TABLE 1.
Effects of mutations, truncations and insertions in APC Repeat 3 region on the interaction with β-catenin. p-APC indicates phosphomimetic mutations, APC indicates non-phosphorylated form. Presence or deletion of subdomains a, b and c is indicated in subscript. FL indicates flexible linker, RL indicates rigid linker, PP indicates polyproline linker (see also Figure 5A), and the site of linker insertion is indicated in subscript. In the schematics, the key contacting residues are denoted in circles (phosphomimetics) and triangles (lysine-binding residues). ND indicates not detectable. The values of K d , ΔG and Hill coefficient are derived from the fluorescence anisotropy experiments. The on- and off-rates and time constant (τ, calculated from the fast off-rates) were determined by stopped-flow fluorescence experiments. ND indicates not detectable. The SE error come from at least three replicates.
| APC fragment | Schematic of APC R3 region | K d (nM) | ΔG (kcal mol−1) | Hill coefficient | k on (µM−1s−1) | k off fast (s−1) | k off slow (s−1) | τ (s) |
|---|---|---|---|---|---|---|---|---|
| APCabc |
|
16.5 ± 5.1 | −10.6 ± 0.1 | 1.09 ± 0.1 | 49.2 ± 5.1 | 0.46 ± 0.06 | 0.11 ± 0.02 | 1.54 ± 0.23 |
| APCab |
|
89.9 ± 26.6 | −9.6 ± 0.2 | 1.3 ± 0.5 | 13.3 ± 1.7 | 1.12 ± 0.31 | 0.24 ± 0.032 | 0.66 ± 0.18 |
| APCbc |
|
196 ± 18.7 | −9.2 ± 0.1 | 1.1 ± 0.1 | 14.8 ± 1.5 | 0.73 ± 0.06 | ND | 0.96 ± 0.08 |
| APCb |
|
246 ± 3.5 | −9.0 ± 0.1 | 0.9 ± 0.1 | — | — | — | — |
| pAPCabc |
|
5.3 ± 0.6 | −11.3 ± 0.1 | 2.0 ± 0.2 | 207 ± 11 | 0.07 ± 0.01 | 0.0021 ± 0.003 | 9.51 ± 0.53 |
| pAPCc |
|
160 ± 37.0 | −9.2 ± 0.2 | 1.1 ± 0.2 | — | — | — | — |
| APCabcD1486S |
|
2054 ± 213 | −7.8 ± 0.1 | 0.8 ± 0.1 | ND | ND | ND | ND |
| pAPCabcD1486S |
|
21.4 ± 1.2 | −10.5 ± 0.1 | 1.2 ± 0.3 | 151 ± 14 | 2.89 ± 0.01 | 0.400 ± 0.002 | 0.24 ± 0.006 |
| APCabcFL1500 |
|
28.3 ± 5.9 | −10.3 ± 0.1 | 1.0 ± 0.1 | 24.1 ± 7.0 | 0.45 ± 0.04 | 0.10 ± 0.003 | 1.54 ± 0.13 |
| APCabcRL1500 |
|
31.2 ± 3.6 | −10.3 ± 0.1 | 0.9 ± 0.1 | 18.1 ± 1.6 | 0.89 ± 0.02 | 0.223 ± 0.002 | 0.80 ± 0.14 |
| pAPCabcFL1500 |
|
10.9 ± 2.4 | −10.6 ± 0.1 | 1.8 ± 0.1 | 101.0 ± 9.6 | 0.16 ± 0.18 | 0.0085 ± 0.0015 | 4.45 ± 1.16 |
| pAPCabcRL1500 |
|
10.8 ± 2.9 | −10.9 ± 0.2 | 2.0 ± 0.3 | 102.5 ± 7.9 | 0.21 ± 0.02 | 0.0122 ± 0.0016 | 3.52 ± 0.78 |
| pAPCabcPP1500 |
|
16.4 ± 2.5 | −10.6 ± 0.1 | 1.2 ± 0.2 | 89.7 ± 5.1 | 0.44 ± 0.035 | 0.018 ± 0.002 | 1.59 ± 0.24 |
| APCabcFL1478 |
|
21.5 ± 3.5 | −10.5 ± 0.1 | 1.4 ± 0.2 | 83.5 ± 2.4 | 0.77 ± 0.038 | 0.177 ± 0.007 | 0.90 ± 0.04 |
| pAPCabcFL1478 |
|
18.7 ± 0.17 | −10.5 ± 0.1 | 1.2 ± 0.2 | — | — | — | — |