Thermodynamic stability of wild-type and mutant p53 core domain

Abstract

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...

Transcriptional regulation by p53: one protein, many possibilities

“…Mutations at some positions (e.g. R273) decrease the binding ability of p53 by simply eliminating direct contacts between the protein and DNA, 21,26 while some other mutations (e.g. R175, G245, R249, R282) diminish binding by destabilizing the tertiary structure of p53 DBD.…”

Reactivation of mutant p53: molecular mechanisms and therapeutic potential

“…Mutations result in lowering of the melting temperature by 5-101, which is sufficient to tip the balance towards the unfolded state at physiological temperature (Bullock et al, 1997). The extent of unfolding varies depending on the site and the nature of the mutation.…”

Strategies for manipulating the p53 pathway in the treatment of human cancer

“…(C) Thermodynamic stability. A thermodynamically unstable class of mutant protein has a relatively high degree of intrinsic instability with respect to cis-acting protein-folding pathways [143] and can be further subdivided into distinct thermodynamically defined groups [144]. The purple arrow represents the shift in equilibrium promoted by mutation from the folded active tetramer (light-grey squares) to the unfolded inactive tetramer (lilac squares).…”

“…One class of p53 mutant containing a point mutation within the core domain (prototype His"(&) cannot be activated for DNA binding by the allosteric Cterminal modifications and have a relatively high degree of intrinsic thermodynamic instability [143,144]. A second class of p53 mutant protein with a thermodynamic stability similar to wild-type p53 has the capacity to bind to DNA sequencespecifically, but cannot be activated from the latent state by phosphorylation ( [146] ; prototypes His#($ and Lys#)&).…”

“…One class of compounds, comprising the benzoquinone ansamycin class of antitumour fungal antibiotics (geldanamycin), inhibits the HSP90 (yellow)-dependent chaperone holoenzyme complex unfolding of p53 protein [165,184,185] and forms a precedent for developing therapeutically relevant agents that modulate chaperone-dependent anti-apoptotic pathways [182,183]. A second class of compounds (prototype being CP-31,398) presumably binds directly to native p53 protein and prevents its unfolding and inactivation by stabilizing the intrinsic thermoinstability (∆) of the tetramer [143,168,169,171,174,263].…”