Various substrates were moved out via binding with MRP4 transporters. The export of inhibitors and bile acids can confer drug resistance and bile recirculation, respectively. Three MRP1 proteins and a P-gp, used as the four PDB templates, showed an open inward-facing conformation with inner cavity, which appears to be suitable for substrate uptake. These templates provided a possible structural foundation to perform in silico prediction of ligand binding by docking simulation. Thus, we investigated ligands and their binding sites for CsMRP4 using two methods. First, probable ligands were analyzed based on the identification of analogs with similar binding sites as the solved structures using COACH . Then, Mg2+ and ATP were predicted to bind to NBD1 and NBD2 of CsMRP4 based on data for HsMRP1  (Table 1 and Fig. 2a). Mg2+ is necessary for ATP hydrolysis and results in the formation of Mg-ATP dimers . Moreover, the cyclic peptide inhibitor, QZ59-SSS (a.k.a. OZ-VAL or 2 J8), was predicted to bind to CsMRP4 according to the ligand-bound pockets of three experimentally characterized P-glycoproteins [43–45] (Table 1). Among them, Ile at the position 1093 was commonly involved in coordinating the inhibitor.Table 1Ligand and ligand-binding residues predicted using COACHLigandRegionConsensus binding residuesPDB templateATPNBD1W412, T420, V440, G441, C442, G443, K444, S445, S446, Q4732CBZ_AMg2+ NBD1S445, Q4732CBZ_AAMP-PNPa NBD2Y1185, A1192, T1212, G1213, A1214, G1215, K1216, S1217, S1218, V1227, Q1258, E1338, H13692ONJ_AMg2+ NBD2S1217, Q1258, D1337, E1338, V13674FWI_B2J8CavityL94, P98, M101, S348, L864, I1093, V10974M2T_AQZ59-VALCavityP98, I10934Q9J_A0JZCavityY349, L353, I1093, I1118, V11223G61_B aAMP-PNP is an ATP analogue 