Table 1. Ligand library of BRCT used for binding affinity exploration and study of flexibility of binding site.
Tetrapeptides | |||
No. | Sequence | IC50 (μM) a | ΔΔGexp (kcal/mol) |
P1 | Ac-pSPTF-COOH | 1.0±0.2 | 0 |
P2 | Ac-pSPVF-COOH | 1.6±0.3 | 0.28 |
P3 | Ac-pSPVF-CONH2 | 3.2±0.8 | 0.69 |
P4 | Ac-pSPTF-CONH2 | 4.6±0.9 | 0.91 |
P5 | Ac-pSPIF-CONH2 | 7.1±1.4 | 1.17 |
P6 | Ac-pSPTY-CONH2 | 14.9±2.8 | 1.61 |
P7 | Ac-pSATF-CONH2 | 15.0±1.7 | 1.61 |
P8 | Ac-pSPLF-CONH2 | 18.4±1.8 | 1.74 |
P9 | Ac-pSPSF-CONH2 | 30.1±7.2 | 2.03 |
P10 | Ac-pSPAF-CONH2 | 35.0±7.9 | 2.12 |
P11 | Ac-γcEPTF-CONH2 | 52.8±1.6 | 2.36 |
P12 | Ac-pSAAF-CONH2 | 98.4±23.1 | 2.74 |
P13 | Ac-pSPPF-CONH2 | >250 | >3.29 |
P14 | Ac-pTPTF-CONH2 | >250 | >3.29 |
Compounds | |||
No. | Structure | IC50 (μM) | ΔΔGexp (kcal/mol) |
C1 | See Fig 2 | 0.31 | -0.70 |
N1 | See Fig 2 | >250 | >3.29 |
D1 | See Fig 2 | N/A | N/A |
Long peptides | |||
No. | Sequence | Kd (μM) b | ΔGexp (kcal/mol) |
L1 | ISRSTpSPTFNKQ | 0.9 | -8.30 |
L2 | PTRVSpSPVFGA | 3.7 | -7.46 |
L3 | AAYDIpSQVFPFA | 0.4 | -8.78 |
L4 | PQpSPTFPEAG | 5.2 | -7.25 |
The major binding residues, pSer and Phe (P+3), are in bold. The relative binding free energy for ligand X (X = P2–P14, C1, N1) to ligand P1 is approximated using the half maximal inhibitory concentration IC50 as ΔΔGexp = RT ln IC50(X)/IC50(P1) based on equation ΔG = RT ln Kd = RT ln(IC50 + 0.5Cenzyme) ≈ RT ln IC50 [44, 45]. Binding free energies for L1–L4 are calculated through equation ΔGexp = RT ln (Kd).