Mechanism of initiation of aggregation of p53 revealed by Φ-value analysis

摘要

Many oncogenic mutations inactivate the tumor suppressor p53 by destabilizing it, leading to its rapid aggregation. Small molecule drugs are being developed to stabilize such mutants. The kinetics of aggregation of p53 is deceptively simple. The initial steps in the micromolar concentration range follow apparent sigmoidal sequential first-order kinetics, with rate constants k1 and k2. However, the aggregation kinetics of a panel of mutants prepared for Φ-value analysis has now revealed a bimolecular reaction hidden beneath the observed first-order kinetics. Φu measures the degree of local unfolding on a scale of 0–1. A number of sequential Φu-values of ∼1 for k1 and k2 over the molecule implied more than one protein molecule must be reacting, which was confirmed by finding a clear concentration dependence at submicromolar protein. Numerical simulations showed that the kinetics of the more complex mechanism is difficult, if not impossible, to distinguish experimentally from simple first order under many reaction conditions. Stabilization of mutants by small molecules will be enhanced because they decrease both k1 and k2. The regions with high Φu-values point to the areas where stabilization of mutant proteins would have the greatest effect.