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 ΔΔG_exp = RT ln IC50(X)/IC50(P1) based on equation ΔG = RT ln K_d = RT ln(IC50 + 0.5C_enzyme) ≈ RT ln IC50 [44, 45]. Binding free energies for L1–L4 are calculated through equation ΔG_exp = RT ln (K_d).

^a IC50 values of P1–P14 were taken from ref [24]. IC50 values of C1 was taken from ref [27].

^b K_d values of L1–L4 were taken from ref [46], ref [47], ref [48] and ref [49], respectively.