Some 50% of human cancers are associated with mutations in the core domain of the tumor suppressor p53. Many mutations are thought just to destabilize the protein. To assess this and the possibility of rescue, we have set up a system to analyze the stability of the core domain and its mutants. The use of differential scanning calorimetry or spectroscopy to measure its melting temperature leads to irreversible denaturation and aggregation and so is useful as only a qualitative guide to stability. There are excellent two-state denaturation curves on the addition of urea that may be analyzed quantitatively. One Zn 2؉ ion remains tightly bound in the holo-form of p53 throughout the denaturation curve. The stability of wild type is 6.0 kcal (1 kcal ‫؍‬ 4.18 kJ)͞mol at 25°C and 9.8 kcal͞mol at 10°C. The oncogenic mutants R175H, C242S, R248Q, R249S, and R273H are destabilized by 3.0, 2.9, 1.9, 1.9, and 0.4 kcal͞mol, respectively. Under certain denaturing conditions, the wildtype domain forms an aggregate that is relatively highly f luorescent at 340 nm on excitation at 280 nm. The destabilized mutants give this f luorescence under milder denaturation conditions.The tumor suppressor protein p53 is a sequence-specific transcription factor that functions to maintain the integrity of the genome (1). On its induction in response to DNA damage, p53 promotes cell cycle arrest in G 1 phase (2) and apoptosis if DNA repair is not possible (3). Negative regulation occurs by the synthesis and subsequent binding of the oncoprotein Mdm2 to the transactivation domain of p53. This targets it for degradation and ensures that the cellular stability of p53 is low (4, 5). About 50% of human cancers and 95% of lung cancers are associated with mutations in p53. The majority of these map to its core domain, which is responsible for binding DNA (6). The crystal structure of the core domain bound to DNA has been determined (7). A number of the tumorigenic mutants affect residues that contact the DNA, but many are not directly involved in binding and appear to affect the thermodynamic stability of the protein (8, 9). p53 is a possible target for cancer therapy, including drugs that can stabilize it or using superstable p53 variants that would be suitable for gene therapy applications. There is a lack of quantitative information on the stability of p53 on which to base experiments measuring its change in stability on mutation. Data tend to be restricted so far to measurements of the temperature dependence of transactivation or PAb 1620 binding (8, 9), a monoclonal antibody specific for the native state of wild-type p53 (10). These suggest that p53 is relatively unstable. We find in this study that the core domain denatures irreversibly with temperature, and so the T m measured by differential scanning calorimetry or spectroscopy cannot be used quantitatively for analyzing structure-activity relationships of p53. We have turned instead to studying the stability of the isolated core domain by using urea-mediated denaturation, which is of p...

We have designed a p53 DNA binding domain that has virtually the same binding affinity for the gadd45 promoter as does wild-type protein but is considerably more stable. The design strategy was based on molecular evolution of the protein domain. Naturally occurring amino acid substitutions were identified by comparing the sequences of p53 homologues from 23 species, introducing them into wild-type human p53, and measuring the changes in stability. The most stable substitutions were combined in a multiple mutant. The advantage of this strategy is that, by substituting with naturally occurring residues, the function is likely to be unimpaired. All point mutants bind the consensus DNA sequence. The changes in stability ranged from ؉1.27 (less stable Q165K) to ؊1.49 (more stable N239Y) kcal mol ؊1 , respectively. The changes in free energy of unfolding on mutation are additive. Of interest, the two most stable mutants (N239Y and N268D) have been known to act as suppressors and restored the activity of two of the most common tumorigenic mutants. Of the 20 single mutants, 10 are cancerassociated, though their frequency of occurrence is extremely low: A129D, Q165K, Q167E, and D148E are less stable and M133L, V203A and N239Y are more stable whereas the rest are neutral. The quadruple mutant (M133LV203AN239YN-268D), which is stabilized by 2.65 kcal mol ؊1 and T m raised by 5.6°C is of potential interest for trials in vivo.

Conformationally compromised oncogenic mutants of the tumor suppressor protein p53 can, in principle, be rescued by small molecules that bind the native, but not the denatured state. We describe a strategy for the rational search for such molecules. A nine-residue peptide, CDB3, which was derived from a p53 binding protein, binds to p53 core domain and stabilizes it in vitro. NMR studies showed that CDB3 bound to p53 at the edge of the DNA binding site, partly overlapping it. The fluorescein-labeled peptide, FL-CDB3, binds wild-type p53 core domain with a dissociation constant of 0.5 M, and raises the apparent melting temperatures of wild-type and a representative oncogenic mutant, R249S core domain. gadd45 DNA competes with CDB3 and displaces it from its binding site. But this competition does not preclude CDB3 from being a lead compound. CDB3 may act as a ''chaperone'' that maintains existing or newly synthesized destabilized p53 mutants in a native conformation and then allows transfer to specific DNA, which binds more tightly. Indeed, CDB3 restored specific DNA binding activity to a highly destabilized mutant I195T to close to that of wild-type level.