Stability Studies of p53 Tumour Suppressor Mutants
p53 is extremely susceptible to mutations that compromise the integrity of its global fold and lead to loss of function. We have analysed the stability and DNA binding activity of mutants of the core domain of p53 to investigate the mechanism of restoring function to tumorigenic mutants by second-site suppressor mutations. The cancer mutants were destabilized by 3.34 (V143A), 1.69 (R249S) and 1.22 (G245S) kcal mol-1. The suppressors N239Y and N268D were 1.37 and 1.21 kcal mol-1 more stable than wild type, respectively; H168R was 2.75 kcal mol-1 less stable, while T123A was as stable as wild type. Double mutant cycles show that N239Y and N268D act as "global stability" suppressors by increasing the stability of G245S and V143- the free energy changes are additive. Conversely, the suppressor H168R is specific for the R249S mutation: despite destabilizing wild type, H168R has virtually no effect on the stability of the R249S mutant, but does restores its binding affinity for the gadd45 promoter. NMR structural comparisons of R249S/H168R and R249S/T123A/H168R with wild-type and R249S proteins, show that H168R reverts some of the structural changes induced by R249S. These results have implications for possible drug therapy aimed at restoring the function of tumorigenic mutants of p53: the function of mutants such as V143A and G245S is theoretically possible to restore by small molecules that simply bind to and hence stabilize the native structure, whereas R249S requires alteration of its mutant native structure.
Panka V. Nikolova, Kam-Bo Wong, Brian Dedecker, Julia Henckel and Alan. R. Fersht
Cambridge University Chemical Laboratory and