Table 1.
Binding and kinetics parameters of various VH domains against eIF4E
| Kd (nM) | kon (S−1M−1) | koff (S−1) | |
|---|---|---|---|
| VH-1A2 | 115.2 ± 4.4 | 3.43 × 105 ± 3.75 × 104 | 3.78 × 10−2 ± 3.17 × 10−3 |
| VH-1C5 | 154.3 ± 70.3 | 2.85 × 105 ± 8.5 × 104 | 4.11 × 10−2 ± 6.99 × 10−3 |
| VH-1C5D104A (VH-M4) | 4.8 ± 1.7 | 2.46×106 ± 3.89 × 104 | 1.18 × 10−2 ± 3.90 × 10−3 |
| VH-1C5D104A/S108R | 0.94 ± 0.024 | 2.63 × 106 ± 2.98 × 105 | 2.48 × 10−3 ± 3.79 × 10−4 |
| VH-1C5D104A/F120I (VH-S2) | 0.64 ± 0.06 | 1.18 × 106 ± 1.85 × 105 | 7.64 × 10−4 ± 1.80 × 10−4 |
| VH1-C5D104A/S108R/F120I (VH-S4) | 0.057 ± 0.004 | 5.18 × 106 ± 8.55 × 105 | 2.96 × 10−4 ± 6.47 × 10−5 |
| 4E-BP14ALA (1:1) | 0.60 ± 0.14 | 1.05 × 1010 ± 1.48 × 109 | 5.93 ± 0.56 |
| kon1, kon2 | koff1, koff2 | ||
| 4E-BP14ALA (2 state) | 0.68 ± 0.13 | 8.35 × 106 ± 4.90 × 106,1.70 × 10−2 ± 1.08 × 10−2 | 3.19 × 10−4 ± 7.91 × 10−5, 1.84 × 10−4 ± 7.00 × 10−5 |
Kd, kon and koff values were derived from SPR experiments using either single cycle or multi cycle injection experiments against eIF4E amine coupled to a CM5 sensorchip. Parameters were derived using a 1:1 site binding model. However, with regards to 4E-BP14ALA, binding parameters were initially determined using a 1:1 site binding model, which generated a poor fit and physically irrelevant Kon and Koff values beyond the detection limit of the machine. This analysis was superseded with a 2-state analysis, which generated more meaningful kinetic parameters. The use of a 2-state model is supported by the following reasons: (1) 4E-BP14ALA undergoes a disorder to order transition upon binding eIF4E and, (2) it interacts at eIF4E through 2 binding sites. Binding and kinetic data values (mean ± standard error) shown in the table were calculated from n = 2 independent experiments. Data shown for 4E-BP14ALA was calculated from n = 3 independent experiments. Source data is provided in the Source Data File.