Thermodynamics of p53 binding to hdm2(1-126): effects of phosphorylation and p53 peptide length.
Upon exposure to DNA-damaging agents, the p53 tumor suppressor protein is stabilized and activated, leading to cell cycle arrest, DNA repair, or apoptosis. One of the major factors that regulates the level and the transcriptional activity of p53 is the hdm2 oncoprotein. hdm2 binds to the N-terminal transactivation domain of p53 to block the transcriptional activity of p53 directly. hdm2 also functions as the E3 ligase that ubiquitinates p53 for proteasome degradation. Fluorescence anisotropy was employed to measure directly the binding of hdm2(1-126) to a p53 N-terminal peptide labeled with Oregon Green (an analogue of fluorescein). Phosphorylation of Ser15 and Ser2O did not affect the binding of the p53 peptide to hdm2. Thrl8 phosphorylation, on the other hand, reduced the binding by at least 20-fold. This suggests that phosphorylation of Thr18 could be a regulatory mechanism that disrupts the hdm2-p53 complex, thus activating p53 in response to DNA damage. The effect of p53 peptide length on binding to hdm2 was also measured quantitatively. Interestingly, p53(18-26) exhibits 10-fold higher affinity to hdm2 than do longer peptides (20- or 35-mer). This result may reflect a strong entropic barrier to binding for the longer peptides.
Lai
Z
Z
Chemical Enzymology, DuPont Pharmaceuticals Company, Wilmington, Delaware 19840, USA. zhihong.lai@dupontpharma.com
Auger
K R
KR
Manubay
C M
CM
Copeland
R A
RA
D018360:Crystallography, X-Ray; D005454:Fluorescence Polarization; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008958:Models, Molecular; D009687:Nuclear Proteins; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2; D013816:Thermodynamics; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000378:metabolism*
D046911:Macromolecular Substances; D009687:Nuclear Proteins; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D016159:Tumor Suppressor Protein p53; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2