Cell Biochemistry and Biophysics

, Volume 44, Issue 3, pp 475–489 | Cite as

NMR structure of the p63 SAM domain and dynamical properties of G534V and T537P pathological mutants, identified in the AEC syndrome

  • Daniel O. Cicero
  • Mattia Falconi
  • Eleonora Candi
  • Sonia Mele
  • Bruno Cadot
  • Almerinda Di Venere
  • Stefano Rufini
  • Gerry Melino
  • Alessandro Desideri
Original Article

Abstract

The p63 protein is crucial for epidermal development, and its mutations cause the extrodactyly ectodermal dysplasia and cleft lip/palate syndrome. The three-dimensional solution structure of the p63 sterile α-motif (SAM) domain (residues 505–579), a region crucial to explaining the human genetic disease ankyloblepharon-ectodermal dysplasia-clefting syndrome (AEC), has been determined by nuclear magnetic resonance spectroscopy. The structure indicates that the domain is a monomer with the characteristic five-helix bundle topology observed in other SAM domains. It includes five tightly packed helices with an extended hydrophobic core to form a globular and compact structure. The dynamics of the backbone and the global correlation time of the molecule have also been investigated and compared with the dynamical properties obtained through molecular dynamics simulation. Attempts to purify the pathological G534V and T537P mutants, originally identified in AEC, were not successful because of the occurrence of unspecific proteolytic degradation of the mutated SAM domains. Analysis of the structural dynamic properties of the G534V and T537P mutants through molecular dynamics simulation and comparison with the wild type permits detection of differences in the degree of free-dom of individual residues and discussion of the possible causes for the pathology.

Index Entries

p63 p53 family ankyloblepharon-ectodermal dysplasia-clefting syndrome ectodermal dysplasia and cleft lip/palate syndrome NMR structure molecular dynamics structure destabilization 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Yang, A., Kaghad, M., Wang, Y., et al. (1998) p63, a p53 homolog at 3q27–29, encodes multiple products with transactivating, death inducing, and dominant-negative activities. Mol. Cell 2, 305–316.PubMedCrossRefGoogle Scholar
  2. 2.
    Kaghad, M., Bonnet, H., Yang, A., et al. (1997) Monoallelically expressed gene related to p53 at 1p35, a region frequently deleted in neuroblastoma and other cancer. Cell 90, 809–819.PubMedCrossRefGoogle Scholar
  3. 3.
    Donehower, L. A. and Bradley, A. (1993) The tumor suppressor p53. Biochim. Biophys. Acta 1155, 181–205.PubMedGoogle Scholar
  4. 4.
    Yang, A. and McKeon, F. (2000) P63 and p73: p53 mimics, menaces and more. Nat. Rev. Mol. Cell Biol. 1, 199–207.PubMedCrossRefGoogle Scholar
  5. 5.
    Yang, A., Kaghad, M., Caput, D., and McKeon, F. (2002) On the shoulders of giants: p63, p73 and the rise of p53. Trends Genet. 18, 90–95.PubMedCrossRefGoogle Scholar
  6. 6.
    Melino, G., Lu, X., Gasco, M., Crook, T., and Knight, R. A. (2003) Functional regulation of p63 and p73: development and cancer. Trands Biochem. Sci. 28, 663–670.CrossRefGoogle Scholar
  7. 7.
    De Laurenzi, V., Costanzo, A., Barcaroli, D., et al. (1998) Two new p73 splice variants gamma and delta, with different transcriptional activity. J. Exp. Med. 188, 1763–1768.PubMedCrossRefGoogle Scholar
  8. 8.
    Zhu, J., Jiang, J., Zhou, W., and Chen, X. (1998) The potential tumor suppressor p73 differentially regulates cellular p53 target genes. Cancer Res. 58, 5061–5065.PubMedGoogle Scholar
  9. 9.
    Di Como, C. J., Gaiddon, C., and Prives, C. (1999) p73 function is inhibited by tumor-derived p53 mutants in mammalian cells. Mol. Cell. Biol. 19, 1438–1449.PubMedGoogle Scholar
  10. 10.
    Melino, G., De Laurenzi, V., and Vousden, K. H. (2002) p73: friend or foe in tumorigenesis. Nat. Rev. Cancer 2, 605–615.PubMedCrossRefGoogle Scholar
  11. 11.
    Yang, A., Schweitzer, R., Sun, D., et al. (1999) p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 398, 714–718.PubMedCrossRefGoogle Scholar
  12. 12.
    Mills, A. A., Zheng, B., Wang, X. J., Vogel, H., Roop, D. R., and Bradley, A. (1999) p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 398, 708–713.PubMedCrossRefGoogle Scholar
  13. 13.
    Yang, A., Walker, N., Bronson, R., et al. (2000) p73-deficient mice have neurological, pheromonal and inflammatory defects but lack spontaneous tumours. Nature 404, 99–103.PubMedCrossRefGoogle Scholar
  14. 14.
    Van Bokhoven, H. and McKeon, F. (2002) Mutations in the p53 homolog p63: allele-specific developmental syndromes in humans. Trends Mol. Med. 8, 133–139.PubMedCrossRefGoogle Scholar
  15. 15.
    Bork, P. and Koonin, E. V. (1998) Predicting functions from protein sequences-where are the bottlenecks. Nat. Genet. 18, 313–318.PubMedCrossRefGoogle Scholar
  16. 16.
    Thanos, C. D. and Bowie, J. U. (1999) p53 family members p63 and p73 are SAM domain containing proteins. Protein. Sci. 8, 1708–1710.PubMedCrossRefGoogle Scholar
  17. 17.
    Schultz, J., Ponting, C. P., Hofmann, K., and Bork, P. (1997) SAM as a protein interaction domain involved in development regulation. Protein Sci. 6, 249–253.PubMedCrossRefGoogle Scholar
  18. 18.
    Stapleton, D., Balan, I., Pawson, Y., and Sicheri, F. (1999) The crystal structure of an Eph receptor SAM domain reveals a mechanism for modular dimerization. Nat. Struct. Biol. 6, 44–49.PubMedCrossRefGoogle Scholar
  19. 19.
    Thanos, C. D., Goodwill, K. E., and Bowie, J. U. (1999) Oligomeric structure of the human EphB2 receptor SAM domain. Science 283, 833–836.PubMedCrossRefGoogle Scholar
  20. 20.
    Chi, S. W., Ayed, A., and Arrowsmith, C. H. (1999) Solution structure of a conserved C-terminal domain of p73 with structural homology to the SAM domain. EMBO J. 18, 4438–4445.PubMedCrossRefGoogle Scholar
  21. 21.
    Wang, W. K., Bycroft, M., Foster, N. W., Buckle, A. M., Fersht, A. R., and Chen, Y. W. (2001) Structure of the C-terminal SAM domain of human p73. Acta Crystallogr. D Biol. Crystallogr. 57, 545–551.PubMedCrossRefGoogle Scholar
  22. 22.
    Thanos, C. D. and Bowie, J. U. (1999) p53 Family members p63 and p73 are SAM domain-containing proteins. Protein Sci. 8, 1708–1710.PubMedGoogle Scholar
  23. 23.
    Falconi, M., Melino, G., and Desideri, A. (2004) Molecular dynamics simulation of the C-terminal sterile alpha-motif domain of human p73(:evidence of a dynamical relationship between helices 3 and 5. Biochem. Biophys. Res. Commun. 316, 1037–1042.PubMedCrossRefGoogle Scholar
  24. 24.
    McGrath, A. J., Duijf, P. H. G., Doetsch, V., et al. (2001) Hay-Wells syndrome is caused by heterozygous missense mutations in the SAM domain of p63. Hum. Mol. Gen. 10, 221–229.PubMedCrossRefGoogle Scholar
  25. 25.
    Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J. and Bax, A. (1995) NMR pipe: a multidimensional spectral processing based on UNIX pipes. J. Biomol. NMR 6, 277–293.PubMedCrossRefGoogle Scholar
  26. 26.
    Johnson, B. A. and Blevins, R. A. (1994) NMR VIEW—a computer program for the visualization and analysis of NMR Data. J. Biomol. NMR 4, 603–614.CrossRefGoogle Scholar
  27. 27.
    Bax, A. and Grzesiek, S. (1993) Methodological advances in protein NMR. Acc. Chem. Res. 26, 131–137.CrossRefGoogle Scholar
  28. 28.
    Ikura, M., Kay, L. E., and Bax, A. (1991) Improved three-dimensional 1H-13C-1H correlation spectroscopy of a 13C-labeled protein using constant-time evolution. J. Biomol. NMR 1, 299–304.PubMedCrossRefGoogle Scholar
  29. 29.
    Bazzo, R., Cicero, D. O., and Barbato, G. (1995) A new 3D HCACO Pulse sequence with optimized Resolution and Sensitivity. Application to the 21 kDa protein human interleukin-6. J. Magn. Reson. B 107, 189–191.PubMedCrossRefGoogle Scholar
  30. 30.
    Clore, G. M., Bax, A., Driscoll, P. C., Wingfield, P. T., and Gronenborn, A. M. (1990) Assignment of the side-chain 1H-and 13C resonances of interleukin-1 beta using double and triple resonance heteronuclear three-dimensional NMR spectroscopy. Biochemistry 29, 8172–8184.PubMedCrossRefGoogle Scholar
  31. 31.
    Wishart, D. S. and Sykes, B. D. (1994) Chemical shift as a tool for structure determination. Methods Enzymol. 239, 363–392.PubMedCrossRefGoogle Scholar
  32. 32.
    Kuboniwa, H., Grzesiek, S., Delaglio, F., and Bax, A. (1994) Measurement of HNNA J coupling in calcium free calmodulin using new 2D and 3D water flip back methods. J. Biomol. NMR 4, 871–878.PubMedCrossRefGoogle Scholar
  33. 33.
    Hansen, M. R., Rance, M. and Pardi, A. (1998) Tunable alignment of macromolecules by filamentous phage yields dipolar coupling interactions. Nat. Struct. Biol. 5, 1065–1074.PubMedCrossRefGoogle Scholar
  34. 34.
    Ottinger, M., Delaglio, F., and Bax, A. (1998) Measurement of J and Dipolar couplings from simplified two-dimensional NMR spectra. J. Magn. Reson. 131, 373–378.CrossRefGoogle Scholar
  35. 35.
    Koenig, B. W., Hu, J. S., Ottiger, M., Bose, S., Hendler, R. W., and Bax, A. (1999) NMR measurement of dipolar couplings in proteins aligned by transient binding to purple membrane fragments. J. Am. Chem. Soc. 121, 1385–1386.CrossRefGoogle Scholar
  36. 36.
    Schwieters, C. D., Kuszewski, J. J., Tjandra, N., and Clore, G. M. (2003) The Xplor-NIH NMR molecular structure determination package. J. Magn. Reson. 160, 65–73.PubMedCrossRefGoogle Scholar
  37. 37.
    Laskowski, R. A., Rullmann, J. A. C., MacArthur, M. W., Kaptein, R., and Thornton, J. M. (1996) AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J. Biomol. NMR 8, 477–486.PubMedCrossRefGoogle Scholar
  38. 38.
    stone, M. J., Fairbrother, W. J., Palmer, A. G., Reizer, J., Saier, M. H., and Wright P. E. (1992) Backbone dynamics of the Bacillus subtilis Glucose Permease II. A domain determined from 15N relaxation measurements. Biochemistry 31, 4394–4406.PubMedCrossRefGoogle Scholar
  39. 39.
    Orekhov, V. Y., Nolde, D. E., Golovanov, A. P., Korzhenev, P. M., and Arseniev, A. S. (1995) Processing of heteronuclear NMR relaxation data with the new software DASHA. Appl. Magn. Reson. 9, 581–588.CrossRefGoogle Scholar
  40. 40.
    Kay, L. E., Torchia, D. A., and Bax, A. (1989) Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: Application to Staphylococcal Nuclease. Biochemistry 28, 8972–8979.PubMedCrossRefGoogle Scholar
  41. 41.
    Guex, N. and Peitsch, M. C. (1997) SWISS-MODEL and the Swiss-Pdb Viewer: an environment for comparative protein modeling. Electrophoresis 18, 2714–2723.PubMedCrossRefGoogle Scholar
  42. 42.
    Cornell, W. D., Cieplak, P., Bayly, C. I., et al. (1995) A second generation force field for the simulation of proteins, nucleic acids, and organic molecules. J. Am. Chem. Soc. 117, 5179–5197.CrossRefGoogle Scholar
  43. 43.
    Jorgensen, W. L. (1981) Transferable intermolecular potential functions for water alcohols and ethers: application to liquid waters. J. Am. Chem. Soc. 103, 335–340.CrossRefGoogle Scholar
  44. 44.
    Berendsen, H. J. C., Postma, J. P. M., van Gusteren, W. F., Di Nola, A., and Haak, J. R. (1984) Molecular dynamics with coupling to an external bath. J. Comput. Phys. 81, 3684–3690.Google Scholar
  45. 45.
    Darden, T., York, D., and Pedersen, L. (1993) Particle mesh Ewald-an N.log(n) method for Ewald sums in large systems. J. Chem. Phys. 98, 10,089–10,092.CrossRefGoogle Scholar
  46. 46.
    Cheatham, T. E., Miller, J. L., Fox, T., Darden, T. A., and Kollman, P. A. (1995) Molecular dynamics simulation on solvated biomolecular systems: the particle mesh Ewald method leads to stable trajectories of DNA, RNA and proteins J. Am. Chem. Soc. 117, 4193–4194.CrossRefGoogle Scholar
  47. 47.
    Ryckaert, J. P., Ciccotti, G., and Berendsen H. J. C. (1977) Numerical integration of the Cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes. J. Comput. Phys. 23, 327–341.CrossRefGoogle Scholar
  48. 48.
    Kabsch, W. and Sander, C. (1983) Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22, 2577–2637.PubMedCrossRefGoogle Scholar
  49. 49.
    Kneller, G. (1991) Superposition of molecular structures using quaternions. Mol. Sim. 7, 113–119.CrossRefGoogle Scholar
  50. 50.
    Farrow, N., Muhandiram, D. R., Singer, A. U., et al. (1994) Backbone dynamics of a free and phospopeptide-complexed Src homology domain studied by 15N NMR relaxation. Biochemistry 33, 5984–6003.PubMedCrossRefGoogle Scholar
  51. 51.
    Lipari, G. and Szabo, A. (1982) Model free-approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules: Theory and range of validity. J. Am. Chem. Soc. 104, 4546–4559.CrossRefGoogle Scholar
  52. 52.
    Serra-Pages, C., Kedersha, N. L., Fazikas, L., Medley, Q., Debant, A., and Streuli, M. (1995) The LAR transmembrane protein tyrosine phosphatase and a coiled-coil LAR interacting protein co-localize at focal adhesions. EMBO J. 14, 2827–2838.PubMedGoogle Scholar
  53. 53.
    Falconi, M., Parrilli, L., Battistoni, A., and Desideri, A. (2002) Flexibility in monomeric Cu,Zn superoxide dismutase detected by limited proteolysis and molecular dynamics simulation. Proteins 47, 513–520.PubMedCrossRefGoogle Scholar
  54. 54.
    Polverino de Laureto, P., Taddei, N., Frare, E., et al. (2003) Protein aggregation and amyloid fibril formation by an SH3 domain probed by limited proteolysis. J. Mol. Biol. 334, 129–141.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Daniel O. Cicero
    • 1
  • Mattia Falconi
    • 2
  • Eleonora Candi
    • 3
  • Sonia Mele
    • 1
  • Bruno Cadot
    • 3
  • Almerinda Di Venere
    • 3
  • Stefano Rufini
    • 2
  • Gerry Melino
    • 3
  • Alessandro Desideri
    • 2
  1. 1.Department of Science and Chemical TechnologiesUniversity of Rome “Tor Vergata”RomeItaly
  2. 2.INFM and Department of BiologyUniversity of Rome “Tor Vergata”RomeItaly
  3. 3.Biochemistry Laboratory, IDI-IRCCS,c/o Department of Experimental Medicine and Biochemical SciencesUniversity of Rome “Tor Vergata”RomeItaly

Personalised recommendations