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Factors governing loss and rescue of DNA binding upon single and double mutations in the p53 core domain.

ABSTRACT: The mutation of R273-->H in the p53 core domain (p53-CD) is one of the most common mutations found in human cancers. Although the 273H p53-CD retains the wild-type conformation and stability, it lacks sequence-specific DNA binding, a transactivation function and growth suppression. However, mutating T284-->R in the 273H p53-CD restores the DNA binding affinity, and transactivation and tumour suppressor functions. Since X-ray/NMR structures of DNA-free or DNA-bound mutant p53-CD molecules are unavailable, the factors governing the loss and rescue of sequence-specific DNA binding in the 273H and 273H+284R p53-CD, respectively, are unclear. Hence, we have carried out molecular dynamics (MD) simulations of the wild-type, single mutant and double mutant p53-CD, free and DNA bound, in the presence of explicit water molecules. Based on the MD structures, the DNA-binding free energy of each p53 molecule has been computed and decomposed into component energies and contributions from the interface residues. The wild-type and mutant p53-CD MD structures were found to be consistent with the antibody-binding, X-ray and NMR data. The predicted DNA binding affinity and specificity of both mutant p53-CDs were also in accord with experimental data. The non-detectable DNA binding of the 273H p53-CD is due mainly to the disruption of a hydrogen-bonding network involving R273, D281 and R280, leading to a loss of major groove binding by R280 and K120. The restoration of DNA binding affinity and specificity of the 273H+284R p53-CD is due mainly to the introduction of another DNA-binding site at position 284, leading to a recovery of major groove binding by R280 and K120. The important role of water molecules and the DNA major groove conformation as well as implications for structure-based linker rescue of the 273H p53-CD DNA-binding affinity are discussed.

SUBMITTER: Wright JD

PROVIDER: S-EPMC101848 | biostudies-literature | 2002 Apr

REPOSITORIES: biostudies-literature

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Factors governing loss and rescue of DNA binding upon single and double mutations in the p53 core domain.

Wright Jon D JD Noskov Sergey Yu SY Lim Carmay C

Nucleic acids research 20020401 7

The mutation of R273-->H in the p53 core domain (p53-CD) is one of the most common mutations found in human cancers. Although the 273H p53-CD retains the wild-type conformation and stability, it lacks sequence-specific DNA binding, a transactivation function and growth suppression. However, mutating T284-->R in the 273H p53-CD restores the DNA binding affinity, and transactivation and tumour suppressor functions. Since X-ray/NMR structures of DNA-free or DNA-bound mutant p53-CD molecules are una ...[more]

PMID: 11917017

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Project description:To gain insights into the mechanisms by which certain second-site suppressor mutations rescue the function of a significant number of cancer mutations of the tumor suppressor protein p53, X-ray crystallographic structures of four p53 core-domain variants were determined. These include an oncogenic mutant, V157F, two single-site suppressor mutants, N235K and N239Y, and the rescued cancer mutant V157F/N235K/N239Y. The V157F mutation substitutes a smaller hydrophobic valine with a larger hydrophobic phenylalanine within strand S4 of the hydrophobic core. The structure of this cancer mutant shows no gross structural changes in the overall fold of the p53 core domain, only minor rearrangements of side chains within the hydrophobic core of the protein. Based on biochemical analysis, these small local perturbations induce instability in the protein, increasing the free energy by 3.6?kcal?mol(-1) (15.1?kJ?mol(-1)). Further biochemical evidence shows that each suppressor mutation, N235K or N239Y, acts individually to restore thermodynamic stability to V157F and that both together are more effective than either alone. All rescued mutants were found to have wild-type DNA-binding activity when assessed at a permissive temperature, thus pointing to thermodynamic stability as the critical underlying variable. Interestingly, thermodynamic analysis shows that while N239Y demonstrates stabilization of the wild-type p53 core domain, N235K does not. These observations suggest distinct structural mechanisms of rescue. A new salt bridge between Lys235 and Glu198, found in both the N235K and rescued cancer mutant structures, suggests a rescue mechanism that relies on stabilizing the ?-sandwich scaffold. On the other hand, the substitution N239Y creates an advantageous hydrophobic contact between the aromatic ring of this tyrosine and the adjacent Leu137. Surprisingly, the rescued cancer mutant shows much larger structural deviations than the cancer mutant alone when compared with wild-type p53. These suppressor mutations appear to rescue p53 function by creating novel intradomain interactions that stabilize the core domain, allowing compensation for the destabilizing V157F mutation.

Project description:BackgroundThe tumour suppressor protein p53 protein has a core domain that binds DNA and is the site for most oncogenic mutations. This domain is quite unstable compared to its homologs p63 and p73. Two key residues in the core domain of p53 (Tyr236, Thr253), have been mutated in-silico, to their equivalent residues in p63 (Phe238 and Ile255) and p73 (Phe238 and Ile255), with subsequent increase in stability of p53. Computational studies have been performed to examine the basis of instability in p53.ResultsMolecular dynamics simulations suggest that mutations in p53 lead to increased conformational sampling of the phase space which stabilizes the system entropically. In contrast, reverse mutations, where p63 and p73 were mutated by replacing the Phe238 and Ile255 by Tyr and Thr respectively (as in p53), showed reduced conformational sampling although the change for p63 was much smaller than that for p73. Barriers to the rotation of sidechains containing aromatic rings at the core of the proteins were reduced several-fold when p53 was mutated; in contrast they increased when p73 was mutated and decreased by a small amount in p63. The rate of ring flipping of a Tyrosine residue at the boundary of two domains can be correlated with the change in stability, with implications for possible pathways of entry of agents that induce unfolding.ConclusionA double mutation at the core of the DNA binding domain of p53 leads to enhanced stability by increasing the softness of the protein. A change from a highly directional polar interaction of the core residues Tyr236 and Thr253 to a non-directional apolar interaction between Phe and Ile respectively may enable the system to adapt more easily and thus increase its robustness to structural perturbations, giving it increased stability. This leads to enhanced conformational sampling which in turn is associated with an increased "softness" of the protein core. However the system seems to become more rigid at the periphery. The success of this methodology in reproducing the experimental trends in the stability of p53 suggests that it has the potential to complement structural studies for rapidly estimating changes in stability upon mutations and could be an additional tool in the design of specific classes of proteins.

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Factors governing loss and rescue of DNA binding upon single and double mutations in the p53 core domain.

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Factors governing loss and rescue of DNA binding upon single and double mutations in the p53 core domain.

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