No abstract

The oncoprotein MDM2 inhibits the tumor suppressor protein p53 by binding to the p53 transactivation domain. The p53 gene is inactivated in many human tumors either by mutations or by binding to oncogenic proteins. In some tumors, such as soft tissue sarcomas, overexpression of MDM2 inactivates an otherwise intact p53, disabling the genome integrity checkpoint and allowing cell cycle progression of defective cells. Disruption of the MDM2/p53 interaction leads to increased p53 levels and restored p53 transcriptional activity, indicating restoration of the genome integrity check and therapeutic potential for MDM2/p53 binding antagonists. Here, we show by multidimensional NMR spectroscopy that chalcones (1,3-diphenyl-2-propen-1-ones) are MDM2 inhibitors that bind to a subsite of the p53 binding cleft of human MDM2. Biochemical experiments showed that these compounds can disrupt the MDM2/p53 protein complex, releasing p53 from both the p53/MDM2 and DNA-bound p53/MDM2 complexes. These results thus offer a starting basis for structure-based drug design of cancer therapeutics.

Conformational stability is a prerequisite for the physiological activity of the tumor suppressor protein p53. p53 protein can be allosterically activated for DNA binding by phosphorylation or through noncovalent interaction with proteins such as DnaK, the Escherichia coli homologue of the heat shock protein Hsp70. We present in vitro evidence for a rapid temperature-dependent change in the conformation and tetrameric nature of wild-type p53 upon incubation at 37°C, which correlates with a permanent loss in DNA binding activity. We show that p53 is allosterically regulated for stabilization of the wild-type conformation and DNA binding activity at 37°C by binding of two classes of ligands to regulatory sites on the N and C terminus of the molecule through which an intrinsic instability of p53 is neutralized. Deletion of the domain conferring instability at the C terminus is sufficient to confer enhanced stability to the total protein. DnaK binding to the C terminus can profoundly protect p53 at 37°C from a temperature-dependent loss of the DNA binding activity but does not renature or activate denatured p53. In contrast, another activator of the DNA binding activity of latent p53, the monoclonal antibody PAb421, which also interacts with the C terminus of the protein, is not able to protect p53 from thermal denaturation. Two monoclonal antibodies to the N terminus of p53, PAb1801 and DO-1, do not activate the latent DNA binding function of p53 but can protect the p53 wild-type conformation at 37°C. Thus, activation of the DNA binding function of p53 is not synonymous with protection from thermal denaturation, and therefore, both of these pathways may be used in cells to control the physiological activity of p53. The protection of p53 conformation from heat denaturation by interacting proteins suggests a novel mechanism by which p53 function could be regulated in vivo.

The p53 protein is a transcription factor that acts as the major tumor suppressor in mammals. The core DNAbinding domain is mutated in about 50% of all human tumors. The crystal structure of the core domain in complex with DNA illustrated how a single core domain specifically interacts with its DNA consensus site and how it is inactivated by mutation. However, no structural information for the tetrameric full-length p53-DNA complex is available. Here, we present novel experimental insight into the dimerization of two p53 core domains upon cooperative binding to consensus DNA in solution obtained by NMR. The NMR data show that the p53 core domain itself does not appear to undergo major conformational changes upon addition of DNA and elucidate the dimerization interface between two DNA-bound core domains, which includes the short H1 helix. A NMR-based model for the dimeric p53 core-DNA complex incorporates these data and allows the conclusion that the dimerization interface also forms the actual interface in the tetrameric p53-DNA complex. The significance of this interface is further corroborated by the finding that hot spot mutations map to the H1 helix, and by the binding of the putative p53 inhibitor 53BP2 to this region via one of its ankyrin repeats. Based on symmetry considerations it is proposed that tetrameric p53 might link non-contigous DNA consensus sites in a sandwich-like manner generating DNA loops as observed for transcriptionally active p53 complexes.The tumor suppressor gene p53 is the most frequent site of genetic alterations found in human tumors (1) and acts as the major tumor suppressor in mammals. In addition to non-transcriptional functions, p53 acts primarily as a transcriptional activator, that regulates the expression of several genes involved in cell cycle arrest, cellular senescence, anti-angiogenesis, and apoptosis (reviewed in Refs. 2-4). Recently, two homologues of p53, p63 and p73, were discovered, coding for a variety of different isoforms. These three p53 family members play distinct roles in differentiation, development, and tumor suppression (reviewed in Ref. 5). p53 possesses a modular architecture with an N-terminal transactivation domain (TAD), 1 a strongly conserved core DNA-binding domain (DBD), a tetramerization domain (TD), and a regulatory C terminus (6, 7). Tetrameric p53 binds specifically to a DNA consensus sequence consisting of two consecutive palindromic 10-bp halfsites, where each half-site is formed by two head-to-head quarter-sites (8 -12). The isolated TD forms a symmetric dimer of dimers (13-15), and contrasting models have been proposed that describe how the DBDs of each dimer are attached to DNA, namely with either consecutive or alternating arrangements (16). The p53 DBD comprises several hot spot regions for mutation that inactivate p53 in more than half of all human tumors (1). Therefore, wild-type and mutant p53 DBDs have been the focus of various studies (17-21). The crystal structure of the p53 DBD in complex with DNA (10) showed that almost all known...

The p53 protein is the major tumor suppressor in mammals. The discovery of the p53 homologs p63 and p73 defined a family of p53 members with distinct roles in tumor suppression, differentiation, and development. Here, we describe the biochemical characterization of the core DNA-binding domain of a human isoform of p63, p63-␦, and particularly novel features in comparison with p53. In contrast to p53, the free p63 core domain did not show specific binding to p53 DNA consensus sites. However, glutathione S-transferase-fused and thus dimerized p63 and p53 core domains had similar affinity and specificity for the p53 consensus sites p21, gadd45, cyclin G, and bax. Furthermore, the fold of p63 core was remarkably stable compared with p53 as judged by differential scanning calorimetry (T m ‫؍‬ 61°C versus 44°C for p53) and equilibrium unfolding ([urea] 50% ‫؍‬ 5.2 M versus 3.1 M for p53). A homology model of p63 core highlights differences at a segment near the H1 helix hypothetically involved in the formation of the dimerization interface in p53, which might reduce cooperativity of p63 core DNA binding compared with p53. The model also shows differences in the electrostatic and hydrophobic potentials of the domains relevant to folding stability.