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.
Similar content being viewed by others
References
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.
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.
Donehower, L. A. and Bradley, A. (1993) The tumor suppressor p53. Biochim. Biophys. Acta 1155, 181–205.
Yang, A. and McKeon, F. (2000) P63 and p73: p53 mimics, menaces and more. Nat. Rev. Mol. Cell Biol. 1, 199–207.
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.
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.
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.
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.
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.
Melino, G., De Laurenzi, V., and Vousden, K. H. (2002) p73: friend or foe in tumorigenesis. Nat. Rev. Cancer 2, 605–615.
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.
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.
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.
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.
Bork, P. and Koonin, E. V. (1998) Predicting functions from protein sequences-where are the bottlenecks. Nat. Genet. 18, 313–318.
Thanos, C. D. and Bowie, J. U. (1999) p53 family members p63 and p73 are SAM domain containing proteins. Protein. Sci. 8, 1708–1710.
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.
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.
Thanos, C. D., Goodwill, K. E., and Bowie, J. U. (1999) Oligomeric structure of the human EphB2 receptor SAM domain. Science 283, 833–836.
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.
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.
Thanos, C. D. and Bowie, J. U. (1999) p53 Family members p63 and p73 are SAM domain-containing proteins. Protein Sci. 8, 1708–1710.
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.
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.
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.
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.
Bax, A. and Grzesiek, S. (1993) Methodological advances in protein NMR. Acc. Chem. Res. 26, 131–137.
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.
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.
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.
Wishart, D. S. and Sykes, B. D. (1994) Chemical shift as a tool for structure determination. Methods Enzymol. 239, 363–392.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Guex, N. and Peitsch, M. C. (1997) SWISS-MODEL and the Swiss-Pdb Viewer: an environment for comparative protein modeling. Electrophoresis 18, 2714–2723.
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.
Jorgensen, W. L. (1981) Transferable intermolecular potential functions for water alcohols and ethers: application to liquid waters. J. Am. Chem. Soc. 103, 335–340.
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.
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.
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.
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.
Kabsch, W. and Sander, C. (1983) Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22, 2577–2637.
Kneller, G. (1991) Superposition of molecular structures using quaternions. Mol. Sim. 7, 113–119.
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.
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.
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.
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.
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.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Cicero, D.O., Falconi, M., Candi, E. et al. NMR structure of the p63 SAM domain and dynamical properties of G534V and T537P pathological mutants, identified in the AEC syndrome. Cell Biochem Biophys 44, 475–489 (2006). https://doi.org/10.1385/CBB:44:3:475
Issue Date:
DOI: https://doi.org/10.1385/CBB:44:3:475