Table 4.
Variations in binding affinities (Kd) and free energies (ΔΔG) between BCL-xL and p53 upon mutagenesis of interfacial residues*
| p53 N-D-T mutant | Wild Type | K120E | H178D | E180K | D186A | R248Q | C277W | H178D E180K | Bac-p53 |
|---|---|---|---|---|---|---|---|---|---|
| Kd (μM) | 1.3±0.1 | 150±20 | 41±2 | 13±1 | 27±3 | 24±1 | 22±2 | 210±20 | 1.5±0.4 |
| ΔΔG Wild Type** | 0 | +2.81 | +2.04 | +1.36 | +1.80 | +1.73 | +1.67 | +3.01 | n.d. |
| BCL-xL mutant | Wild Type | K20A | R103E | F105A | H113A | I114A | D156K | E158K | K20A I114A | E158K I114A |
|---|---|---|---|---|---|---|---|---|---|---|
| Kd (μM) | 1.9±0.4 | 36±2 | 46±7 | 26±3 | 35±5 | 42±7 | 8±1 | 7±1 | >100 | >100 |
| ΔΔG Wild Type** | 0 | +1.74 | +1.89 | +1.55 | +1.72 | +1.83 | +0.85 | +0.77 | >+2.3 | >+2.3 |
*
Binding between wild-type or mutant p53 N-D-T and wild-type fluorescent BCL-xL (F-BCL-xL) was measured by fluorescence polarization titrations (top rows). Binding between wild-type or mutant BCL-xL and wild-type p53 N-D-T was measured by competition titrations against F-BCl-xL (bottom rows). Error ranges indicate uncertainties of fit for curves obtained from the average of three independent titrations.
**
Determined as follows: ΔΔG = −RT*ln(Kd mut/Kd wild type); values in Kcal/mol