mechanism of folding and assembly of a small tetrameric protein domain from tumor suppressor p53 , 1999, Nature Structural Biology.

[38]  A. Fersht,et al.  Nine hydrophobic side chains are key determinants of the thermodynamic stability and oligomerization status of tumour suppressor p53 tetramerization domain , 1998, The EMBO journal.

[39]  Y. Sung,et al.  Transactivation Ability of p53 Transcriptional Activation Domain Is Directly Related to the Binding Affinity to TATA-binding Protein (*) , 1995, The Journal of Biological Chemistry.

[40]  A. Fersht,et al.  Thermodynamic stability of wild-type and mutant p53 core domain. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[41]  P E Wright,et al.  Structural studies of p21Waf1/Cip1/Sdi1 in the free and Cdk2-bound state: conformational disorder mediates binding diversity. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[42]  A. Gronenborn,et al.  Four p53 DNA-binding domain peptides bind natural p53-response elements and bend the DNA. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[43]  G. Marius Clore,et al.  Refined solution structure of the oligomerization domain of the tumour suppressor p53 , 1995, Nature Structural Biology.

[44]  A. Levine,et al.  Surfing the p53 network , 2000, Nature.

[45]  P. Jeffrey,et al.  Crystal structure of a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations. , 1994, Science.

[46]  G. Böhm,et al.  Quantitative analysis of protein far UV circular dichroism spectra by neural networks. , 1992, Protein engineering.

[47]  A. Fersht,et al.  Rescuing the function of mutant p53 , 2001, Nature Reviews Cancer.

[48]  David J. Weber,et al.  Structure of the negative regulatory domain of p53 bound to S100B(ββ) , 2000, Nature Structural Biology.

[49]  D. Woods,et al.  Regulation of p53 function. , 2001, Experimental cell research.

[50]  Kyou-Hoon Han,et al.  Local Structural Elements in the Mostly Unstructured Transcriptional Activation Domain of Human p53* , 2000, The Journal of Biological Chemistry.

[51]  J Milner,et al.  Flexibility: the key to p53 function? , 1995, Trends in biochemical sciences.

[52]  C. Prives,et al.  The C-terminus of p53: the more you learn the less you know , 2001, Nature Structural Biology.

[53]  C. Prives,et al.  The N Terminus of p53 Regulates Its Dissociation from DNA* , 2000, The Journal of Biological Chemistry.

[54]  F. King,et al.  Hsp70 interactions with the p53 tumour suppressor protein , 2001, The EMBO journal.

[55]  J. E. Stenger,et al.  p53 domains: identification and characterization of two autonomous DNA-binding regions. , 1993, Genes & development.