To further explain the energy differences between

. To further explain the energy differences between Group-A and Group-B, the average distances between ligands and the zinc cation were obtained from the last 1ns trajectory. It turns out that the members of group A were within the coordination distance of the zinc ion, whereas those of group B stayed out of the distance limit of 2.4Å. These average distances of the ligands to zinc versus the internal electrostatic interaction energies and the predicted binding free energies are shown in . As the distance between ligand and zinc decreases, Δ and becomes more negative. As a result, the members in Group-A exhibited stronger inhibitory activities than those in Group-B. This peculiar relationship between the ligand-zinc distance and the electrostatic interaction energy strongly suggests that coordination with zinc is a crucial ipi-145 in the ligand-binding process. . The chemical structures of the representative NASIDs shown in are quite similar. They are comprised of two ring moieties (moiety A and moiety B), which are connected by the carbonyl linker (except fenoprofen). The interaction patterns were represented by average conformations, which were generated by averaging 100 snapshots from the last 1ns MD trajectory. However, they exhibited diverse conformations and binding modes as simulation progresses. This may be attributed to the flexibility of the protein. To better understand the intrinsic features of the ligand-binding site which account for both the energetic and binding difference of these molecules, the ligand-binding site of GLOI is subdivided into four subsites (A, B, C, and D) as shown in . Subsite A consists of Leu69B, Leu92B, Met179A, Met183A, and Phe71B and is represented by bulky hydrophobic groups. Subsite B contains the zinc ion and other residues which are responsible for coordination. Subsite C is located in the flexible loop area and displays fluctuating conformation. It consists of Lys150A, Gly155A, Lys156A, Met157A, Leu160A, and Phe162A. Subsite D is formed by polar residues (Arg122A, Asn103B, and Arg37B) as well as nonpolar residues (Phe67B and Trp170A). . The binding mode of indomethacin (at 10ps intervals for the final 1ns) is depicted in . On top of it, the carbonyl linker between moiety A and B forms a coordination bond of 2.24Å with the zinc ion. This has been recognized as the key element of ligand binding. The -methylphenyl of indomethacin (moiety A) is buried inside subsite A, surrounded by Leu160A, Met179A, Leu69B, and Phe62B. However, the pocket is not fully occupied comparing with curcumin (), which has a larger group of moiety A to saturate the pocket. The B moiety of indomethacin is bulky enough to take up both subsites C and D, delimited by Phe162A, Met35B, Met65B, Phe67B, and Thr101B. These interactions were also observed in NMR titration experiments. Besides, the carboxyl groupof moiety B points toward the opening of the active site pocket and establishes weak hydrogen bonds or electrostatic interactions with Arg37B located in subsite D. However, the carboxyl groupof moiety B of indomethacin does not make strong contact with those electropositive groups because trichocysts is not long and flexible enough. . In comparison, the other ligands studied here tend to contact with subsite C or subsite D exclusively. This explains why indomethacin possesses the most favorable Δ (−38.88kcal/mol) in binding. As shown in , the moiety B of curcumin binds to subsite C, leaving subsite D unoccupied. Tolmetin binds to GLOI in a similar manner as indomethacin, except that the size of ring B is smaller than that of indomethacin and subsite C is not filled, which results in weaker van der Waals interactions (Δ=−31.31kcal/mol). A flexible loop (152–159) was described to be responsible for the binding of ligands in our previous study. The loop is located over the active site, and is proposed to be open in the absence of a bound ligand. To illustrate this mechanism, both the trajectories of the protein with bound ligand and the one were analyzed. The open and close of the pocket can be monitored by the distance between the CA atoms of Lys156A and Ile64B, which are located on the two opposite sides of the opening of the pocket. The distances are monitored in two different systems, as shown in . It turns out that the indomethacin-GLOI complex exhibits a shorter distance (about 2Å) than the -protein, indicating that the loop tends to be in a more closed conformation with indomethacin. The B moiety of indomethacin is bulky and rigid, and occupies both subsite C and subsite D, which suggests that bulky and rigid components in moiety B are required to stabilize the flexible loop.