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Mechanism of An Anticancer Peptide Rescuing p53 from Degradation by COP1
Author(s) -
Gao Meng,
Huang Yongqi
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.lb28
Subject(s) - alanine , chemistry , mutagenesis , amino acid , peptide , docking (animal) , ubiquitin ligase , biochemistry , alanine scanning , ubiquitin , stereochemistry , biophysics , mutation , biology , gene , medicine , nursing
A 28 amino‐acid peptide derived from azurin (amino acids 50–77, p28) preferentially enters human cancer cells and induces apoptosis or growth inhibition. Previous studies have revealed that p28 exerts its anticancer activity mainly by interacting with the p53 DNA binding domain (DBD) and interfering in the p53 degradation signaling pathway. However, the interacting mechanisms between p28 and p53‐DBD are missing and how p28 inhibits p53‐DBD ubiquitination remains largely unknown. In this work, we characterized the interacting mechanisms between p28 and p53‐DBD by a combination of NMR, X‐ray crystallography, ITC and computer simulations. Interactions between p28 and p53‐DBD were mainly mediated by hydrophobic contacts. Structural models of the p28/p53‐DBD complex obtained with NMR datadriven docking indicated that p28 adopted an α‐helix upon binding to p53‐DBD. Val59, Val60, Thr61, Tyr72 and Leu73 of p28 made extensive interactions with the L1 loop and S7–S8 loop of p53‐DBD. Site‐directed mutagenesis and ITC characterization showed that single alanine substitution of the above mentioned hydrophobic residues reduced the binding affinity by more than one order of magnitude. GST pull‐down assays showed that the interactions between p53‐ DBD and the E3 ligase COP1 were blocked by p28. Since alanine substitution of key hydrophobic residues reduced the binding affinity of p28 with p53‐DBD, alanine mutated p28 were less efficient in blocking the p53‐DBD/COP1 interaction. Determination of the p28/p53‐ DBD complex structure is in progress, which will allow us to rationally design p28 derived peptides exhibiting higher affinity with p53‐DBD and stronger anticancer activity in the future. Support or Funding Information This work was supported by National Natural Science Foundation of China (Grant number: 21603121, Y.H.) and Hubei University of Technology (M. G. and Y.H.). This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .