Figure 2. Water molecules in the simulated ligand binding and a putative allosteric binding site.

(A) Ligand RMSD with respect to the native pose as a function of time, for each binding simulation; each conformation is superimposed on the X-ray structures of the protein. Poses a–f refer to non-native PP1 poses, while N refers to the native poses of PP1 and dasatinib. Specifically, b–e refer to poses in which PP1 remains for 1 µs or more, while a and f refer to poses immediately preceding the native pose. In Simulation 2, PP1 binds via an alternative pathway that circumvents pose a. Poses e and f are observed only in Simulation 2, one in the ATP-binding site and the other in the extended ATP-binding site adjacent to the αC helix. (B) Water density (blue surfaces) in the native pose and in intermediate pose a, from a PP1 binding simulation. The simulated water density for the native pose, particularly that of the enclosed water (indicated by the red arrow), mirrors that of the X-ray structure. In pose a, which immediately precedes binding, a single layer of water molecules separates the ligand from the binding pocket surface. (C) A close-up of the G-loop site occupied by PP1, which is underformed and too small to accommodate PP1 in the X-ray structure from which the simulation was initiated (PDB entry 1Y57). (D) In an intermediate state preceding dasatinib binding by 130 ns, a layer of water molecules separates dasatinib from the protein. In both (B) and (C), the protein is clipped to show the otherwise enclosed region.