Single-molecule analysis of interaction between p53TAD and MDM2 using aerolysin nanopores

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Title
Single-molecule analysis of interaction between p53TAD and MDM2 using aerolysin nanopores
Author(s)
Sohee Oh; Mi-Kyung LeeSeung-Wook Chi
Bibliographic Citation
Chemical Science, vol. 12, no. 16, pp. 5883-5891
Publication Year
2021
Abstract
Protein?protein interactions (PPIs) are regarded as important, but undruggable targets. Intrinsically disordered p53 transactivation domain (p53TAD) mediates PPI with mouse double minute 2 (MDM2), which is an attractive anticancer target for therapeutic intervention. Here, using aerolysin nanopores, we probed the p53TAD peptide/MDM2 interaction and its modulation by small-molecule PPI inhibitors or p53TAD phosphorylation. Although the p53TAD peptide showed short-lived (<100 ms) translocation, the protein complex induced the characteristic extraordinarily long-lived (0.1 s ∼ tens of min) current blockage, indicating that the MDM2 recruitment by p53TAD peptide almost fully occludes the pore. Simultaneously, the protein complex formation substantially reduced the event frequency of short-lived peptide translocation. Notably, the addition of small-molecule PPI inhibitors, Nutlin-3 and AMG232, or Thr18 phosphorylation of p53TAD peptide, were able to diminish the extraordinarily long-lived events and restore the short-lived translocation of the peptide rescued from the complex. Taken together, our results elucidate a novel mechanism of single-molecule sensing for analyzing PPIs and their inhibitors using aerolysin nanopores. This novel methodology may contribute to remarkable improvements in drug discovery targeted against undruggable PPIs.
ISSN
2041-6520
Publisher
Royal Soc Chem
DOI
http://dx.doi.org/10.1039/d1sc00386k
Type
Article
Appears in Collections:
Division of Biomedical Research > Disease Target Structure Research Center > 1. Journal Articles
Division of Biomedical Research > 1. Journal Articles
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