TABLE 1.
Summary of dissociation constants for ASPP2-CT and iASPP-CT binding to various constructs of p53 determined by NMR, ITC, or fluorescence
The quaternary states of the p53 constructs that contain the oligomerization domain are indicated: monomer (m), dimer (d), and tetramer (t). NMR data were recorded at 17 °C, whereas the ITC and fluorescence data were collected at 12 °C.
| p53 variants | ASPP variants | Kd (NMR) | Kd (ITC) | Kd (fluorescence) |
|---|---|---|---|---|
| μm | μm | μm | ||
| p53-PCD | ASPP2-CT | 1.3 ± 0.2a | 1.5 ± 0.1a | |
| p53-PCD | iASPP-CT | 76.7 ± 10.0a | —b | |
| p53-PCD2F | ASPP2-CT | 2.2 ± 0.6c | 2.3 ± 0.3a | 2.8 ± 0.6a |
| p53-PCD2F | iASPP-CT | 107.5 ± 12.1c | —b | —b |
| p53-PCD2F-L | ASPP2-CT | 2.2 ± 0.9c | 3.0 ± 0.5c | |
| p53-PCD2F-L | iASPP-CT | 11.1 ± 1.7c | 7.7 ± 5.9c | |
| p53-PCD2F-L-OD (t) | ASPP2-CT | 2.7 ± 0.6c | ||
| p53-PCD2F-L-OD (t) | iASPP-CT | 7.6 ± 1.9c | ||
| p53-PCD2F-L-OD-BD (t) | ASPP2-CT | 1.7 ± 0.2, 33 ± 8c,d | 1.9 ± 0.3c | |
| p53-PCD2F-L-OD-BD (t) | iASPP-CT | —e | 4.6 ± 1.2c | |
| p53-PCD2F-L-OD(L344R)-BD (d) | ASPP2-CT | 1.9 ± 0.2, 29 ± 7c,d | 1.7 ± 0.3c | |
| p53-PCD2F-L-OD(L344R)-BD (d) | iASPP-CT | —e | 3.3 ± 1.5c | |
| p53-PCD2F-L-OD(L344P)-BD (m) | ASPP2-CT | 1.5 ± 0.7c | 1.4 ± 0.3, 18 ± 5c,d | 1.6 ± 0.2c |
| p53-PCD2F-L-OD(L344P)-BD (m) | iASPP-CT | 4.3 ± 1.1c | —e | 3.0 ± 0.6c |
| p53-L | ASPP2-CT | 39.7 ± 4.8c | ||
| p53-L | iASPP-CT | 15.9 ± 2.0c | ||
| p53-L-OD-BD (t) | ASPP2-CT | 38.9 ± 5.0c | ||
| p53-L-OD-BD (t) | iASPP-CT | 17.8 ± 0.9c |
a
Kd values were obtained in the presence of 50 mm NaCl.
b
ITC and fluorescence experiments were not performed for these pairs due to weak interaction.
c
Kd values were obtained in the presence of 150 mm NaCl.
d
The titration curve was best fitted to two sequential binding events.
e
ITC data using iASPP-CT were not analyzed, because the sample precipitated during the experiment.