Single-Molecule Nanopore Profiling of p53-TAD Conformational Dynamics, Interactions, and Inhibition

Researchers have developed a novel nanopore-based method to study the conformational dynamics and interactions of intrinsically disordered proteins (IDPs) at the single-molecule level. The technique was applied to the transactivation domain of tumor suppressor p53, a protein of significant importance in cancer biology. The study revealed that the nanopore platform, MspA, can capture p53-TAD and distinguish up to six different current states, with the protein frequently transitioning between them, showcasing a complex conformational landscape.

The nanopore was also capable of detecting the impact of a cancer-associated double mutational variant, N29K/N30D, of p53-TAD. By integrating experimental results with steered molecular dynamics simulations, the researchers found that the mutant sampled compact conformational states more often than the wild-type protein, aligning with earlier NMR studies.

Moreover, the MspA platform allowed for real-time observation of the interaction between the E3 ligase MDM2 and p53-TAD, and how this binding was inhibited by the anti-cancer compound epigallocatechin gallate (EGCG). The findings showed that EGCG stabilizes one of the six conformational states sampled by p53-TAD, offering a mechanistic rationale for its inhibitory effect on the protein.

In summary, this study highlights the potential of the single-molecule nanopore platform for the label-free monitoring of IDP conformational dynamics, modulation, binding, and inhibition at an unprecedented level of detail.